host-pathogen interactions of porphyromonas gingivalis
TRANSCRIPT
Host-Pathogen Interactions of
Porphyromonas gingivalis
Jiamin Aw
ORCID 0000-0002-0973-9376
Submitted in total fulfilment of the requirement of the degree of
Doctor of Philosophy
April 2018
Melbourne Dental School
The University of Melbourne
i
Declaration
This is to certify that:
(i) the thesis comprises only my original work towards the PhD, except when indicated
in the Preface,
(ii) due acknowledgment has been made in the text to all other material used,
(iii) the thesis is fewer than 100,000 words in length, exclusive of tables, maps,
bibliographies and appendices.
Jiamin Aw
Melbourne Dental School
The University of Melbourne
April 2018
ii
Preface
In accordance with the regulations of The University of Melbourne, I acknowledge that some of
the work presented in this thesis was collaborative. Specifically:
(i) In Chapter 6, cryo-electron microscope images of P. gingivalis W50 and P. gingivalis
ΔPG0382 were acquired by Dr. Yu-Yen Chen (The University of Melbourne).
The remainder of this thesis comprises only my original work.
iii
Publications
The work presented in this thesis has given rise to the following publication:
Aw, J., Scholz, G.M., Huq, N.L., Huynh, J., O’Brien‐Simpson, N.M., Reynolds, E.C. (2018), “Interplay
between Porphyromonas gingivalis and EGF signalling in the regulation of CXCL14”, Cellular
Microbiology, e12837
The work done during my PhD also contributed to the following publications:
Scholz, G.M., Heath, J.E., Aw, J. and Reynolds, E.C. (2018), “ Regulation of the petidoglycan
amidase PGLYRP2 in epithelial cells by IL-36”, Infection and Immunity, doi:
10.1128/IAI.00384-18
Huynh, J., Scholz, G.M., Aw, J. and Reynolds, E.C. (2017), “Interferon Regulatory Factor 6
Promotes Keratinocyte Differentiation in Response to Porphyromonas gingivalis”, Infection and
Immunity, Vol. 85 No. 5, pp. 1-12.
Huynh, J., Scholz, G.M., Aw, J., Kwa, M.Q., Achuthan, A., Hamilton, J.A. and Reynolds, E.C. (2016),
“IRF6 Regulates the Expression of IL-36 by Human Oral Epithelial Cells in Response to
Porphyromonas gingivalis”, The Journal of Immunology, Vol. 196 No. 5, pp. 2230–2238.
Kwa, M.Q., Huynh, J., Aw, J., Zhang, L., Nguyen, T., Reynolds, E.C., Sweet, M.J., Hamilton, J.A.,
Scholz, G.M. (2014), “Receptor-interacting protein kinase 4 and interferon regulatory factor 6
function as a signalling axis to regulate keratinocyte differentiation”, Journal of Biological
Chemistry, Vol. 289 No. 45, pp. 31077-31087.
iv
Abstract
Periodontal health is supported by various host immune defence mechanisms, which act in
concert to maintain host-microbe homeostasis. However, breakdown of homeostasis can lead to
the development of chronic periodontitis, an inflammatory disease that causes the destruction
of periodontal tissues. Pattern recognition receptors, including Toll-like receptors (TLRs),
enable the detection of microorganisms and subsequent activation of the host immune
response. The modular intracellular Toll/Interleukin-1 receptor (TIR) domain of TLRs forms
heterotypic interactions with intracellular adaptor proteins, such as MAL and MYD88, to
activate downstream signalling pathways to regulate the transcription of inflammatory genes
(e.g. cytokines and chemokines). P. gingivalis is a major periodontal pathogen and can disrupt
homeostasis between the host and tooth-accreted subgingival biofilm (plaque) by dysregulating
the immune response. The extracellular gingipain proteases (Kgp and RgpA/B) produced by
P. gingivalis are potent virulence factors, which can stimulate as well as proteolytically degrade
host immunomodulatory factors. Consequently, an otherwise host-protective immune response
can become destructive and cause tissue pathology when dysregulated by P. gingivalis.
This thesis has characterised two facets of the interaction between the host and P. gingivalis.
The first part of this thesis investigated the molecular regulation of the orphan chemokine,
CXCL14, by P. gingivalis in oral epithelial cells (e.g. OKF6 cells). By using an isogenic P. gingivalis
gingipain (Kgp/Rgp) protease-deficient mutant and a cysteine protease inhibitor, the
stimulation of CXCL14 expression was shown to be mediated by the gingipain proteases. Given
this finding, a role for protease-activated receptors (PARs) in the regulation of CXCL14
expression was investigated. Gene silencing experiments revealed that P. gingivalis-stimulated
CXCL14 expression occurs in a PAR-3-dependent manner. Notably, CXCL14 expression was
found to be transcriptionally repressed in response to epidermal growth factor-induced
activation of the MEK-ERK1/2 pathway. However, P. gingivalis can overcome the repression of
CXCL14 via the gingipain protease-mediated degradation of EGF. Therefore, P. gingivalis not
only directly stimulates CXCL14 expression via PAR-3, but also promotes its expression by
antagonising EGF signalling.
The functions of CXCL14, including its ability to regulate inflammatory gene expression and oral
epithelial cell migration, were also investigated. No evidence was obtained to indicate that
CXCL14 regulates inflammatory gene expression in oral epithelial cells (e.g. OKF6 cells) or
macrophages (e.g. RAW 264.7 cells), or oral epithelial cell migration. Furthermore, CXCL14 did
not induce changes in the expression levels of inflammatory genes when injected into the mouse
gingiva. However, CXCL14 was shown in vitro to potently kill oral Streptococcus species.
v
Significantly, P. gingivalis was resistant to CXCL14 killing, most likely because the gingipain
proteases can degrade CXCL14. Therefore, the dysregulation of CXCL14 by P. gingivalis may
potentially destabilise the proportions of bacterial species in the tooth-accreted biofilm and
thereby promote biofilm dysbiosis.
In the second part of this thesis, the expression and function of TIR domain-containing proteins
(Tcps) by P. gingivalis was investigated. Other bacterial pathogens have been shown to express
Tcps as a mechanism of molecular mimicry to subvert TLR-mediated immune responses.
Bioinformatics analysis identified eleven putative Tcps (e.g. PG0382) in nine different strains.
Further analyses revealed that the putative TIR domain in the C-terminal half of PG0382
contains sequence features similar to TLR adaptor proteins and bacterial Tcps. Homology
modelling of PG0382 suggests that the domain may adopt a TIR-like structure. Like other
bacterial Tcps, PG0382 is predicted to also contain a coiled-coil motif in the N-terminal half of
the protein, which may facilitate homodimerisation. Functional characterisation of PG0382 by
transient expression in human HEK293T cells and analysis by Western blotting and
immunofluorescence confocal microscopy revealed that the N-terminal half of PG0382 appears
to largely dictate its subcellular localisation. Moreover, co-expression of PG0382 strongly
reduced MAL and MYD88 protein levels. Therefore, P. gingivalis PG0382 may have potential
immunomodulatory functions.
An isogenic P. gingivalis PG0382-deficient mutant (P. gingivalis ΔPG0382) was created to
establish a role for PG0382 in modulating the host immune response to P. gingivalis. Phenotypic
characterisation of the mutant indicates that PG0382 is not important for P. gingivalis growth,
formation of an electron-dense surface layer, or gingipain protease activity. The absence of
PG0382 did not affect the inflammatory gene response of oral epithelial cells (e.g. OKF6 cells)
towards P. gingivalis. However, P. gingivalis ΔPG0382 stimulated a weaker inflammatory
response in macrophages (e.g. RAW 264.7 cells). A peritoneal infection model in mice was used
to further investigate a role for PG0382 in modulating the host immune response to P. gingivalis.
However, the absence of PGO382 did not affect the recruitment of neutrophils and
inflammatory monocytes in response to P gingivalis infection. Further studies will be required
to determine whether PG0382 has a role in P. gingivalis immune subversion.
This thesis has identified and defined novel interactions between host-derived and P. gingivalis-
derived factors in modulating the immune response. Moreover, it provides a molecular basis for
exploring potential roles for CXCL14 and P. gingivalis PG0382 in the development and
progression of chronic periodontitis.
vi
Acknowledgements
This thesis is the culmination of my PhD journey, which would not have been possible or as
enjoyable without the support and encouragement of numerous people. I would like to start by
expressing my deepest gratitude to my supervisor Associate Professor Glen Scholz, whose
enthusiasm for scientific research knows no bounds. His unwavering belief in my abilities
encouraged me to challenge myself as a researcher. Without his patience and knowledge, I
would not have been able to achieve all that I have over the past few years. I would also like to
extend my thanks to my co-supervisor Professor Neil O’ Brien-Simpson for his
thought-provoking discussions. A special thanks to Professor Eric Reynolds for giving me the
opportunity to undertake my PhD under the Oral Health CRC.
My sincerest thanks go to my colleagues at the Melbourne Dental School who have offered their
valued technical support and advice. I have greatly benefited the support of Christine Seers, who
helped me devise a strategy for creating the P. gingivalis PG0382-deficient mutant. I would like
to thank Yu-Yen Chen for acquiring the cryo-EM images of P. gingivalis. I would also like to
express my gratitude towards Katrina Walsh and Alexis Gonzalez who have shared their
insightful knowledge about flow cytometry. Thank you to Dhana Gorasia, Michelle Glew and
Dina Chen for their technical advice in the lab, and also to Laila Huq, who have brightened up
the student room with her cheeriness. A special thanks to Jacqueline Heath, for the time we have
spent at the Bio21 BRF developing a mouse model, which seemed impossible at the time. And
also, for your insightful knowledge into life and research; your passion and determination is
truly inspirational.
I am also thankful to have met some wonderful friends over the past few years. I am especially
grateful to Mei Qi Kwa and Jennifer Huynh, who not only shown me the ropes in the lab when I
first started in my Honours, but also shared their contagious love of research (and cute animals)
with me. I would also like to thank Li-Ming Bhutta and Ben Huang for their support and
encouragement throughout this journey.
Importantly, this work would not have been possible without the support of my family. Mummy
and Baba, I wish to thank you for supporting my pursuit in this quest. You have both given me
the courage and belief to conquer the impossible. Last but not least, to my best friend and
partner, John Huang. Thank you for being extraordinarily tolerant and supportive. You were my
pillar of strength when I faltered; this journey would not have been completed without you.
vii
Table of Contents
Declaration ............................................................................................................................................................................i
Preface ................................................................................................................................................................................... ii
Publications ....................................................................................................................................................................... iii
Abstract ............................................................................................................................................................................... iv
Acknowledgements ........................................................................................................................................................ vi
List of Figures ................................................................................................................................................................... xii
List of Tables .................................................................................................................................................................... xiv
Abbreviations ................................................................................................................................................................... xv
Chapter 1 Introduction ................................................................................................................................................ 1
1.1 Overview of the Oral Microbial Habitat ............................................................................................... 2
Formation of the Oral Biofilm......................................................................................................... 2
1.2 Periodontal Disease ...................................................................................................................................... 5
Overview ................................................................................................................................................. 5
Bacterial Aetiology of Chronic Periodontitis ............................................................................ 5
Periodontal Disease and Systemic Health ................................................................................. 7
1.3 Host Immune Response in Periodontitis ............................................................................................. 8
Innate Immune Response in Periodontitis ............................................................................... 8
1.3.1.1 Oral Epithelial Cells ............................................................................................................................ 9
1.3.1.2 Neutrophils ......................................................................................................................................... 10
1.3.1.3 Macrophages ...................................................................................................................................... 12
1.3.1.4 Dendritic Cells .................................................................................................................................... 13
Adaptive Immune Response in Periodontitis ....................................................................... 14
1.3.2.1 T Lymphocytes .................................................................................................................................. 14
1.3.2.2 B Lymphocytes .................................................................................................................................. 15
Molecular Mediators of Host Immunity .................................................................................. 16
1.3.3.1 Complement System ....................................................................................................................... 16
1.3.3.2 Antimicrobial Mediators ............................................................................................................... 17
1.3.3.3 Cytokines ............................................................................................................................................. 19
1.3.3.4 Chemokines ........................................................................................................................................ 21
1.4 Pattern Recognition Receptors ............................................................................................................. 23
Toll-like Receptors ........................................................................................................................... 24
1.4.1.1 TLR Distribution and Subcellular Localisation .................................................................... 24
1.4.1.2 TLR Structural Organisation ........................................................................................................ 25
viii
Leucine-Rich Repeat Motif ................................................................................................... 25
Toll/Interleukin-1 Receptor (TIR) Domain .................................................................. 27
1.4.1.3 Toll-like Receptor Signal Transduction................................................................................... 28
1.4.1.4 TIR domain-containing Adaptor Proteins .............................................................................. 31
Myeloid Differentiation Primary Response 88 (MYD88) ........................................ 31
MYD88 Adaptor-Like (MAL) ............................................................................................... 31
TIR-domain-containing Adaptor-inducing Interferon- (TRIF) .......................... 32
TRIF-related Adaptor Molecule (TRAM) ........................................................................ 32
Sterile Alpha and Armadillo-motif-containing (SARM) ........................................... 32
NOD-like Receptors ......................................................................................................................... 33
Protease-activated Receptors ...................................................................................................... 37
1.5 Microbial Subversion of TLR Signalling ............................................................................................ 38
PAMP Modification .......................................................................................................................... 38
Targeting TLR Signalling Proteins ............................................................................................. 39
1.6 Bacterial TIR Domain-containing Proteins ...................................................................................... 40
Structural Properties of Bacterial Tcps ................................................................................... 41
Interference with TLR signalling by Bacterial Tcps ........................................................... 41
Functional Consequences of Bacterial Tcps .......................................................................... 42
1.7 Immune Subversion by Porphyromonas gingivalis ...................................................................... 42
P. gingivalis Gingipain Proteases ................................................................................................ 43
1.7.1.1 Gingipain Proteases and Immune Subversion ..................................................................... 43
1.7.1.2 Gingipain Proteases and Tissue Destruction ........................................................................ 44
Fimbriae ............................................................................................................................................... 45
Atypical Lipopolysaccharides ...................................................................................................... 46
SerB Phosphatase ............................................................................................................................. 47
1.8 Research Objectives .................................................................................................................................. 47
Chapter 2 Materials and Methods ........................................................................................................................ 48
2.1 Materials ........................................................................................................................................................ 49
Tissue culture reagents .................................................................................................................. 49
Bacterial culture reagents ............................................................................................................. 49
General reagents and chemicals ................................................................................................. 49
Molecular biology reagents .......................................................................................................... 49
Molecular cloning ............................................................................................................................. 49
Quantitative real-time PCR probes............................................................................................ 50
SDS-PAGE and Western Blotting ................................................................................................ 50
ix
Antibodies ............................................................................................................................................ 50
2.2 In vitro methods ......................................................................................................................................... 51
Cell culture........................................................................................................................................... 51
2.2.1.1 OKF6/TERT-2 cells .......................................................................................................................... 51
2.2.1.2 HEK293T cells.................................................................................................................................... 51
2.2.1.3 RAW 264.7 cells ................................................................................................................................ 51
Bacterial strains and culture conditions ................................................................................. 51
2.2.2.1 P. gingivalis .......................................................................................................................................... 51
2.2.2.2 Streptococcus strains ....................................................................................................................... 52
2.2.2.3 E. coli ...................................................................................................................................................... 52
Challenging of OKF6 cells and RAW264.7 cells with P. gingivalis ................................ 52
RNA interference-mediated gene silencing ........................................................................... 52
RNA purification ............................................................................................................................... 52
Reverse transcription ..................................................................................................................... 53
Quantitative real-time PCR ........................................................................................................... 53
CXCL14 Enzyme-linked immunosorbent assays (ELISA) ................................................ 53
In vitro proteolytic degradation of EGF and CXCL14......................................................... 54
LTQ Orbitrap Elite mass spectrometry .................................................................................... 54
Antibacterial assays ......................................................................................................................... 55
Wound healing assay ...................................................................................................................... 55
Transfection of HEK293T cells ................................................................................................... 55
Cell lysis ................................................................................................................................................ 55
Co-immunoprecipitation Assay .................................................................................................. 55
SDS-PAGE ............................................................................................................................................. 56
Western Blotting ............................................................................................................................... 56
Immunofluorescence staining and confocal microscopy ................................................. 56
Construction of PG0382 mammalian expression vectors................................................ 57
2.2.19.1 Polymerase Chain Reaction ....................................................................................................... 57
2.2.19.2 Bacterial transformation ............................................................................................................ 57
Generation of P. gingivalis PG0382-deficient mutant (ΔPG0382) ................................ 58
2.2.20.1 Splice Overlap Extension Polymerase Chain Reaction (SOE PCR) ............................ 58
2.2.20.2 Electroporation of P. gingivalis ................................................................................................ 58
Gingipain proteinase assay ........................................................................................................... 59
2.3 Bioinformatics methods .......................................................................................................................... 59
Sequence alignments....................................................................................................................... 59
x
Structural modelling........................................................................................................................ 59
2.4 Mouse studies .............................................................................................................................................. 59
Mice ........................................................................................................................................................ 59
Intragingival injection .................................................................................................................... 60
Mouse gingival RNA extraction ................................................................................................... 60
Intraperitoneal infection ............................................................................................................... 60
Flow cytometric analysis ............................................................................................................... 61
2.5 Statistical analysis ...................................................................................................................................... 61
Chapter 3 The regulation of CXCL14 in oral epithelial cells by Porphyromonas gingivalis.......... 68
3.1 Introduction ................................................................................................................................................. 69
3.2 Results............................................................................................................................................................. 70
P. gingivalis stimulates CXCL14 expression in human oral epithelial cells in a
TLR2-independent manner .......................................................................................................................... 70
Gingipain proteases mediate the stimulation of CXCL14 expression by P. gingivalis
.....................................................................................................................................................................73
PAR-3-dependent regulation of P. gingivalis-stimulated CXCL14 gene expression
.....................................................................................................................................................................75
EGFR signalling negatively regulates CXCL14 transcription in a MEK-dependent
manner .....................................................................................................................................................................78
P. gingivalis gingipain proteases antagonise the negative regulation of CXCL14
gene expression by EGF .................................................................................................................................. 82
3.3 Discussion ..................................................................................................................................................... 84
Chapter 4 Function of CXCL14 .............................................................................................................................. 90
4.1 Introduction ................................................................................................................................................. 91
4.2 Results............................................................................................................................................................. 92
Effects of CXCL14 on inflammatory gene expression in macrophages ...................... 92
Effects of CXCL14 on inflammatory gene expression in oral epithelial cells ........... 93
Effects of CXCL14 on inflammatory gene expression in the mouse gingiva ............ 95
Effect of CXCL14 on oral epithelial cell migration .............................................................. 97
Bactericidal activity of CXCL14 against oral bacteria ........................................................ 99
Gingipain protease-dependent degradation of CXCL14 by P. gingivalis .................. 101
Identification of CXCL14 peptides resulting from Kgp digestion ............................... 102
4.3 Discussion ................................................................................................................................................... 103
Chapter 5 Identification and characterisation of P. gingivalis TIR domain-containing proteins
.....................................................................................................................................................................109
5.1 Introduction ............................................................................................................................................... 110
5.2 Results........................................................................................................................................................... 110
xi
Identification of putative P. gingivalis TIR domain-containing proteins ................. 110
PG0382 is predicted to contain a coiled-coil motif ........................................................... 116
Comparison of PG0382 with mammalian TLRs and adaptor proteins .................... 117
Comparison of PG0382 with bacterial Tcps ........................................................................ 121
Structural prediction of PG0382 .............................................................................................. 123
Expression of PG0382 in HEK293T cells .............................................................................. 125
PG0382 expression causes the loss of MAL and MYD88 in HEK293T cells ........... 130
Lack of complex formation between PG0382TD and MAL............................................ 134
Subcellular localisation of PG0382TD, MAL and MYD88 ............................................... 135
5.3 Discussion ................................................................................................................................................... 137
Chapter 6 Investigation of PG0382 and host inflammation .................................................................... 141
6.1 Introduction ............................................................................................................................................... 142
6.2 Results........................................................................................................................................................... 142
Generation of an isogenic P. gingivalis PG0382-deficient mutant .............................. 142
Phenotypic characterisation of P. gingivalis ΔPG0382.................................................... 146
Gingipain protease activity of P. gingivalis ΔPG0382 ...................................................... 148
Stimulation of inflammatory gene responses in oral epithelial cells by P. gingivalis
ΔPG0382 ............................................................................................................................................................. 149
Stimulation of inflammatory gene responses in macrophages by P. gingivalis
ΔPG0382 ............................................................................................................................................................. 150
Innate immune response to P. gingivalis ΔPG0382 in mice .......................................... 151
6.3 Discussion ................................................................................................................................................... 159
Chapter 7 General Discussion .............................................................................................................................. 163
7.1 Summary ...................................................................................................................................................... 164
7.2 Implications of a dysregulated CXCL14 response for chronic inflammation and
microbial dysbiosis ................................................................................................................................................ 164
7.3 CXCL14 in tissue regeneration ........................................................................................................... 167
7.4 A potential role for PG0382 in immune subversion .................................................................. 167
7.5 The TIR domain as a primordial microbial signalling module .............................................. 169
7.6 Bacterial Tcps as therapeutic agents for inflammation............................................................ 170
7.7 Conclusion ................................................................................................................................................... 171
Bibliography .................................................................................................................................................................. 172
Appendix ......................................................................................................................................................................... 204
xii
List of Figures
Figure 1.1 The effects of subgingival plaque on chronic periodontitis. ..................................................... 4
Figure 1.2 Structural arrangement of Toll-like receptors. ........................................................................... 26
Figure 1.3 Crystal structure of the TLR3 LRR motif. ....................................................................................... 27
Figure 1.4 Crystal structure of the TLR1 and TLR2 TIR domains. ............................................................ 28
Figure 1.5 TLR signalling pathways. ...................................................................................................................... 30
Figure 1.6 Structural arrangement of TIR domain-containing adaptor proteins. .............................. 33
Figure 1.7 Structural arrangement of NOD-like receptors. .......................................................................... 35
Figure 1.8 NOD-like receptor signalling pathways. ......................................................................................... 36
Figure 3.1 P. gingivalis stimulates CXCL14 gene expression in a TLR2-independent manner. .... 71
Figure 3.2 P. gingivalis stimulates CXCL14 gene expression in an IRAK-1 and IRF6-independent
manner. .............................................................................................................................................................................. 72
Figure 3.3 P. gingivalis gingipain proteases stimulate CXCL14 expression. ......................................... 74
Figure 3.4 P. gingivalis stimulates PAR-2 gene expression in oral epithelial cells. ............................ 75
Figure 3.5 PAR-3-dependent regulation of P. gingivalis-inducible CXCL14 expression. ................. 77
Figure 3.6 EGF differentially regulates cytokine expression. ...................................................................... 79
Figure 3.7 EGF suppresses CXCL14 transcription via MEK signalling. ................................................... 81
Figure 3.8 P. gingivalis antagonises the regulation of CXCL14 by EGF. ................................................... 83
Figure 3.9 A proposed model for the regulation of CXCL14 gene expression in oral epithelial
cells by P. gingivalis. ..................................................................................................................................................... 88
Figure 4.1 CXCL14 stimulation of mouse macrophage RAW 264.7 cells................................................ 93
Figure 4.2 CXCL14 stimulation of OKF6 cells..................................................................................................... 94
Figure 4.3 Effects of CXCL14 on inflammatory gene expression in the mouse gingiva. ................... 96
Figure 4.4 Effect of CXCL14 on OKF6 cell migration. ...................................................................................... 98
Figure 4.5 Bactericidal activity of CXCL14. ....................................................................................................... 100
Figure 4.6 Gingipain protease-mediated degradation of CXCL14. .......................................................... 101
Figure 5.1 Phylogenetic tree of P. gingivalis TIR domain-containing proteins. ................................. 112
Figure 5.2 Predicted protein domains of annotated P. gingivalis TIR domain-containing proteins.
............................................................................................................................................................................................. 115
Figure 5.3 COILS analysis output for PG0382 .................................................................................................. 116
Figure 5.4 Amino acid sequence of PG0382. .................................................................................................... 117
Figure 5.5 Multiple sequence alignment of PG0382 TIR domain with TLR TIR domains. ............ 119
Figure 5.6 Multiple sequence alignment of PG0382 TIR domain with TLR adaptor proteins TIR
domains. .......................................................................................................................................................................... 120
Figure 5.7 Multiple sequence alignment of PG0382 TIR domain with TIR domains of known
bacterial TIR domain-containing proteins. ....................................................................................................... 122
xiii
Figure 5.8 PG0382 structural prediction. .......................................................................................................... 124
Figure 5.9 Construction of pEF-V5-PG0382 and pEF-V5-PG0382ΔTD expression vectors. ........ 126
Figure 5.10 Ectopic expression of V5-PG0382, V5-PG0382ΔTD, and V5-PG0382TD in HEK293T
cells. ................................................................................................................................................................................... 128
Figure 5.11 Subcellular localisation of ectopically expressed PG0382, PG0382ΔTD, and
PG0382TD in HEK293T cells. ................................................................................................................................. 129
Figure 5.12 Effects of PG0382 on MAL and MYD88 expression. .............................................................. 131
Figure 5.13 Effects of PG0382 on MAL and MYD88 mRNA levels in HEK293T cells. ..................... 132
Figure 5.14 Concentration-dependent effects of PG0382 on MAL and MYD88 protein expression.
............................................................................................................................................................................................. 133
Figure 5.15 Analysis of PG0382TD and MAL interaction by co-immunoprecipitation. ................. 134
Figure 5.16 Co-localisation of PG0382TD with MAL and MYD88 by immunofluorescence. ........ 136
Figure 6.1 Strategy for the generation of an isogenic P. gingivalis PG0382-deficient mutant. ... 144
Figure 6.2 Gel electrophoresis analysis of PCR products. ........................................................................... 145
Figure 6.3 Growth rates of wildtype P. gingivalis and P. gingivalis ΔPG0382. ................................... 146
Figure 6.4 Phenotypic characterisation of P. gingivalis ΔPG0382. .......................................................... 147
Figure 6.5 P. gingivalis gingipain protease activity. ...................................................................................... 148
Figure 6.6 Effects of P. gingivalis ΔPG0382 on inflammatory responses of oral epithelial cells.149
Figure 6.7 Effects of P. gingivalis ΔPG0382 on inflammatory cytokine responses of macrophages.
............................................................................................................................................................................................. 151
Figure 6.8 FACs Gating strategy for identifying innate immune cells. .................................................. 152
Figure 6.9 Activation of macrophages in response to P. gingivalis. ........................................................ 154
Figure 6.10 Recruitment of inflammatory monocytes in response to P. gingivalis.......................... 156
Figure 6.11 Recruitment of neutrophils in response to P. gingivalis. .................................................... 158
xiv
List of Tables
Table 1.1 Human antimicrobial mediators of host defence. ........................................................................ 19
Table 1.2 Roles of major groups of cytokines in host defence. ................................................................... 21
Table 1.3 Roles of chemokines in host defence. ................................................................................................ 23
Table 1.4 Toll-like receptor subcellular localisation and ligands. ............................................................. 24
Table 1.5 PARs activating proteases, cellular expression and key functions. ...................................... 38
Table 1.6 Bacterial Tcps and immune subversion. .......................................................................................... 40
Table 2.1 List of plasmids used in this study. .................................................................................................... 62
Table 2.2 PCR primers used for constructing PG0382 mammalian expression vectors. ................ 63
Table 2.3 PCR primers used to generate PCR products for constructing ermF cassette. ................ 64
Table 2.4 SOE PCR reaction thermal cycling conditions. .............................................................................. 66
Table 2.5 Flow cytometric analysis antibody cocktails. ................................................................................ 67
Table 4.1 CXCL14 peptides identified by MS from Kgp digestion. .......................................................... 102
Table 5.1 Annotated TIR domain-containing proteins of P. gingivalis strains. .................................. 111
Table 5.2 Amino acid sequence identity (%) between P. gingivalis TIR domain-containing
proteins. ........................................................................................................................................................................... 113
Table 5.3 Amino acid sequence identity (%) between P. gingivalis TIR domain-containing
proteins TIR domains. ............................................................................................................................................... 114
Table 5.4 Amino acid sequence identity (%) between PG0382 TIR domain and TLR TIR domains.
............................................................................................................................................................................................. 118
Table 5.5 Amino acid sequence identity (%) between PG0382 TIR domain and TIR domains of
TLR protein adaptors. ................................................................................................................................................ 120
Table 5.6 Amino acid sequence identity (%) between PG0382 TIR domain and bacterial Tcp TIR
domains. .......................................................................................................................................................................... 121
Table 5.7 Identification of PG0382 TIR domain structural homologs using FUGUE. ...................... 123
xv
Abbreviations
AP-1 Activator protein 1
BCR B cell receptor
BLAST Basic Local Alignment Search Tool
bFGF Basic fibroblast growth factor
BPE Bovine pituitary extract
cAMP Cyclic adenosine monophosphate
CCL C-C motif ligand
CCR C-C motif receptor
CRP C-reactive protein
Cryo-EM Cryo-electron microscopy
CXCL C-X-C motif ligand
ECL Enhanced chemiluminescence
EDSL Electron surface dense layer
EGF Epidermal growth factor
EGFR Epidermal growth factor receptor
FBS Foetal bovine serum
FcR Fc receptor
G-CSF Granulocyte-colony stimulating factor
HSP Heat shock protein
ICAM Intracellular adhesion molecule
IFN Interferon
IGF-1 Insulin-like growth factor
IKK IκB kinase
IAPs Inhibitor of apoptosis
IL Interleukin
IL-1Ra IL-1 receptor antagonist
xvi
IL-1RacP IL-1 receptor accessory protein
IRAK Interleukin-1 receptor-associated kinase
IRF Interferon regulatory factor
KGF Keratinocyte growth factor
Kgp Lysine-specific gingipain
LPS Lipopolysaccharide
LRR Leucine-rich repeat
MAPK Mitogen-activated protein kinase
MCP-1 Monocyte chemotactic protein-1
MMP Matrix metalloproteinase
MAL Myeloid differentiation primary response adaptor-like
MYD88 Myeloid differentiation primary response 88
NETs Neutrophil extracellular traps
NF-κB Nuclear factor kappa B
NK Natural killer
NLR NOD-like receptor
NO Nitric oxide
NDP Nucleotide
NTPase Nucleoside-triphosphatase
NTP Nucleoside triphosphate
PAD Peptidyl-arginine deiminase
PAMP Pathogen-associated molecular pattern
PDGF Platelet-derived growth factor
PI3K Phosphoinositide 3-kinase
PKA Protein kinase A
Poly(I:C) Polyinosinic:polycytidylic acid
PRR Pattern recognition receptor
RANKL Nuclear factor κ-B ligand
xvii
RANTES Regulated on activation, normal T cell expressed and secreted
Rgp Arginine-specific gingipain
RIP Receptor-interacting serine/threonine-protein kinase
RLR RIG-I-like receptors
SARM Sterile α and TIR motif containing protein
SFD-1 Stromal-derived factor-1
SMURF1 SMAD specific E3 ubiquitin protein ligase 1
STAT Signal transducer and activator of transcription
TAB TAK-1 associated binding protein
TAK Transforming growth factor-β activated kinase
TBK TANK-binding kinase
Tcp TIR domain-containing protein
TCR T cell receptor
TERT-2 Telomerase reverse transcriptase 2
TGF Transforming growth factor
TIR Toll/Interleukin-1 receptor domain
TIRAP Toll-interleukin 1 receptor domain-containing adaptor protein
TLCK N--Toysl-L-Lysine chloromethyl ketone hydrochloride
TLR Toll-like receptor
TNF Tumour necrosis factor
TNFR Tumour necrosis factor receptor
TRAF TNF receptor-associated factor
TRAM TRIF-related adaptor molecule
TRIF TIR-domain-containing adaptor-inducing interferon-β
VCAM Vascular cell adhesion molecule
1
Introduction
2
1.1 Overview of the Oral Microbial Habitat
The mucosal surfaces of the human body are inhabited by a myriad of microorganisms, and the
oral cavity provides an excellent niche for the establishment of unique microbial communities.
The mucosal surfaces of the oral cavity (e.g. gingiva, tongue, palate and cheek) and the
non-shedding surface of the teeth provide distinct habitats to sustain the growth of different
microbial communities that form biofilms. A combination of environmental (e.g. nutrient
availability, pH and temperature) and host-derived (e.g. antimicrobial peptides) factors create
an ecological niche that dictates the distribution of bacterial species at various sites in the oral
cavity (Carlsson, 1997). The ecological properties of the oral cavity are complex, dynamic and
highly variable in individuals. Normal physiology, genetics and lifestyle factors (e.g. diet) can
influence the composition of these microbial ecosystems. In health, a symbiotic relationship is
maintained between the host and oral commensal species; microbial inhabitants partake an
active role in shaping and maintaining health by acting as antigenic stimulants to facilitate
homeostatic immunity (Marsh and Devine, 2011). However, changes in the microenvironment
can lead to the proliferation of pathogens or potential pathogens (pathobionts) within the
biofilm and cause disease (Hajishengallis and Lamont, 2012).
Formation of the Oral Biofilm
As indicated above, microorganisms can form organised and highly dynamic communities on
biotic and abiotic substrates, known as biofilms (O’Toole et al., 2000). The mucosal and hard
surfaces of the oral cavity are favourable sites for biofilm formation. Microorganisms within oral
biofilms are constantly exposed to environmental changes, such as pH fluctuation, nutrient
availability and host-produced enzymes (e.g. lysozyme) (Carlsson, 1997; O’Toole et al., 2000).
The formation of a biofilm allows participating microorganisms to be more resistant to physical
and biochemical forces within the oral cavity. Over time, the biofilm develops into a coordinated
microbial community, which during health shares a homeostatic relationship with the host.
The oral microbiome is a consortium of over 700 microbial species that make up different
dynamic polymicrobial communities (Aas et al., 2005). The microenvironment dictates the
composition of microbial species within biofilms on the teeth, tongue, and the buccal, upper and
lower mucosal surfaces within the oral cavity (Aas et al., 2005). Supragingival plaque occupies
the smooth surface of the teeth, whilst subgingival plaque is localised to the gingival crevice
along the tooth enamel and the sulcular epithelium (Fig. 1.1). The oxygenated environment
around the teeth benefits the growth of aerobic bacterial species within supragingival plaque
(e.g. Lactobacillus and Actinomyces species). Contrastingly, subgingival plaque is predominantly
comprised of facultative and strict anaerobic bacterial species (e.g. Fusobacterium nucleatum,
Porphyromonas gingivalis and Prevotella intermedia) (Zijnge et al., 2010).
3
The formation of the tooth-accreted biofilm is a sequential and organised process involving
various microbial species (Kolenbrander et al., 2010; O’Toole et al., 2000). The initial primary
colonisers are the pioneering bacterial species that interact with host proteins to build a
microbial monolayer. Gram-positive bacterial species, including Streptococci and Actinomyces
species (e.g. Streptococcus gordonii and Actinomyces naeslundii), form the oral microbiota of
subgingival plaque (Yao et al., 2003; Palmer et al., 2003; Li et al., 2004; Dige et al., 2009). The
early colonisers express adhesins (e.g. antigen I/II polypeptides and amylase-binding protein
produced by oral Streptococci) to form attachments to the gingival epithelium (O’Toole et al.,
2000; Rogers et al., 2001). Co-aggregation between primary colonisers and other bacterial
species that cannot directly colonise the gingival surface is essential for biofilm formation.
F. nucleatum acts as a central player in facilitating biofilm formation by promoting
co-aggregation (Al-Ahmad et al., 2007). The F. nucleatum radD adhesin can interact with
primary colonising species, such as Streptococcus sanguinis and A. naeslundii, to facilitate mixed-
species biofilm formation (Kaplan et al., 2009). In addition, co-aggregation-mediated
interactions facilitated by F. nucleatum promotes the survival of late colonising obligate
anaerobes in oxygenated environments (Bradshaw et al., 1998). P. gingivalis, Treponema
denticola and Tanerella forsythia are late colonising species that are commonly associated with
chronic periodontitis (Socransky et al., 1998). In collaboration, bacterial species within oral
biofilms therefore form highly organised and structured communities.
4
Figure 1.1 The effects of subgingival plaque on chronic periodontitis. Subgingival plaque is localised to the gingival crevice along the tooth enamel and the sulcular epithelium. The junctional epithelium is the region where the epithelium connects to the tooth and is highly susceptible to bacterial invasion, therefore it is maintained by a high level of neutrophil infiltration. A normal balance between the oral commensal and host immune response is required to maintain host-biofilm homeostasis in the healthy periodontium (left). The accumulation of subgingival plaque (right) can induce chronic periodontitis and results in the destruction of the epithelial tissue and alveolar bone resorption. Figure taken from (Darveau, 2010).
5
1.2 Periodontal Disease
Overview
Periodontal disease is a spectrum of inflammatory conditions that are associated with the
tissues supporting the teeth, otherwise known as the periodontium. Periodontal conditions can
be categorised as: (i) gingivitis, (ii) chronic periodontitis, and (iii) aggressive periodontitis
(Highfield, 2009). Gingivitis is associated with mild inflammation of the periodontium without
the loss of connective tissue. Gingivitis can transit to chronic periodontitis, where there is a
breakdown of gingival tissue, periodontal ligament detachment, and in severe cases, alveolar
bone resorption. Aggressive periodontitis shares similar characteristics with chronic
periodontitis, with the exception of a faster rate of disease progression and occurs more
frequently in juveniles (Highfield, 2009).
Epidemiological studies suggest that there are potential links between chronic periodontitis and
various systemic conditions, such as cardiovascular disease and rheumatoid arthritis (Seymour
et al., 2007). Although the nature of the association of chronic periodontitis with these other
important diseases are yet to be fully determined, it is thought that the continual activation of
the immune system, instigated by subgingival biofilm, compromises the immune system and
leads to the destruction of host tissues.
Bacterial Aetiology of Chronic Periodontitis
Chronic periodontitis is a complex, multifactorial disease that can be attributed to lifestyle
(e.g. diet and smoking) and genetic factors (e.g. familial neutropenia) (Pihlstrom et al., 2005).
The oral biofilm is also a predominant driver in the development and progression of chronic
periodontitis. Numerous studies have documented qualitative and quantitative changes in oral
biofilm (plaque) composition in the transition from health to disease (Hong et al., 2015; Marsh,
1994; Paster et al., 2001; Socransky et al., 1998). The conception of different hypotheses for the
development of periodontitis has evolved over the years. However, each hypothesis highlights
the importance of host-biofilm homeostasis to maintain periodontal health.
The Non-Specific Plaque Hypothesis was developed to describe a correlation between plaque
accumulation and disease development. It was believed that an increase in bacterial load in
plaque exceeded a containment threshold, and thus initiated disease onset (Theilade, 1986).
Therefore, disease prevention was focused on maintenance of oral hygiene and the physical
removal of plaque (e.g. tooth brushing and scaling). The subsequent isolation and identification
of disease-associated microorganisms led to the Specific Plaque Hypothesis, in which specific
bacterial species in plaque were associated with either health or disease (Loesche, 1979;
Loesche et al., 1977). A landmark study by Socransky et al. showed that specific bacterial
6
species could be grouped into six clusters based on their association with health and disease. Of
the six clusters, the “red complex”, comprised of P. gingivalis, T. forsythia and T. denticola, was
most frequently associated with disease (Socransky et al., 1998). A number of recent studies
have also demonstrated, in a more comprehensive and detailed manner, significant differences
in oral microbiota composition between periodontal health and disease (Griffen et al. 2012;
Abusleme et al., 2013; Hong et al., 2015). Indeed, advances in DNA sequencing and
bioinformatics technologies have provided greater insight into the bacterial aetiology of chronic
periodontitis by enabling analysis of microbial community composition. In addition to the “red
complex” bacterial species, these more recent DNA-based sequencing studies suggest that
bacterial species from the Spirochaetes and Synergistetes taxa are also likely to be associated
with the pathogenesis of chronic periodontitis (Griffen et al., 2012; Abusleme et al., 2013). As
such, it has been proposed that antibiotic treatment against specific bacterial species could be
employed to treat chronic periodontitis (Slots & Rams, 1990; Cionca et al., 2009). However, a
conservative and highly selective approach would need to be undertaken to avoid the possible
selection and overgrowth of antibiotic-resistant pathogens (Slots and Ting, 2002).
The Ecological Plaque Hypothesis, proposed by Marsh and colleagues, emphasised the interplay
between the oral microenvironment and microflora in disease development and progression
(Marsh, 1994, 2003). Changes in ecological factors (e.g. pH and nutrients) can select for the
bacterial species composition of the oral biofilm. For instance, the consumption of high sugar
content foods increases the growth of acid-tolerant bacterial species, such as
Streptococcus mutans, which can readily metabolise dietary sugars to acid (Becker et al., 2002).
Similarly, metabolic activity from plaque microbiota can also alter the environment and
contribute to host-biofilm destabilisation. Facultative anaerobes, such as F. nucleatum, can
metabolise oxygen to create a reducing microenvironment that assists the colonisation and
growth of strict anaerobes, including P. gingivalis (Diaz et al., 2002). Thus, the environment and
microbial growth are co-dependent, and disease can ensue when one or the other is disrupted.
The Polymicrobial Synergy and Dysbiosis hypothesis is an extension of the Ecological Plaque
Hypothesis, and emphasises the synergistic contribution of microorganisms in dysbiosis to
disease development. The model describes a role for keystone pathogens, such as P. gingivalis,
in initiating environmental changes that disrupts microbial homeostasis. As a low abundance
species, P. gingivalis appears to have a disproportionate effect on plaque microbiota virulence
(Hajishengallis et al., 2011). The pathogenicity of P. gingivalis comes from its ability to
manipulate the host immune response to subvert defence mechanisms (refer to Section 1.7 for
further details). P. gingivalis creates an inflammatory state that facilitates nutrient acquisition,
whilst inhibiting host bacterial killing mechanisms to ensure its survival (Hajishengallis et al.,
7
2012). By impairing the host immune response to the biofilm, P. gingivalis causes an imbalance
in microbial distribution and shifts microbiota homeostasis to dysbiosis. As such,
microorganisms that are otherwise commensals can act in a synergistic manner to stimulate
chronic inflammation and promote disease. The importance of a polymicrobial biofilm in the
development of chronic periodontitis was demonstrated in germ-free mice, whereby
P. gingivalis failed to promote disease in the absence of other bacterial species (Hajishengallis et
al., 2011). Accordingly, the Polymicrobial Synergy and Dysbiosis model highlights the
participation of dysbiotic microbial communities in disease pathogenesis and illustrates
keystone pathogens as a trigger for dysbiosis. Therefore, therapeutic intervention targeting
specific keystone pathogens is a potential avenue to prevent disease development.
Periodontal Disease and Systemic Health
Epidemiological studies suggest that there are potential links between chronic periodontitis and
various systemic conditions (Genco and Van Dyke, 2010; de Pablo et al., 2009; Seymour et al.,
2007). Bacterial and inflammatory factors derived from the oral cavity appear to influence the
progression of systemic diseases (Hajishengallis, 2015; Seymour et al., 2007), whereby
periodontal pathogens or inflammatory by-products can enter the systemic circulation and act
as stimulatory factors that contribute to the development of systemic disease.
Atherosclerosis results from the accumulation of fatty deposits on the vasculature and can lead
to various systemic complications (e.g. aneurysms and myocardial infarction). C-reactive
protein (CRP), an acute-phase protein produced by the liver, is a classical indicator of systemic
inflammation. Increased CRP levels have been detected in patients with chronic periodontitis
and ongoing periodontal therapy can successfully return CRP to baseline levels (D’Aiuto et al.,
2005; Seinost et al., 2005). Periodontal pathogens can stimulate inflammation in the vasculature
to promote atheroma formation. P. gingivalis, which has been detected in atherosclerotic
plaques, can cause increased expression of vascular adhesion molecules (e.g. intracellular
adhesion molecule-1 (ICAM-1) and vascular cell-adhesion molecule-1 (VCAM-1)) by endothelial
cells to promote leukocyte (e.g. monocytes) recruitment and inflammation in the vasculature
(Haraszthy et al., 2000; Khlgatian et al., 2002). Significantly, P. gingivalis can stimulate the
uptake of low-density lipoprotein by macrophages and foam cell formation, a classical hallmark
of atherosclerotic lesions (Qi et al., 2003). Elevated levels of inflammatory mediators
(e.g. Interleukin-1 (IL-1, IL-6 and tumour necrosis factor (TNF)), secreted as a result of
periodontitis, are also correlated with increased CRP production by the liver (Loos et al., 2000;
Noack et al., 2001). Thus, the combination of bacterial dissemination and increased levels of
inflammatory cytokines in the systemic circulation may contribute to the risk of exacerbating
atherosclerosis.
8
Periodontitis may also be associated with exacerbating rheumatoid arthritis, an inflammatory
condition affecting the underlying structures of bone in synovial tissues. DNA from various oral
bacterial species (e.g. P. gingivalis and T. denticola) have been detected in synovial fluid from
patients with rheumatoid arthritis (Moen et al., 2006), and some patients suffering from
rheumatoid arthritis have been successfully treated with antibiotics (Ogrendik, 2009). The
peptidyl-arginine deiminase (PAD) secreted by P. gingivalis can citrullinate host proteins, which
may lead to the generation of autoantibodies and hence contribute to the development of
rheumatoid arthritis (Koziel et al., 2014; Wegner et al., 2010). In addition, P. gingivalis proteases
have also been suggested to enhance tissue breakdown in arthritis by degrading host
extracellular matrix proteins, including laminin, fibronectin and collagen (Imamura et al., 2003;
Ruggiero et al., 2013).
Associations between poor maternal periodontal health and increased risk of adverse
pregnancy outcomes have also been reported (López et al., 2002; Mitchell-Lewis et al., 2001).
Inflammatory mediators (e.g. IL-1 and TNF) and/or bacterial endotoxins (e.g. bacterial
lipopolysaccharide (LPS)) arising from periodontal infection may enter the systemic circulation
and transverse the placental barrier to trigger increased prostaglandin and TNF levels, which
might then promote preterm birth (Gibbs, 2001). Women with pregnancy-associated gingivitis
who received periodontal treatment during pregnancy had significant reductions in rates of
preterm birth and/or low birth weight (Jeffcoat et al., 2001; López et al., 2005). Chronic
periodontitis may therefore also contribute to the pathogenesis of systemic disease; however,
further studies will be required to confirm causality links, as there are possibly alternate
underlying mechanisms that may cause predisposition to some systemic conditions.
1.3 Host Immune Response in Periodontitis
Although a controlled immune response is essential to prevent infection, dysbiosis in the oral
biofilm can hinder immune-mediated microbial clearance. Consequently, the persistence of
immune cells and continual production of inflammatory mediators can contribute to tissue
destruction and delay the resolution of inflammation.
Innate Immune Response in Periodontitis
The innate immune system is comprised of physical, cellular and bioactive components, which
defend the body upon exposure to potential pathogens (e.g. microbes and fungi). As the first
point of contact, the oral epithelium acts as a barrier to prevent the breach of microbes.
Additionally, oral fluids, such as saliva and gingival crevicular fluid, contain antimicrobial
proteins (e.g. lysozyme) that can inhibit the overgrowth of the biofilm. At the molecular level,
innate immune cells patrol tissues within the human body to promote the rapid clearance of
9
potential pathogens. Importantly, innate immune cells also initiate the activation of the adaptive
immune system through antigen presentation and other mechanisms. The immune system is
reinforced by immune mediators, including complement proteins, cytokines, chemokines and
antimicrobial proteins, which facilitate the clearance of pathogens. Together, the innate immune
system is essential for initiating and establishing host defence against infection.
1.3.1.1 Oral Epithelial Cells
The myriad of microorganisms occupying the oral cavity makes it particularly susceptible to
infection. The oral cavity, which is covered by keratinised and non-keratinised epithelial cells,
depending on anatomical location, acts as a mechanical barrier to bacterial infection (Squier and
Kremer, 2001). The regions that are exposed to greatest physical forces (e.g. gingiva and hard
palate), such as during mastication, are covered by keratinised epithelium that is tightly
attached to the underlying connective tissue. Contrastingly, the soft palate and buccal regions of
the oral cavity are covered with non-keratinising epithelium for greater flexibility, which is
required for chewing and speech.
The oral mucosa has a stratified architecture characterised by an innermost basal layer that is in
direct contact with the underlying basement membrane, comprised of extracellular matrix
proteins (e.g. collagen and fibronectin) and growth factors (e.g. transforming growth factor
(TGF-). The basal layer is followed by the spinous and granular layers. Keratinised epithelium,
such as that covering the hard palate, has an additional keratin layer, which resembles the
epidermis of the skin. Cells at the surface of the oral mucosa are maintained through constant
replacement from a reservoir of stem cells in the basal layer, which undergo a specialised
differentiation program as they migrate towards the superficial layer of the epithelium (Potten
and Morris, 1988). Differentiating cells entering the spinous layer undergo a change in the types
of keratins expressed. For instance, keratin 5 and 14 are downregulated and keratin 1 and
keratin 10 are expressed to form tonofibrils (Fuchs and Green, 1980; Roop et al., 1987). Cells
from the spinous layer undergo further differentiation and express filaggrin to facilitate the
bundling of keratins into macrofibrils for the formation of a protein scaffold (Dale et al., 1997).
Additionally, transglutaminases catalyse the crosslinking of structural envelope proteins
(e.g. involucrin and loricrin) to further reinforce the cornified structure (Thacher and
Rice, 1985). Thus, a mechanically robust scaffold is established within the cornified cell
envelope to provide vital barrier function.
The epithelium is further strengthened by the intercellular linking of proteins at tight junctions
and adherens junctions. Tight junctions localised towards the apical surface of the epithelium
are maintained by a number of proteins, including claudin, occludin and zonula occludens. They
10
maintain the apico-basal polarity of the epithelial layer and regulate the paracellular passage of
ions and water molecules (Kirschner et al., 2013; Yuki et al., 2007). Adherens junctions localised
basal to tight junctions provide strong mechanical links between adjacent cells to reinforce
epithelial cohesion. Adherens proteins, such as E-cadherin, are anchored to intracellular actin
filaments to protect the epithelia from shear forces (Niessen, 2007). Thus, the presence of
junctional proteins fortifies the epithelium and limits the transmigration of pathogens.
However, the breakdown of proteins in tight and adherens junctions, for example by
P. gingivalis and T. denticola proteases, may provide a gateway for bacterial spread deeper into
the underlying tissues (Chi et al., 2003; Katz et al., 2002).
The junctional epithelium localised at the bottom of the gingival sulcus is comprised of
non-keratinising squamous epithelium consisting of basal and suprabasal layers. Junctional
epithelial cells express lower levels of E-cadherin (Hatakeyama et al., 2006), which increases the
intercellular space and enhances tissue permeability. Importantly, the more permeable nature
of the junctional epithelium permits greater occupation by immune cells, including neutrophils,
and thus contributes to a reservoir of neutrophil-associated antimicrobial peptides to promote
tissue homeostasis (refer to Section 1.3.1.2 and 1.3.3 for further details).
In addition to acting as a mechanical barrier against microbes, the oral epithelium also forms an
immunological front. Antimicrobial molecules (e.g. defensins) are secreted constitutively, and in
an inducible manner, as part of the host-protective mechanism against microorganisms in the
oral cavity (Dale and Fredericks, 2005) (refer to Section 1.3.3.2 for further details). Importantly,
oral epithelial cells also functionally connect the innate immune response to the adaptive
immune response during infection. In particular, they express Pattern Recognition Receptors
(PRR) that stimulate an immune response upon the recognition of specific microbial patterns
(refer to Section 1.4 for further details). PRR activation leads to intracellular signalling cascades
that promote the expression of pro-inflammatory genes (e.g. IL-8 and IL-6) to alert and direct
the cells of the innate and adaptive immune system to the site of infection. Our laboratory
recently undertook a preliminary transcriptomics-based experiment to identify
novel P. gingivalis-inducible genes in oral epithelial cells, and one of the genes identified
encodes the orphan chemokine, CXCL14. In summary, the oral epithelium not only provides
barrier protection, it also produces immunomodulatory factors that recruit and activate
immune cells.
1.3.1.2 Neutrophils
The junctional epithelium adjacent to the tooth is highly susceptible to microbial invasion
(Fig. 1.1), and therefore it is maintained by a high level of neutrophil infiltration (Tsukamoto et
11
al., 2012). The fundamental role of neutrophils is to prevent the establishment of infection by
releasing cytotoxic granules to eliminate pathogens. The granules can contain antimicrobial
peptides (e.g. -defensins and lipocalin) and/or reactive oxygen metabolites (e.g. nitric oxide
and superoxide) (Faurschou and Borregaard, 2003; Rice et al., 1987). In addition, neutrophil
extracellular traps (NETs) are comprised of chromatin fibres and bactericidal components
(e.g. elastase and myeloperoxidase), which impede bacterial spread and limit secondary damage
to surrounding tissues (Cooper et al., 2013).
The close association of chronic periodontitis with defective neutrophil recruitment and
function reflects the importance of neutrophils in maintaining periodontal health. Individuals
with neutropenia typically have a greater incidence of periodontitis, and treatment with
colony-stimulating factor-3 (CSF-3; otherwise known as G-CSF) can rescue neutrophil counts to
facilitate bacterial clearance in the periodontium (Deasy et al., 1980; Hastürk et al., 1998). The
constitutive transepithelial migration of neutrophils across the junctional epithelium acts as a
unified antimicrobial front (Darveau, 2010; Faurschou and Borregaard, 2003). An increasing
gradient of ICAM-1 and IL-8 expression from the basal to superficial layers of the junctional
epithelium ensures a constant influx of neutrophils at the epithelium-plaque interface (Tonetti
et al., 1998). Neutrophils are also actively recruited from the blood to the site of infection during
inflammation by the chemokines CXCL2 and CXCL5, as part of the immune defence mechanism
(Kolaczkowska and Kubes, 2013; Phillipson et al., 2006). In addition, the distribution of NETs on
gingival surfaces and in gingival crevicular exudates also provides host protection by limiting
bacterial adherence and invasion of gingival tissues (Krautgartner and Vitkov, 2008; Vitkov et
al., 2009). In summary, neutrophils not only provide host defence upon infection, they also play
an integral protective role at steady state to maintain periodontal health.
The inflammatory nature of the neutrophil response to the oral biofilm can also destabilise
tissue homeostasis and thereby contribute to the onset of periodontitis. Bacterial persistence in
the gingival sulcus can lead to the inappropriate recruitment and activation of neutrophils. As a
result, neutrophils respond to microbes in the biofilm by releasing tissue-degrading proteins,
such as matrix metalloprotease (MMP)-8, MMP-9 and elastase (Shin et al., 2008). The excessive
production of these proteins can cause tissue destruction and delay the wound healing process.
For instance, the cleavage of platelet derived growth factor (PDGF) by elastase can compromise
growth signals in periodontal ligament cells (Nemoto et al., 2005). Consistently, individuals
displaying hyperactive neutrophil function, for example, as a result of a gene polymorphism in
the neutrophil Fc gammareceptor IIa (FcRIIa), are predisposed to developing severe
periodontitis (Nicu et al., 2007). Therefore, the dysregulation of neutrophil responses can
contribute to the pathogenesis of periodontitis.
12
1.3.1.3 Macrophages
Macrophages facilitate the inflammatory response by producing inflammatory mediators, and
phagocytosing microbes to clear cellular debris to promote the wound healing response.
Macrophages can be broadly classified into two major phenotypes: (i) classically-activated M1
macrophages, and (ii) alternatively-activated M2 macrophages. M1 macrophages are involved in
inflammation and are activated by bacterial components (e.g. LPS) to stimulate a
pro-inflammatory response, whilst M2 macrophages have anti-inflammatory characteristics,
and are typically activated by IL-4, IL-10 and IL-13, and produce IL-10, TGF- and IL-1 receptor
antagonist (IL-1Ra) (Gordon, 2003; Stein et al., 1992).
Once activated, M1 macrophages express cytokines (e.g. TNF, IL-12 and IL-6) and chemokines
(e.g. CCL2, CXCL10 and CXCL11) that activate and recruit additional immune cells to aid in the
clearance of pathogens (Murray and Wynn, 2012). Another major role M1 macrophages has
during infection is phagocytosis, to facilitate bacterial clearance (Huynh and Grinstein, 2007).
Contrastingly, M2 macrophages are involved in suppressing inflammation, whereby their
secretion of IL-10 antagonises M1 macrophages (Murray and Wynn, 2012). In addition, M2
macrophages produce growth factors, including PDGF and TGF-, which increase the expression
of tissue inhibitors of metalloproteinases (TIMPs), as well as the activation of extracellular
matrix producing myofibroblasts, to promote wound healing (Barron and Wynn, 2011; Murray
and Wynn, 2012).
Increased numbers of pro-inflammatory macrophages in gingival tissue from periodontitis
patients suggests that macrophages play a role in the pathogenesis of periodontitis (Lappin et
al., 2000). An excessive bacterial load (plaque) in periodontitis can cause the chronic
recruitment and activation of macrophages. Furthermore, the pro-inflammatory mediators
produced by macrophages contribute to the excessive recruitment of other immune cells (e.g.
neutrophils and T lymphocytes), and hence promote chronic inflammation. Periodontal
infection of mice with P. gingivalis induces macrophage secretion of TNF and IL-1 classical
pro-inflammatory cytokines and whose levels correlate with periodontal disease progression
(Graves and Cochran, 2003). Together, TNF and IL-1 stimulate a broad range of immunological
effects, including orchestrating the migration of leukocytes as part of the physiological response
to infection (refer to Section 1.3.3.3 for further details). In periodontitis, a physiological
response can transit to disease because the constant recruitment of leukocytes induces chronic
inflammation. In addition, the cytokines secreted into the inflammatory milieu by macrophages
can contribute to alveolar bone resorption. For example, IL- and TNF can stimulate receptor
activator of nuclear factor kappa-B ligand (RANKL) gene expression in osteoblasts, which
13
promotes the development of bone-resorbing osteoclasts (i.e. osteoclastogenesis) (Hofbauer et
al., 1999). Importantly, mice in which macrophages had been depleted using clodronate-loaded
liposomes were protected from P. gingivalis-induced alveolar bone resorption (Lam et al.,
2014). Thus, the excessive production of cytokines arising from the chronic recruitment of
macrophages can sustain inflammation that is detrimental to the host.
1.3.1.4 Dendritic Cells
Dendritic cells are professional antigen-presenting cells that engage with naïve T lymphocytes
to stimulate their activation (Banchereau and Steinman, 1998; Croft et al., 1992). Dendritic cells
can also mediate B lymphocyte antibody responses through cytokine production (de Saint-Vis et
al., 1998). At steady-state, dendritic cells maintain tissue homeostasis by inducing immune
tolerance towards commensal species, whilst still ensuring effective defence against pathogens
(Steinman et al., 2003). When activated by microbial components (e.g. LPS and bacterial DNA),
dendritic cells release an array of chemokines (e.g. CCL5 and CCL2) to attract additional
immune cells (De Smedt et al. 1996; Sparwasser et al. 1998; Sallusto et al. 1999). The chemokine
receptor CCR7 is upregulated upon dendritic cell activation and responds to CCL19 and CCL21
produced by stromal cells in secondary lymphoid organs (e.g. spleen and lymph nodes)
(Kellermann et al., 1999; Lin et al., 1998). Consequently, activated dendritic cells migrate to
lymphoid tissues to activate and communicate antigenic information to lymphocytes.
Furthermore, dendritic cells provide immunoregulation to prevent the excessive activation of
lymphocytes during infection. Mice depleted of Langerhan cells (using a diphtheria toxin
receptor-based system) were used to study the role of dendritic cells in the P. gingivalis-induced
mouse model of periodontitis. The mice exhibited enhanced tooth loss, which was associated
with increased T lymphocyte-dependent interferon-(IFN-) production and reduced
T regulatory cell numbers (Arizon et al., 2012). Therefore, dendritic cells provide host
protection by regulating the adaptive immune response to prevent chronic inflammation.
The dysbiotic tooth-accreted biofilm in periodontitis can dysregulate dendritic cell activity.
Periodontal pathogens, such as P. gingivalis, can modulate dendritic cell function, and thereby
cause a weakened immunostimulatory response (Kanaya et al., 2004, 2009). P. gingivalis does
so, at least in part, by stimulating IL-10 secretion from dendritic cells to suppress inflammation
(Jotwani et al., 2003; Pulendran et al., 2001). Interestingly, increased IL-10 expression can also
cause the chronic recruitment of dendritic cells by enhancing the expression of chemokine
receptor, CCR6, by immature dendritic cells, and thus promote their recruitment in a
CXCL12-dependent manner into gingival tissues during periodontitis (Dieu-Nosjean et al., 2001).
Consequently, this feedback loop may lead to the chronic recruitment of dendritic cells.
Dendritic cells also contribute to periodontal tissue destruction by producing increased levels of
14
MMPs (e.g. MMP-9) in response to P. gingivalis (Jotwani et al., 2010). Consequently, oral
pathogens can compromise dendritic cell function and skew dendritic cells towards a host-
destructive response.
Adaptive Immune Response in Periodontitis
The adaptive immune system generates a highly specialised response tailored to specific
pathogens. Cells of the innate immune system (e.g. dendritic cells) engage with naïve T and
B lymphocytes to stimulate their proliferation and differentiation into effector and memory
cells. Effector T and B lymphocytes recognise target antigens and can induce cell-mediated and
humoral immunity, respectively. Both T and B lymphocytes also differentiate into memory cells,
which are retained to induce a faster response upon a subsequent encounter with the same
antigen. Although the adaptive immune system is important for host protection, the presence of
excessive numbers of T and B lymphocytes in periodontal lesions strongly suggest that they also
contribute to the pathogenesis of chronic periodontitis (Teng, 2003).
1.3.2.1 T Lymphocytes
T lymphocytes recognise processed peptide antigens, presented by cells of the innate immune
system (e.g. dendritic cells), via the T cell receptor (TCR). T lymphocytes can differentiate into
CD8+ cytotoxic or CD4+ helper T lymphocytes. Cytotoxic T lymphocytes execute immunity by
triggering apoptosis of infected cells, and secreting granules containing cytotoxic components
(e.g. perforin, granzymes and granulysin) and inflammatory cytokines (e.g. IFN- and TNF) to
mediate pathogen clearance (Henkart, 1994; Kägi et al., 1994). In contrast, T helper
lymphocytes are involved in coordinating the immune response. Based on their cytokine
profiles, T helper lymphocytes are divided into different subsets, including Th1, Th2 and Th17.
Th1 cells produce IFN-, IL-2 and TNF to promote cell-mediated immunity and
phagocyte-mediated inflammation, whilst Th2 cells produce IL-4, IL-6 and IL-10 to drive an
antibody-mediated humoral immune response (Kidd, 2003; Mosmann and Coffman, 1989).
Th17 cells produce various cytokines, including IL-17 and IL-22, which primarily act on
epithelial cells to induce the expression of antimicrobial proteins (e.g. -defensins and S100
proteins) (Harrington et al., 2005; Liang et al., 2006). Regulatory T lymphocytes are a T helper
subset that exert regulatory activity by secreting anti-inflammatory cytokines (e.g. IL-10 and
TGF-) to dampen/inhibit immune responses. Together, the proportions of T lymphocyte
subsets can dictate the type of adaptive immune response that is mounted, and an imbalance
can contribute to inflammatory and autoimmune diseases.
Increased numbers of T lymphocytes in periodontal lesions is correlated with the progression of
gingivitis to periodontitis (Liu et al., 2010; Yamazaki et al., 1995). Early periodontal lesions
15
exhibit a typical Th1 cytokine profile characterised by high IFN- levels, which promotes
phagocytic activity and cell-mediated immunity (Dutzan et al., 2009). However, the detection of
increased IL-4 and IL-6 levels in advanced periodontal lesions suggested that there is a shift
from a Th1 to Th2 response in chronic periodontitis (Lappin et al., 2001). Ex vivo analysis of
T lymphocytes from patients with chronic periodontitis displayed reduced cell-mediated
immune function (Ivanyi and Lehner, 1970). Therefore, it was proposed that Th1 cytokines
were important for establishing a stable lesion, whereas the persistence of the tooth-accreted
biofilm (plaque) led to a Th2 dominant response. However, the Th1/Th2 paradigm in
periodontitis was challenged when several studies demonstrated contradictory levels of Th1
and Th2 cytokines in periodontal lesions (Berglundh, Liljenberg and Lindhe, 2002; Takeichi et
al., 2000). The identification of the Th17 lineage led to the re-examination of the Th1/Th2
paradigm. The high levels of the Th17 cytokines IL-17 and IL-22 in inflamed periodontal tissues
and gingival crevicular fluid suggested that Th17 cells might mediate host protection by
promoting neutrophil chemotaxis and antimicrobial defence (Liang et al., 2006; Vernal et al.,
2005; Yu et al., 2007). Indeed, IL-17 receptor-deficient mice showed enhanced alveolar bone
loss in a P. gingivalis-induced mouse model of periodontitis (Yu et al., 2007). Although this
suggests that Th17 cells have host-protective functions in periodontitis, IL-17-mediated
neutrophil infiltration has been implicated in driving alveolar bone resorption in ageing mice
(Eskan et al., 2012). Despite emerging studies demonstrating a role for Th17 cells in the
pathogenesis of periodontitis, the roles of Th17 cells in host-protection and host-destruction
need to be further examined.
1.3.2.2 B Lymphocytes
B lymphocytes mediate humoral immunity by secreting antibodies. B lymphocytes are activated
upon antigen engagement of the B cell receptor (BCR). In addition, cytokines produced by
activated Th2 cells (e.g. IL-4) also promote B lymphocyte activation and differentiation. Once
activated, B lymphocytes proliferate and differentiate into plasma cells, which produce
antibodies with unique antigen-binding specificities. Antibodies can opsonise bacteria by
binding to target antigens present on the surface of bacteria and thereby promote their
clearance by phagocytes (e.g. macrophages).
B lymphocytes constitute a major inflammatory infiltrate in established periodontal lesions
(Gemmell and Seymour, 1998). The importance of humoral immunity was demonstrated when
B lymphocyte-deficient rats were shown to have increased alveolar bone resorption when
infected with P. gingivalis (Hou et al., 2000; Klausen et al., 1989). Elevated levels of periodontal
bacteria-specific antibodies in individuals affected by gingivitis suggest there is an antibody-
driven response to plaque (Ebersole et al., 2001; Lamster et al., 1990). The ability of anti-sera
16
containing high titre, anti-P. gingivalis antibodies from patients with periodontitis to inhibit
bone resorption in vitro suggested that the antibodies mediate a protective immune response
(Meghji et al., 1993). Interestingly, a separate study found that anti-P. gingivalis antibodies
exhibited reduced opsonisation capabilities (Cutler et al., 1991). Clinical studies indicate that
high titres of antibodies do not necessarily confer protection, as anti-P. gingivalis antibodies
from patient sera can display varying avidities (Lopatin and Blackburn, 1992). The degradation
of opsonising antibodies by P. gingivalis proteases (e.g. gingipain proteases) may also influence
the immunoreactivity of antibodies (Vincents et al., 2011).
Importantly, the persistence of a tooth-accreted biofilm (plaque) in chronic periodontitis can
lead to the generation of auto-reactive antibodies. Increased levels of autoantibodies targeting
components of the extracellular matrix (e.g. type I collagen and laminin) have been detected in
periodontal lesions, and can contribute to localised tissue destruction in periodontitis
(Berglundh et al. 2002; De-Gennaro et al. 2006). Antibodies specific for bacterial antigens
produced during periodontitis that can cross-react with self-antigens might result in an
autoimmune response. For example, antibodies against P. gingivalis GroEL, a homolog of the
human heat shock protein 60 (HSP60), have been shown to cross-react with endothelial HSP60,
resulting in endothelial dysfunction in atherosclerosis (Ford et al., 2005; Seymour et al., 2007;
Tabeta et al., 2000). As such, humoral immunity can potentially be damaging when it exerts
non-specific effects against host antigens.
Molecular Mediators of Host Immunity
The host immune response is supported and regulated by an array of bioactive molecules that
can activate and amplify host inflammation, promote the activation of immune cells and
facilitate the clearance of microorganisms. Importantly, they also regulate the immune response
to protect against the development of chronic inflammatory and autoimmune diseases.
Therefore, alterations in their levels of expression and/or biological activity can have
detrimental effects.
1.3.3.1 Complement System
The complement system is comprised of serum proteins, which are activated by proteolytic
cleavage to facilitate the clearance of microorganisms. A cascade of enzymatic reactions,
involving the successive cleavage of zymogens, mediates the activation and amplification of
complement. Complement proteins circulate the body in an inactive form and can be activated
through multiple pathways, namely the classical, lectin and alternative pathways, which
ultimately converge to generate the same effector molecules (e.g. C3a, C3b and C5a) (Zipfel and
Skerka, 2009). When activated, the components of the complement system can exert multiple
17
immunomodulatory activities, including stimulating inflammation (C3a and C5a) and
opsonisation of microorganisms (C3b) to promote their uptake by phagocytes (e.g. dendritic
cells and macrophages). Other activated complement proteins (C5b, C6, C7, C8 and C9) form a
membrane attack complex that can directly perforate the membranes of some microorganisms,
causing cell lysis (Zipfel and Skerka, 2009).
Elevated levels of activated complement proteins have been detected in gingival crevicular fluid
from patients with periodontitis (Attströum et al., 1975; Patters et al., 1989). Indeed, the
continual activation of the complement system by oral pathogens might stimulate chronic
inflammation by promoting vasodilation to enhance the flow of inflammatory exudate and
chemotaxis of immune cells (Hajishengallis, 2010). Antagonistically, some bacterial proteases
(e.g. gingipain proteases and interpain A) can degrade complement factor C3 to inhibit the
activation of the complement system (Popadiak et al., 2007; Potempa et al., 2009). P. gingivalis
gingipain proteases can also direct signalling crosstalk between complement receptors
(e.g. C5aR) and immune receptors to stimulate an inflammatory response, whilst subverting
host defence (refer to Section 1.7.1.1 for further details). The administration of a C5aR
antagonist in mice was demonstrated to be an effective therapeutic in arresting periodontitis
disease progression by reducing levels of pro-inflammatory and bone resorptive cytokines
(e.g. TNF and IL-1) (Abe et al., 2012). Therefore, the dysregulation of the complement system
can contribute to periodontal inflammation.
1.3.3.2 Antimicrobial Mediators
Antimicrobial proteins provide host protection by killing microorganisms directly or indirectly,
for example, through limiting nutrient acquisition (Table 1.1). Antimicrobial proteins, including
defensins and cathelicidin, are cationic peptides that kill microorganisms directly by binding to
negatively charged membrane components (e.g. LPS and lipoteichoic acids), and thus
compromise bacterial membrane integrity, resulting in cell lysis (Greer et al., 2013). The human
defensins are divided into two subclasses, namely -defensins and -defensins. The -defensins
(e.g. human neutrophil peptide (HNP)-1, HNP-2 and HNP-3) are contained in neutrophilic
granules, and therefore are largely localised to the junctional epithelium, where there is an
influx of neutrophils (Dale et al., 2001). Contrastingly, -defensins (e.g. hBD-1, hBD-2 and hBD-
3) are expressed throughout the stratified oral epithelium to serve as an antimicrobial layer
(Dale and Fredericks, 2005). hBD-1 and hBD 2 are expressed constitutively in the spinous,
granular and cornified layers in the oral epithelium at steady state, however hBD-2 expression
is also upregulated in the presence of oral commensals and pro-inflammatory stimuli (Greer et
al., 2013; Krisanaprakornkit et al., 2000; Mathews et al., 1999). hBD-3 is expressed in the basal
18
layers during health and can extend towards the spinous layer in disease (Dale and Fredericks,
2005; Greer et al., 2013).
Human cathelicidin is a cationic, antimicrobial peptide that is expressed by leukocytes
(e.g. neutrophils and monocytes) and various epithelia following pro-inflammatory stimulation
(Dale et al., 2001). The cathelicidin pro-protein (hCAP18) is processed by host serine proteases
(e.g. proteinase 3 and kallikerin) to produce the active cationic peptide, LL-37 (Sørensen et al.,
2001; Yamasaki, 2006). Like defensins, LL-37 exhibits a broad spectrum of antimicrobial
activity by disrupting microbial membranes. In addition, LL-37 is also chemotactic for
monocytes (Yang et al., 2000). Although defensins and LL-37 are effective in killing various oral
bacteria (e.g. F. nucleatum and P. intermedia) (Greer et al., 2013), bacterial species in the “red
complex” have been found to be less susceptible to the bactericidal activity of -defensins and
LL-37 (Bachrach et al., 2008; Joly et al., 2004; Ouhara et al., 2005). The resistance of T. denticola
to killing by hBD-2 was found to be attributable to the ability of the bacterium to produce a
unique outer membrane lipid, with a lower binding affinity for -defensins (Brissette and
Lukehart, 2007; Schultz et al., 1998), whereas P. gingivalis gingipain proteases can degrade and
thereby inactivate antimicrobial peptides (Devine et al., 1999; Maisetta et al., 2003; McCrudden
et al., 2013). Therefore, the resistance of pathogenic oral microbes to antimicrobial peptides
may contribute to microbial dysbiosis.
Antimicrobial proteins can also inhibit microbial growth by preventing the acquisition of
essential metal ions by microorganisms. Calprotectin is a heterodimer comprised of S100A8 and
S100A9 proteins, and exerts bacteriostatic activity by chelating essential divalent metal ions
(e.g. zinc and manganese) (Corbin et al., 2008). During inflammation, calprotectin levels are
upregulated in periodontal tissues and gingival crevicular fluid, where it has been shown to
facilitate immunity by promoting epithelial barrier function, and thus inhibit P. gingivalis
invasion of epithelial cells (Nakamura et al., 2000; Nisapakultorn et al., 2001). Lactoferrin,
which is present in gingival crevicular fluid and saliva, inhibits bacterial growth by sequestering
iron (Dale and Fredericks, 2005) and exerts growth-inhibitory activity on various oral bacteria
(e.g. S. mutans and P. gingivalis) (Aguilera et al., 1998; Arnold et al., 1980). Additionally,
lactoferrin was shown to inhibit multispecies biofilm formation in vitro (Arslan et al., 2009;
Wakabayashi et al., 2009). Together, antimicrobial mediators prevent the establishment of
bacterial infection by exerting bactericidal activity and inhibiting bacterial growth.
19
Table 1.1 Human antimicrobial mediators of host defence.
Antimicrobial Cellular sources Key immune functions
-defensins: HNP-1 HNP-2 HNP-3 HD5 HD6
Neutrophils Paneth cells
• Permeabilise bacterial membrane. • HNP1-3 stimulate migration of
monocytes
-defensins: hBD-1 hBD-2 hBD-3
Epithelial cells • Permeabilise bacterial membrane • hBD-2 stimulates migration of
macrophages, neutrophils and mast cells via CCR6
Cathelicidin: LL-37
Epithelial cells Monocytes Neutrophils
• Permeabilise bacterial membrane • Stimulate monocyte and neutrophil
chemotaxis
Calprotectin: S100A8 S100A9
Keratinocytes Neutrophils Monocytes Macrophages
• Sequester divalent cations (e.g. zinc and manganese) required for bacterial metabolism
• Stimulate neutrophil chemotaxis
Lactoferrin Neutrophils • Sequester iron required for bacterial metabolism
Adapted from (Kolls et al. 2008).
1.3.3.3 Cytokines
Cytokines are critical immune mediators that coordinate the host inflammatory response
through pleiotropic paracrine and autocrine immunomodulatory effects (Table 1.2). Cytokines
act through specific receptors, which are cell-surface glycoproteins that function as oligomeric
complexes, comprised typically of two to four receptor chains. For instance, the type-1 IL-1
receptor (IL-1R), which recognises IL-1, consists of immunoglobulin-like domains and an
intracellular Toll/Interleukin-1 receptor (TIR) domain. Upon IL-1 binding, IL-1R
heterodimerises with the IL-1 receptor accessory protein (IL-1RAcP) for signal transduction to
occur (Sims and Smith, 2010). Increased levels of TNF and IL-1 are associated with chronic
periodontitis (Graves and Cochran, 2003). They are potent pro-inflammatory cytokines, which
amplify the inflammatory response by stimulating the upregulation of additional pro-
inflammatory genes (Graves and Cochran, 2003). Furthermore, they can enhance the expression
of adhesion molecules, including ICAM-1 and VCAM-1, by endothelial cells to promote leukocyte
recruitment into affected tissues (Moser et al., 1989; Pober et al., 1986). Pro-inflammatory
cytokines (e.g. IL-12 and IL-18) produced by innate immune cells (e.g. macrophages) can also
enhance the activation of T lymphocytes to provide specific immunity against infection
(Trinchieri, 2003). Other cytokines, including IL-6 and IL-7, can stimulate the proliferation and
20
differentiation of B lymphocytes (Muraguchi et al., 1988; Takatsu, 1997). By contrast, IL-10 is an
important anti-inflammatory cytokine, which regulates the immune response by dampening the
production of pro-inflammatory molecules (Couper et al., 2008). In collaboration, the
combination of cytokines in the inflammatory milieu facilitate and coordinate the host immune
response.
Although cytokines are crucial for host defence, they also contribute to inflammatory disease
pathology when dysregulated. A dysregulated immune response maintained by excessive levels
of pro-inflammatory cytokines is a major contributor to the pathology of chronic periodontitis.
Consistently, exogenous administration of TNF in rats was shown to exacerbate inflammation
and increase alveolar bone loss (Gaspersic et al., 2003). The IL-1R and TNF receptor (TNFR)
signalling in a primate model of experimental periodontitis was shown to significantly reduce
alveolar bone loss, which correlated with reductions in the numbers of inflammatory immune
cells and bone-resorbing osteoclasts (Assuma et al., 1998). Furthermore, P. gingivalis can
destabilise the balance between IL-10 and IL-12 levels to promote inflammation. IL-10-deficient
mice were demonstrated to be more susceptible to P. gingivalis-induced alveolar bone loss
because of increased IL-12 production, which resulted in enhanced T lymphocyte-mediated
activity (Sasaki et al., 2004). Thus, the immune response can be compromised when cytokine
signalling networks are dysregulated in chronic periodontitis.
21
Table 1.2 Roles of major groups of cytokines in host defence.
Adapted from (Taylor, 2010).
1.3.3.4 Chemokines
Chemokines play an indispensable role in regulating the directed migration of immune cells
(Table 1.3). Chemokines are typically characterised by the presence of three to four N-terminal
cysteine residues, and further divided into subfamilies based on the position of the first two
cysteine residues (Allen et al., 2007). The majority of chemokines belong to the CC and CXC
subfamilies. The CXC family of chemokines is further divided based on the presence or absence
of a glutamate, leucine and arginine (ELR) motif in the N-terminal region. Chemokines bind to
seven transmembrane G-protein coupled receptors, which are named based on the chemokine
type they bind (e.g. CC receptor (CCR) and CXC receptor (CXCR)). Most chemokine receptors can
Cytokine group Examples Key functions
Pro-inflammatory cytokines
IL-1, IL-6, IL-12, TNF
• Promote primary innate immune response and activation of the inflammatory response
• Facilitate the activation of the adaptive immune response
Anti-inflammatory cytokines
IL-10, IL-13, TGF • Downregulate the immune response and inflammation
Colony-stimulating factors
CSF-1, CSF-2, CSF-3 • Drive myelopoiesis by stimulating proliferation and differentiation of monocytes and granulocytes
Th1 regulatory cytokines
IL-12, IL-18 • Promote Th1 differentiation and activation
Th2 regulatory cytokines
IL-4, IL-5, IL-25 • Promote Th2 differentiation and activation
Th17 regulatory cytokines
IL-17, IL-22, IL-23 • Promote Th17 differentiation and activation
B lymphocyte regulatory cytokines
IL-4, IL-5, IL-6, IL-7 • Promote B lymphocyte proliferation and activation
Growth factors EGF, TGFPDGF • Regulate tissue repair and fibrosis by promoting proliferation, differentiation and migration of fibroblast, endothelial and epithelial cells.
22
be activated by multiple ligands, which enables significant functional redundancy between
chemokines (Allen et al., 2007). For instance, CXCR2, which is highly expressed by neutrophils,
is activated by multiple chemokines, including CXCL1, CXCL2 and CXCL3.
Chemokines possess an array of immunomodulatory functions. Homeostatic chemokines
regulate the migration of immune cells as part of haematopoiesis, which is required for the
formation, development and differentiation of leukocytes. For instance, CXCL12 is produced by
bone marrow stromal cells and regulates the retention of haematopoietic stem cells in the bone
marrow niche (Ara et al., 2003). As their name suggests, inflammatory chemokines are induced
in response to inflammatory stimuli, such as microbial products as well as inflammatory
cytokines. Classical inflammatory chemokines include IL-8 (CXCL8) and CXCL1, which regulate
the recruitment of neutrophils during the early stages of infection. Chemokines also facilitate
the adaptive immune response by directing the recruitment of lymphocytes. For example,
CXCL9 and CXCL10 activate CXCR3 expressed on the cell-surface of naïve T lymphocytes to
promote their migration (Groom and Luster, 2011; Kobayashi, 2006). In addition to regulating
immune cell chemotaxis, some chemokines have also been shown to have bactericidal activity.
These chemokines, including CCL20 and CCL28, contain positively-charged surface amino acids
and appear to act in a similar manner as antimicrobial peptides (e.g. -defensins) to kill bacteria
(Hoover et al., 2002; Yang et al., 2003).
By regulating the types of immune cells recruited and activated, chemokines can influence the
polarisation of immune responses. The upregulation of CCL2 and its receptor, CCR4, in chronic
periodontitis can cause the excessive recruitment of macrophages and exacerbate the
inflammatory response (Garlet et al., 2003; Souto et al., 2014). Elevated chemokine expression
can also contribute to tissue destruction and alveolar bone resorption. Increased levels of
CXCL12 in the gingival crevicular fluid of patients with chronic periodontitis is associated with
enhanced osteoclast bone-resorptive activity and MMP-9 production (Grassi et al., 2004; Havens
et al., 2008). Moreover, the dysregulation of chemokine responses by bacterial pathogens can
lead to a suboptimal immune response. P. gingivalis can inhibit the recruitment of immune cells
by blocking nuclear factor-NF-) activation to suppress chemokine expression (e.g. IL-8)
(Takeuchi et al., 2013). P. gingivalis gingipain proteases can also degrade chemokines, including
IL-8, to inhibit neutrophil chemotaxis (Darveau et al., 1998; Zhang et al. 1999; Stathopoulou et
al. 2009). In addition, the CXCL12 receptor, CXCR4, can also be “hijacked” by P. gingivalis to
dampen the antimicrobial functions of macrophages (refer to Section 1.7.2 for further details).
Consequently, dysregulated chemokine responses arising from microbial dysbiosis can perturb
the balance between protective and destructive immunity.
23
Table 1.3 Roles of chemokines in host defence.
1.4 Pattern Recognition Receptors
Pattern recognition receptors (PRRs) are germline-encoded receptors that are critical for the
detection of conserved pathogen-associated molecular patterns (PAMPs) by the host. The major
families of PRRs include transmembrane Toll-like receptors (TLRs), protease-activated
receptors (PARs), cytosolic NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs). PRRs
differ in their subcellular localisation and ability to recognise specific PAMPs. PRR activation
initiates intracellular signalling cascades, which classically lead to the activation of transcription
factors, including NF-B and Interferon Regulatory Factors (e.g. IRF3), which regulate the
expression of inflammatory genes (e.g. TNF, IL-6, and IFN-). Although not classical PRRs,
protease-activated receptors (PARs) can also detect bacteria by their expression of proteases.
Given that bacterial dysbiosis is a critical driver of chronic periodontitis, the following section
will focus on receptors that are involved in recognising bacteria.
Chemokine Former name Receptor Key immune functions
CXCL1 Gro CXCR2 • Regulate neutrophil migration
CXCL8 IL-8 CXCR1 CXCR2
• Regulate neutrophil migration
CXCL10 IP-10 CXCR3 • Regulate T lymphocyte migration. • Promote Th1 adaptive immunity by
inducing IFN-expression
CXCL12 SDF-1 CXCR4 • Regulate T lymphocyte migration
• Maintenance of haematopoietic stem cells
CCL1 I-309 CCR8 • Regulate Th2 lymphocyte migration
CCL2 MCP-1 CCR2 • Regulate monocyte/macrophage migration
CCL5 RANTES CCR1 • Regulate innate immune cells (e.g. natural killer cells) migration
• Regulate adaptive immune cells (e.g. T lymphocytes) migration
CCL20 MIP-3 CCR6 • Regulate Th17 lymphocyte migration • Antimicrobial activity
24
Toll-like Receptors
The TLR family is comprised of ten members and are expressed in varying cell types (Table 1.4)
(Takeda et al., 2003). The subcellular localisation of TLRs varies to allow optimal recognition of
their cognate ligand. TLRs form homodimers or heterodimers upon activation, and the
intracellular TIR domain facilitates protein-protein interactions with adaptor proteins to induce
a series of signalling cascades. Ultimately, transcription factors (e.g. NF-B and IRFs) are
activated and induce the expression of inflammatory genes to activate and coordinate the
immune response. TLRs also mediate the development of adaptive immunity.
Table 1.4 Toll-like receptor subcellular localisation and ligands.
1.4.1.1 TLR Distribution and Subcellular Localisation
TLRs are widely distributed in various tissues throughout the body. Consistent with their role in
immune surveillance, TLRs are highly expressed in tissues that are exposed to the external
environment (e.g. oral cavity and skin, and respiratory, gastrointestinal and urogenital tracts),
and are therefore potentially more susceptible to infection (Zarember and Godowski, 2002).
Epithelial cells at the apical surface of the oral mucosa express lower levels of TLR1 to TLR9 to
avoid harmful activation of the immune system by constant exposure to commensal microbes
Receptor Subcellular Localisation
Ligand Synthetic agonist
TLR1 Cell surface Triacyl lipopeptides Pam3CSK4
TLR2 Cell surface Lipoproteins Diacyl lipopeptides Triacyl lipopeptides
Pam3CSK4 FSL-1
TLR3 Endosomes Double-stranded RNA Poly I:C
TLR4 Cell surface Lipopolysaccharide TLR5 Cell surface Flagellin
TLR6 Cell surface Diacyl lipopeptides
TLR7 Endosomes Single-stranded RNA Imiquimod
TLR8 Endosomes Single-stranded RNA Imiquimod
TLR9 Endosomes Unmethylated CpG DNA CpG-oligonucleotides
TLR10 Unknown Unknown
25
(Beklen et al., 2008). Peripheral blood leukocytes and spleen cells express the largest
repertoires of TLRs (Zarember and Godowski, 2002). The expression of TLRs can vary between
different cellular subsets, which can change over the course of leukocyte development. For
instance, the expression levels of TLR1, 2, 4, and 5 by immature dendritic cells decrease
following maturation (Visintin et al., 2001). Importantly, the selective expression and activation
of TLRs on dendritic cells imparts functional specialisation and tolerance in immunity (Dalod et
al., 2014).
The TLRs are localised to the cell-surface or intracellular membranes (e.g. endosomes) to
facilitate PAMP recognition. TLRs that are localised to the plasma membrane include: TLR2,
which can heterodimerise with TLR1 and TLR6 to recognise bacterial triacyl and diacyl
lipoproteins, respectively (Takeuchi et al. 2002; Ozinsky et al. 2000); TLR4, which recognises
LPS, and TLR5, which recognises flagellin (Hayashi et al., 2001; Hoshino et al., 1999). The other
TLRs are localised to intracellular compartments (e.g. endosomal membranes) and include:
TLR3, which recognises double-stranded viral RNA (Alexopoulou et al., 2001); TLR7 and TLR8,
which recognise single-stranded RNA, and TLR9, which recognises unmethylated CpG-
containing DNA (Hemmi et al., 2000). The complex regulation and trafficking of TLRs to the cell
surface and endosomal compartments is mediated by specific chaperones (e.g. endoplasmin)
(Gay et al., 2014). The subsequent degradation of activated TLRs is essential to regulate the host
immune response. Ligand binding by cell-surface TLRs, such as LPS with TLR4, triggers
clathrin-mediated endocytosis and subsequent lysosomal degradation (Husebye et al., 2006),
whilst endosomal TLRs (e.g. TLR9) are targeted for proteasomal degradation (Chuang and
Ulevitch, 2004). As such, the trafficking of TLRs to their correct subcellular localisation allows
for optimal ligand recognition as well as the subsequent downregulation of signalling.
1.4.1.2 TLR Structural Organisation
Leucine-Rich Repeat Motif
TLRs are type I transmembrane receptors, and consist of an N-terminal leucine-rich repeat
(LRR) domain, a transmembrane region, and a C-terminal TIR domain (Fig. 1.2). The LRR
domain is comprised of multiple repeats of a conserved “LxxLxLxxN” motif, where “x” denotes a
hydrophobic residue. The conserved leucine residues confer the domain with a horseshoe-like
structure, as the side-chains point inward to form a hydrophobic core (Fig. 1.3). Each TLR has a
distinct LRR domain, which is important for PAMP-binding specificity (Bella et al., 2008).
26
Figure 1.2 Structural arrangement of Toll-like receptors. TLRs are comprised of an extracellular LRR domain that forms a horseshoe-like structure, which binds a cognate ligand. The intracellular Toll-Interleukin-1 (TIR) domain is crucial for facilitating adaptor protein recruitment, through homotypic interactions, for subsequent intracellular signalling.
LRR motif
TIR domain
Phospholipid
bilayer
Transmembrane domain
27
Figure 1.3 Crystal structure of the TLR3 LRR motif. The TLR3 ectodomain (PDB: 3CIG) forms a solenoid structure, comprised of twenty-three LRR repeats. The conserved hydrophobic residues of the LRRs form a tightly packed hydrophobic core, which provides lateral stability.
Toll/Interleukin-1 Receptor (TIR) Domain
The intracellular TIR domain facilitates the binding of specific adaptor proteins (e.g. myeloid
differentiation primary response 88 (MYD88) and MYD88 adaptor like (MAL)), which is
required for subsequent downstream signalling. As its name suggests, the TIR domain shares a
high degree of homology with the cytoplasmic domain of the type-1 IL-1 receptor. The TIR
domain is composed of around 200 amino acid, and typically adopts a flavodoxin-like fold
comprised of five central -sheets (A - E) surrounded by five -helices (A - E) (Fig. 1.4).
The loops adjoining -sheets and -helices are named according to the secondary elements to
which they are connected; for instance, the BB loop connects the B strand and B helix.
Mammalian TIR domains share 20-30% sequence conservation, which provides sufficient
structural diversity to confer signalling specificity (Xu et al., 2000). The TIR domain of TLRs is
conserved in three regions, denoted as box 1, box 2 and box 3 (Watters et al., 2007). Box 1
signifies the start of the TIR domain and is characterised by a conserved (F/Y)DAFISY motif. The
box 2 motif in the BB loop, which has been studied most extensively, is an important structural
determinant for TIR-TIR interactions (Xu et al. 2000). The BB loop is characterised by an
RDxɸ1ɸ2G motif, where “x” denotes any residue, and “ɸ” denotes a hydrophobic residue. The
proline residue at the ɸ2 position has also been shown to be required for TLR signalling
N
C
28
(Poltorak et al., 1998; Tao et al., 2002). For example, the natural mutation of Pro712 to histidine
in TLR4 renders CH3/HeJ mice unresponsive to LPS (Poltorak et al., 1998). Similarly, a Pro681
to histidine mutation in the BB loop of TLR2 impaired signalling in response to Mycobacterium
tuberculosis (Underhill et al., 1999). Further studies revealed that the mutation disrupted the
interaction between TLR2 and MYD88 (Xu et al., 2000). Box 3 is defined by a FW motif, which is
surrounded by basic amino acids. The functional role of box 3 has yet to be fully elucidated;
however, mutagenesis studies indicate that it is required for signalling, and, in the case of IL-1R,
for correct receptor subcellular localisation (Slack et al., 2000). In summary, TIR domains share
conserved features to enable specific TIR-TIR interactions and downstream signalling.
Figure 1.4 Crystal structure of the TLR1 and TLR2 TIR domains. The TIR domain of (A) TLR1 and (B) TLR2 contain a characteristic flavodoxin fold comprised of -sheets surrounded by helices. The box 1, box 2 and box 3 are as indicated by the yellow, green and pink colouring, respectively.
1.4.1.3 Toll-like Receptor Signal Transduction
When activated, TLRs can either homo or heterodimerise through TIR-TIR domain interactions.
Furthermore, the interacting TIR domains provide a platform for subsequent binding by TIR
domain-containing adaptor proteins to initiate downstream signalling. Depending on the
adaptor protein(s) that is recruited, TLRs can signal either through the MYD88-dependent
pathway or TRIF-dependent pathway (Fig. 1.5). The MYD88-dependent pathway is utilised by
all TLRs, with the exception of TLR3, to stimulate downstream signalling. In the case of TLR2
and TLR4, MAL acts as a bridging adaptor to facilitate the recruitment of MYD88 (Fitzgerald et
al., 2001). The protein kinases Interleukin-1 receptor-associated kinase 4 (IRAK-4) and IRAK-1
subsequently interact with MYD88 through their N-terminal death domain (Wesche et al.,
1997). Activated IRAK-1 phosphorylates and thereby activates the E3 ubiquitin ligase, Tumour
A B
29
necrosis factor receptor-associated factor-6 (TRAF6). TRAF6, in conjunction with the ubiquitin
conjugating enzymes, UEV1A and UBC13, catalyses the Lys-63 polyubiquitination of TAB2 and
TAB3, which are regulatory components of the protein kinase, TGF--activated kinase (TAK1).
This causes the activation of TAK1, and brings it into close proximity with the inhibitor nuclear
factor-B (IB) kinase (IKK) complex. The IKK complex regulates the activity of IB, which
sequesters NF-B in the cytoplasm to prevent its activation and translocation into the nucleus.
TAK1-mediated phosphorylation of the IKK complex leads to NF-B activation via the
phosphorylation and subsequent proteasomal degradation of IB. This releases NF-B, allowing
it to translocate into the nucleus to activate inflammatory gene expression (e.g. TNF and IL-6).
TLR3, as well as TLR4, signals through the TIR domain-containing adaptor-inducing
Interferon- (TRIF)-dependent pathway (Yamamoto et al., 2003a); TLR4 specifically requires
the protein adaptor TRIF-related adaptor molecule (TRAM) for the recruitment of TRIF (Jenkins
et al., 2010). TRIF signalling leads to the activation of NF-B and IRF3. TRIF associates with
TRAF6 and induces NF-B activation in a similar manner to the MYD88-dependent pathway. In
addition, TRIF can also stimulate IRF3 activation by forming a complex with TANK-binding
protein kinase-1 (TBK1) and inducible B kinase IKKi, which phosphorylate and thereby
activates IRF3 (Sato et al., 2003). Once activated, IRF3 can then translocate into the nucleus to
transcribe type I interferon and interferon-stimulated genes (e.g. 2'-5'-oligoadenylate
synthetases).
30
Figure 1.5 TLR signalling pathways. All TLRs, except for TLR3, signal via the MYD88-dependent pathway. TLR4 utilises both the MYD88 and TRIF pathways to activate the inflammatory response.
31
1.4.1.4 TIR domain-containing Adaptor Proteins
As indicated above, TIR domain-containing adaptor proteins play a central and critical role in
TLR signalling. The subcellular localisation of TIR domain-containing adaptor proteins is
essential in facilitating and ensuring efficient signal transduction. The initial binding of TIR
domain-containing adaptor proteins (e.g. MYD88 and TRIF) upon TLR activation promotes the
assembly of intracellular signalling platforms for the subsequent recruitment of downstream
adaptor proteins to confer TLR signalling specificity. Post-translational modifications to adaptor
proteins also serve as an additional level of regulation for TLR signalling.
Myeloid Differentiation Primary Response 88 (MYD88)
The crucial role of MYD88 in TLR signalling was demonstrated when MYD88-deficient mice
were shown to be unresponsive to LPS (Kawai et al., 1999). The MYD88 protein has an
N-terminal death domain and a C-terminal TIR domain, which are separated by an intermediate
domain (Fig. 1.6). At steady state, MYD88 is localised throughout the cytosol; however, MYD88
has also been shown to form punctate inclusions when ectopically overexpressed in cells
(e.g. RAW 264.7 mouse macrophages) (Into et al., 2010; Nishiya et al., 2007). When activated,
the N-terminal death domain of MYD88 is essential for its correct subcellular localisation to
facilitate TIR domain interaction with TLRs, and to recruit IRAK family members (e.g. IRAK-4
and IRAK-1) for downstream signalling (Dunne et al., 2003; Nishiya et al., 2007).
MYD88 activity can be modulated to regulate the inflammatory response. An alternative splice
variant of MYD88, lacking the intermediate domain, is expressed in monocytes upon LPS
stimulation (Janssens et al., 2002). This splice variant of MYD88 inhibits LPS-induced NF-B
signalling because it cannot recruit IRAK-4, which is necessary for IRAK-1 phosphorylation and
activation (Burns et al., 2003). MYD88-mediated signalling is also regulated by TGF-, whereby
MYD88 is ubiquitinated and targeted for proteasomal degradation (Lee et al., 2011).
MYD88 Adaptor-Like (MAL)
The importance of MAL for TLR2 and TLR4 signalling was established with MAL-deficient mice
(Horng et al., 2002; Yamamoto et al., 2002). MAL is comprised of an N-terminal
phosphoinositide-binding domain, which can bind phosphatidylinositol-4, 5-biphosphate
(PtdIns(4,5)P2) and thereby localise MAL to the plasma membrane (Kagan et al., 2006), and a
C-terminal TIR domain (Fig. 1.6). The physical association of MAL with the plasma membrane
facilitates its role as a bridging adaptor for MYD88 with TLR2 and TLR4. The electro-negative
charge on the surface of the TIR domain of MAL allows it to interact with the electro-positive
surfaces of the corresponding TIR domains of TLR4 and MYD88 (Dunne et al., 2003). In addition
32
to acting as a bridging adaptor, MAL can direct TLR signalling in a MYD88-independent manner
by recruiting TRAF6 into a signalling complex through its TRAF6-binding domain (Mansell et al.,
2004). MAL is also involved in the negative regulation of TLR2 and TLR4 signalling. The
phosphorylation of MAL by Bruton’s tyrosine kinase in response to TLR2 and TLR4 activation
triggers its interaction with the suppressor of cytokine signalling 1 (SOCS-1) to promote the
subsequent degradation of MAL (Mansell et al., 2006). Thus, not only is MAL involved in signal
transduction required to initiate inflammation, its degradation also forms part of the negative
regulation of TLR responses to limit inflammation.
TIR-domain-containing Adaptor-inducing Interferon- (TRIF)
TRIF is essential for mediating TLR3 and TLR4-activated type I interferon induction (Yamamoto
et al. 2003b). TRIF is directly recruited to TLR3, whilst TRAM is required to facilitate TRIF
recruitment to TLR4 (Yamamoto et al. 2003c). TRIF is characterised by an N-terminal
TRAF6-binding domain, and a C-terminal TIR domain (Fig. 1.6). Mutations in the TRAF6-binding
domain compromises NF-B but not IRF3 activation (Jiang et al., 2004; Sato et al., 2003). TRIF
forms a complex with TBK1 through its N-terminus to mediate IRF3 activation (Sato et al.,
2003). Moreover, studies indicate that NF-B-activating kinase (NAK)-associated protein
(NAP1) is required for TRIF to form a signalling complex with TBK1 (Sasai et al. 2005). TRIF is
also subject to negative regulation, whereby the C-terminal domain of TRAF1 can interact with
the TIR domain of TRIF to inhibit signalling (Su et al., 2006).
TRIF-related Adaptor Molecule (TRAM)
TRAM is similar to MAL, in that it acts as a bridging adaptor for TRIF to interact with TLR4
(Yamamoto et al. 2003c). The TIR domain is localised at the C-terminus of TRAM (Fig. 1.6).
Myristoylation of the second glycine residue of TRAM allows its hydrophobic interaction with
the lipid bilayer to stabilise its association with the plasma membrane (Rowe et al., 2006).
TLR4-mediated TRAM-TRIF signalling occurs following endocytosis of the TLR4 complex
(Kagan et al., 2008). It was proposed that the rearrangement of the TLR4 TIR domain in the
acidic environment of endosomes enables optimal interaction with TRAM (Gangloff, 2012). The
protein kinase C (PKC)-mediated phosphorylation of TRAM at serine-16 is essential for TRAM
signalling (McGettrick et al., 2006). However, the mechanistic involvement of PKC-mediated
phosphorylation of TRAM has yet to be elucidated.
Sterile Alpha and Armadillo-motif-containing (SARM)
SARM was the last TIR domain adaptor protein to be identified, and acts as a negative regulator
of TRIF signalling (Carty et al., 2006). It is comprised of two consecutive sterile alpha motif
33
(SAM) domains, and a TIR domain at the C-terminus (Fig. 1.6). The expression of SARM is
upregulated in response to TLR4 activation and SARM acts as an inhibitor of NF-B and IRF3
activation (Carty et al. 2006). It was proposed that SARM associates with TRIF transiently in the
resting state, and the activation of TLR3 or TLR4 stabilises this interaction to block the
recruitment of TIR domain adaptor proteins to inhibit downstream signalling (O’Neill, 2006).
Although SARM does not affect MYD88-dependent NF-B signalling, it was recently found to
inhibit the MYD88-dependent activation of the transcription factor activator-protein-1 (AP-1)
(Peng et al., 2010). Further studies will be required to clarify the mechanism of SARM inhibition
of TRIF, and to elucidate whether SARM has additional functions in TLR signalling.
Figure 1.6 Structural arrangement of TIR domain-containing adaptor proteins.
NOD-like Receptors
NOD-like receptors (NLRs) are a family cytosolic PRRs that can detect invading pathogens and
their products. The NLR family consists of 23 members, which are expressed in various cell
types, including epithelial cells and immune cells (Franchi et al., 2009). Structurally, NLRs are
characterised by an N-terminal effector domain (e.g. BIR, CARD or Pyrin domain), which
mediates its interaction with other proteins, a central NACHT domain, which mediates
homo-oligomerisation, and a C-terminal LRR domain for bacterial-sensing. Based on their
N-terminal domain, NLRs can be classified into four subfamilies: (i) NLRA, (ii) NLRB, (iii) NLRC
and (iv) NALP (Fig. 1.7). The activation of NLRs is triggered by the LRR binding a PAMP, which
initiates the oligomerisation of the receptor through the NACHT domain for subsequent
recruitment of downstream signalling proteins.
NOD1 and NOD2 are two of the most widely studied members of the NLR family and recognise
bacterial cell wall peptidoglycan fragments, namely, muropeptide (iE-DAP) and muramyl
dipeptide (MDP), respectively (Chamaillard et al., 2003; Inohara et al., 2003). Upon ligand
binding, NOD1 and NOD2 undergo self-oligomerisation and recruit receptor-interacting serine-
MYD88
MAL
TRIF
TRAM
SARM
Death domain
Intermediate domain
TIR domain
TRAF6-binding domain
SAM motif
Key
Phosphoinositide-binding motif
34
threonine kinase 2 (RIP2) via a CARD-CARD interaction (Abbott et al., 2004). Activated RIP2
promotes TRAF-mediated (TRAF2 and/or TRAF5 in the case of NOD1 and TRAF6 in the case of
NOD2) K63-linked ubiquitination TAK1 and the IKK complex. The ensuing co-localisation of
TAK1 and IKK complex leads to the phosphorylation and subsequent activation of the IKK
complex for the degradation of IB, resulting in NF-B activation and nuclear translocation
(Fig. 1.8). RIP2 can also signal with inhibitor of apoptosis (IAPs) to induce NOD-mediated NF-B
activation (Krieg et al., 2009). Notably, NOD2 has also been found to be involved in the negative
regulation of TLRs. For example, co-stimulation of TLR2 and NOD2 with peptidoglycan and
MDP, respectively, inhibited TLR2-induced IL-12 production in splenocytes (Watanabe et al.,
2004).
NLRs also regulate inflammation by forming inflammasomes, multimeric complexes that
regulate the activation of inflammatory caspases, which mediate IL-1 maturation. Pro-IL-1
expression is typically maintained at the transcriptional level by NF-B, whilst the processing
and secretion of mature IL-1is regulated by inflammasome-mediated caspase-1 activation
(Fitzgerald, 2010). NLRP1, NLRP3 and NLRC4 act as cellular sensors for the assembly of
canonical inflammasomes. When activated, NLRP1 and NLRP3 interact with the adaptor protein
apoptosis-associated speck-like protein containing a CARD domain (ASC), which in turn binds
the CARD domain of caspase-1 to facilitate inflammasome assembly (Schroder and Tschopp,
2010). Contrastingly, homo-oligomers of NLRC4 can interact directly with caspase-1
independently of ASC (Schroder and Tschopp, 2010). Consequently, a regulated and controlled
inflammatory response is achieved through both the transcriptional control and post-
translational processing of IL-1.
35
Figure 1.7 Structural arrangement of NOD-like receptors.
NLRA
NLRB
NLRC
NLRP
NOD1 and
NLRC4
NOD2
Key
CARD
NACHT
BIR
Pyrin
LRR
NLRC3 and
NLRC5
Undefined domain
NLRP1
NLRP2 and
NLRP9
NLRP10
NAIP
CIITA
36
Figure 1.8 NOD-like receptor signalling pathways. NOD2 homo-oligomerises upon binding of muramyl dipeptide (MDP) fragments derived from microbes, and recruit downstream signalling adaptor proteins to stimulate a pro-inflammatory response.
37
Protease-activated Receptors
The innate immune system can also sense proteolytic enzymes during infection through
protease-activated receptors (PARs). The family of four PARs are transmembrane G-protein
coupled receptors that regulate an array of responses (Table 1.5). Host- and bacterial-derived
serine proteases cleave PARs to expose an N-terminal sequence, which acts as a tethered ligand
and interacts with the second extracellular loop of the receptor to initiate intracellular signal
transduction (Soh et al., 2010). Synthetic, activating peptides have been designed to mimic the
tethered ligand and stimulate receptor activation. Ligand activation of PARs induces a
conformational change to stabilise the binding of heterotrimeric G proteins, comprised of ,
and subunits. Once activated, the G proteins act as guanine nucleotide exchange factors,
whereby GDP bound by the subunit is converted to GTP. This also results in the dissociation of
the and subunits to induce diverse cellular responses, including inflammation, haemostasis
and thrombosis (Soh et al., 2010). Although PARs are activated by similar mechanisms, they can
regulate different biological outcomes depending on their tissue distribution. Furthermore, the
association of PARs with different G subunits also confer signalling specificity. For instance,
PAR-1 can interact with Gq11, G12/13 and Gi, whilst PAR-2 is coupled to Gq. Following
activation, PARs (with the exception of PAR-1) are subsequently phosphorylated and recognised
by -arrestins to facilitate their internalisation via clathrin-coated pits to dampen signalling
(DeFea et al., 2000; Paing et al., 2002).
PARs have been extensively studied for their role in the coagulation cascade; however, they
have also been shown to be activated by bacterial proteases and activate host inflammatory
responses. The gingipain proteases expressed by P. gingivalis were shown to activate PAR-2 in
gingival epithelial cells and enhance the production of IL-6 (Lourbakos et al., 2001). Moreover,
PARs have also been shown to contribute to the inflammatory response by promoting immune
cell recruitment by stimulating the upregulation of endothelial VCAM-1 and ICAM-1 expression
(Coughlin and Camerer, 2003; Rahman et al., 2002). Consistently, alveolar bone resorption in a
P. gingivalis-induced model of periodontitis was reduced in PAR-2-deficient mice (Holzhausen
et al., 2006). Thus, PARs can serve as a defence mechanism to trigger host inflammation by
sensing bacterial proteases.
38
Table 1.5 PARs activating proteases, cellular expression and key functions.
Adapted from (Ossovskaya and Bunnett 2004).
1.5 Microbial Subversion of TLR Signalling
Many bacterial and viral pathogens have evolved mechanisms to subvert and thereby modulate
the host immune response to their benefit. The innate immune system is particularly attractive
as a target for subversion, as suppressing innate immunity may also compromise adaptive
immunity. Pathogens can subvert the host immune responses through multiple mechanisms,
including PAMP modification and targeting proteins critical for PRR signalling.
PAMP Modification
Structural modifications of membrane-surface PAMPs can mask PRR recognition sites to avoid
immune detection. LPS is a major component of the outer membrane of Gram-negative bacteria.
It is an amphipathic molecule comprised of a hydrophilic polysaccharide chain, denoted as the
O-antigen, a core region, and a hydrophobic lipid A moiety. TLR4 recognises the lipid A
component of LPS, which is conserved in Gram-negative bacteria (Poltorak et al., 1998; Shimazu
et al., 1999). Interestingly, the microenvironment to which Gram-negative bacteria are exposed
may generate LPS species with different inflammatory capacities due to differential
modification of lipid A (e.g. acylation) (Dixon and Darveau, 2005). Highly-acylated lipid A
(e.g. hexa-acylated lipid A of Escherichia coli) is a potent inflammatory stimulus, whereas less-
acylated lipid A (e.g. tetra-acylated lipid A of P. gingivalis) is a weaker inflammatory stimulus
(Barksby et al., 2009). Salmonella typhimurium LPS is subjected to 3-O-deacylation and
Receptor Expression Activating Protease
Activating peptides Key function(s)
PAR-1 Platelets Epithelia Endothelia Fibroblasts Neurons
Thrombin SFLLR-NH2 • Platelet aggregation
• Vascular smooth muscle cell proliferation
• Cytokine secretion
PAR-2 Epithelium Endothelium Fibroblasts Neurons
Trypsin Tryptase Factor VIIa Factor Xa Elastase
SLIGKV-NH2 • Cell migration
• Cell proliferation
PAR-3 Endothelium Thrombin • Cytokine secretion
PAR-4 Platelets Endothelium
Thrombin Trypsin Plasmin
GYPGQV-NH2
AYPGKF-NH2
• Platelet activation • Neutrophil recruitment
39
palmitoylation by the lipid modifying enzymes 3-O-deacylase (PagL) and lipid A
palmitoyltransferase (PagP), respectively (Bishop et al., 2000; Trent et al., 2001). These
modifications were demonstrated to prevent TLR4, and hence NF-B, activation (Kawasaki et
al., 2004). Similarly, Pseudomonas aeruginosa lipid A containing a 3-O-deacylation modification
was also shown to stimulate weaker TLR4-mediated signalling (Stöver et al., 2004).
TLR5 recognises the conserved N-terminal region of flagellin, the main structural component of
flagella. Polymorphisms in the N-terminal region of flagellin can induce weaker TLR5 activation.
For example, the absence of the conserved 89-96 amino acid sequence towards the N-terminus
of flagellin from alpha proteobacteria and epsilon proteobacteria (e.g. Helicobacter pylori and
Campylobacter jejuni, respectively) results in it no longer being detected by TLR5 (Andersen-
Nissen et al., 2005). Bacteria can also avoid TLR5 recognition by modulating flagellin
expression; Listeria monocytogenes downregulates flagellin at physiological temperature (37 °C)
(Kathariou et al., 1995). Thus, modifications to bacterial cell-surface components can
significantly reduce the magnitude of the host immune response elicited.
Targeting TLR Signalling Proteins
As discussed above, TLR adapter proteins are essential for signal transduction by TLRs.
Therefore, pathogens have evolved mechanisms to target TLR adaptor proteins as a strategy to
suppress host immunity. Bacterial pathogens can modulate MAPK signalling by expressing
bacterial phosphatases. Shigella flexneri expresses a phosphatase, OspF, which is delivered into
the host cell cytoplasm via a type III secretion system, where it translocates into the nucleus and
dephosphorylates and inactivates ERK1/2 and p38 MAPK (Arbibe et al., 2007; Zurawski et al.,
2006). Transcriptional analyses indicate that OspF suppresses the expression of early
inflammatory genes, including IL-8 and CCL20 (Arbibe et al., 2007). Consequently, mice infected
with an OspF-deficient S. flexneri mutant developed more severe mucosal lesions associated
with a greater neutrophil influx (Arbibe et al., 2007). Viruses also target components
downstream of TIR-domain-containing adaptors. The vaccinia viral protein, A52R, was
demonstrated to inhibit NF-B activation by interacting with TRAF6, and thereby suppress
TLR3-mediated IL-8 and CCL5 expression (Bowie et al., 2000; Maloney et al., 2005). In addition,
A52R can also stimulate the activation of p38 mitogen-associated protein kinase (MAPK) to
induce the expression of the anti-inflammatory cytokine, IL-10 (Maloney et al., 2005). The
upregulation of IL-10 expression dampens inflammation and cell-mediated immune response,
thus reducing virus elimination by the host. Taken together, the inhibition of TLR signalling is a
key strategy used by pathogens to suppress host defence.
40
1.6 Bacterial TIR Domain-containing Proteins
Some pathogens have evolved TIR domain-containing proteins that can disrupt host TIR-TIR
interactions and thus interfere with TLR signalling (Table 1.6). Bacteria have been found to
express TIR domain-containing proteins (Tcps) that inhibit TLR-induced responses. Using
bioinformatics approaches, Newman et al. identified over 200 bacterial proteins containing a
putative TIR domain (Newman et al., 2006). These bacterial Tcps are found in various bacterial
phyla, including Chlorobi, Proteobacteria and Bacteroidetes (Spear et al., 2009). Phylogenetic
analysis suggests that bacterial Tcps may have evolved through multiple horizontal gene
transfer events (Zhang et al., 2011). Several bacterial Tcps have been characterised and studied
for their ability to inhibit TLR signalling. This section will highlight the involvement of
functionally characterised bacterial Tcps in suppressing TLR signalling.
Table 1.6 Bacterial Tcps and immune subversion.
Protein Bacterium Mode of antagonisation Functional in vivo studies
TcpB Brucella melitensis
• Inhibit TLR4-mediated signalling
• Interact with MAL, MD88 and TLR4
• Downregulate MAL expression
• Facilitate systemic spread of bacteria
TcpC Escherichia coli
• Inhibit TLR2 and TLR4-mediated signalling
• Interact with MYD88
• Required for virulence in mouse model of urinary tract infection
• Facilitate bacterial colonisation
PdTIR Paracoccus denitrifcans
• Interact with TLR4 and MYD88
YpTdp Yersinia pestis • Interact with MYD88 • Interfere with
TLR2-dependent signalling
• Not required for bacterial colonisation
TIRs Staphylococcus aureus
• Inhibit MAL and MYD88-dependent immune response
TlpA Salmonella enterica
• Inhibit TLR signalling • Induce caspase-1-
dependent IL-1 secretion
• Mice infected with TlpA-deficient mutant exhibited reduced lethality
41
Structural Properties of Bacterial Tcps
Bacterial Tcps are typically 200-300 amino acids in length, and have a C-terminal TIR domain
that is comprised of approximately 150-200 amino acids. Initial structural homology modelling
of TcpC of E. coli CFT073, a uropathogenic strain of E. coli and TcpB of Brucella melitensis
suggested that their TIR domains may adopt a fold similar to mammalian TIR proteins (Cirl et
al., 2008). Indeed, the crystal structure of TcpB from B. melitensis and PdTIR from
Paracoccus dentrificans revealed that their TIR domains form a flavodoxin-like fold,
characteristic of human TIR domains (Chan et al., 2009; Snyder et al., 2014). The TIR domains of
TcpB and PdTIR were isolated as monomers; however, their crystal lattice structures revealed
potential dimer interfaces (Chan et al., 2009; Snyder et al., 2014). Contrastingly, the TIR domain
of YpTdp from Yersinia pestis formed dimers in solution, which was mediated by the formation
of two disulphide bonds (Rana et al., 2011). Additionally, the N-terminus of TcpB and PdTIR,
and TlpA from Salmonella enterica, contain an -helical coil-coiled domain, which is proposed to
facilitate protein dimerisation and stabilise the TIR-TIR interaction interface, and may thereby
enhance the inhibition of TLR signalling (Fekonja et al., 2012).
Interference with TLR signalling by Bacterial Tcps
Given the structural similarities between bacterial Tcps and human TIR domains, functional
studies have largely focused on their role as potential virulence factors in suppressing TLR
responses (Table 1.6). Newman et al. provided evidence for the ability of TlpA from S. enterica
serovar Enteritidis to inhibit TLR and IL-1R-mediated NF-B activation (Newman et al., 2006).
Further studies revealed that TcpC and TcpB were also capable of blocking TLR2- and
TLR4-induced NF-B activation (Cirl et al., 2008). The molecular mechanism of TcpB-mediated
inhibition of TLR2 and TLR4 is unclear because different studies identified different binding
partners for TcpB. It was initially proposed that TcpB mimicked MAL function, by co-localising
to the plasma membrane through its phosphoinositide-binding motif, and thereby blocked the
formation of the signalling platform required for MYD88 recruitment (Radhakrishnan et al.,
2009). TcpB has also been shown to interact with MAL, but not MYD88, to promote the
ubiquitination and subsequent degradation of MAL (Sengupta et al., 2010). However, a more
recent study suggested that TcpB can bind to MAL, MYD88 and TLR4, and thus may interfere
with the activation of NF-B (Alaidarous et al., 2014). Like other bacterial Tcps, YpTdp can
inhibit NF-B activation by TLR4 and IL-1R signalling (Spear et al., 2012). The interaction of
YpTdp with MYD88 was dependent on the conserved proline residue (Pro173) in the BB loop of
YpTdp (Spear et al., 2012). It is clear from these studies that bacterial Tcps can interact
differently with mammalian TIR domain proteins. Thus, further studies are required to define
42
the precise mechanism(s) of interactions between bacterial Tcps and mammalian TIR domain
proteins.
Functional Consequences of Bacterial Tcps
Intuitively, the inhibition of TLR signalling by bacterial Tcps would dampen the host
inflammatory response and thus might be advantageous to microbial growth. Indeed, in vitro
studies revealed that TlpA-deficient S. entrica was no longer able to survive and/or replicate in
human THP-1 monocytes. Moreover, mice inoculated with the TlpA-deficient S. entrica mutant
had prolonged survival and reduced bacterial burden, compared to mice infected with the
wildtype strain (Newman et al., 2006). Similarly, in vitro studies also demonstrated the
importance of TcpC for the intracellular survival of E. coli CFT073 in RAW264.7 macrophage-
like cells. Consistently, TcpC-deficient E. coli CFT073 mutant displayed reduced bacterial
colonisation and tissue damage in a mouse model of urinary tract infection (Cirl et al., 2008). A
B. melitensis TcpB-deficient mutant also displayed a reduction in dissemination during the early
stages of infection in mice (Radhakrishnan et al., 2009). In contrast to these observations, host
cytokine responses and bacterial colonisation were comparable for wildtype Y. pestis and a
YpTdp-deficient Y. pestis mutant (Spear et al., 2012). However, Y. pestis growth characteristics
in vitro were affected by YpTdp deficiency, as the YpTdp-deficient mutant exhibited
spontaneous aggregation in broth culture, and was also intolerant of high salinity (Spear et al.,
2012). In summary, bacterial Tcps have been shown to be involved in subverting immune
detection and inhibiting the host inflammatory response to enhance survival and/or replication.
1.7 Immune Subversion by Porphyromonas gingivalis
P. gingivalis is one of the core species of the “red complex” that is closely associated with driving
the development of chronic periodontitis (Hajishengallis et al., 2011, 2012; Socransky et al.,
1998). Subgingival levels of P. gingivalis have been shown to correlate with the clinical
symptoms of chronic periodontitis, including bleeding on probing, periodontal pocket depth,
attachment loss, and alveolar bone resorption (Byrne et al., 2009; Griffen et al., 1998; Socransky
et al., 1998). Effective periodontal treatment is associated with reduced P. gingivalis levels in
subgingival plaque (Haffajee et al., 1997; Kawada et al., 2004). Despite being a low abundance
species, P. gingivalis is capable of dysregulating the host immune response to promote biofilm
dysbiosis and chronic inflammation, which is achieved by the pathogen expressing various
virulence factors (Hajishengallis et al., 2011). Thus, P. gingivalis has been proposed to be a
“keystone pathogen” in chronic periodontitis (Hajishengallis et al., 2012). The following Section
will discuss P. gingivalis virulence factors, and how they impact the host immune response and
thereby promote disease development.
43
P. gingivalis Gingipain Proteases
The extracellular gingipain proteases expressed by P. gingivalis are critical virulence factors.
They are cysteine proteases, and encoded by distinct but related genes to produce the lysine-
specific gingipain, Kgp, and the arginine-specific gingipains, RgpA and RgpB (Potempa et al.,
1995). Kgp and RgpA contain a C-terminal haemagglutinin domain, followed by a catalytic
protease domain. While its protease domain is identical to RgpA, RgpB lacks a haemagglutinin
domain. The gingipain proteases are primarily attached to the outer membrane of P. gingivalis
through LPS-associated modifications; however, they can also be found on outer membrane
vesicles (OMVs) or secreted in a soluble form (Potempa et al., 2003; Veith et al., 2014).
Consequently, the gingipain proteases can exert stimulatory activity on host tissues locally, as
well as at distant sites.
1.7.1.1 Gingipain Proteases and Immune Subversion
The gingipain protease-mediated dysregulation of immune signalling is important for the ability
of P. gingivalis to counter many host defence mechanisms. For example, the gingipain proteases
can proteolytically degrade the TLR4 co-receptor CD14 on monocytes and fibroblasts, thus
making the cells hyporesponsive to LPS (Sugawara et al., 2000; Tada et al., 2002). In addition,
the degradation of RIP2 by the gingipain proteases has also been shown to inhibit NOD1 and
NOD2 signalling (Madrigal et al., 2012). The proteolytic degradation of inflammatory cytokines,
such as TNF and IL-6, by the gingipain proteases may also dampen the host inflammatory
response (Calkins et al., 1998; Banbula et al., 1999). Notably, the degradation of IL-8 by the
gingipain proteases is proposed to cause “local chemokine paralysis” by impairing neutrophil
recruitment (Darveau et al. 1998; Zhang et al. 1999; Stathopoulou et al. 2009). Interestingly,
complement activity may be modulated based on the levels of the gingipain proteases produced
during different stages of P. gingivalis infection. It has been proposed that during the early
stages of infection, P. gingivalis is present in lower numbers and thus the levels of gingipain
proteases are low. This may result in the proteolytic processing of complement C3, C4 and C5
into active fragments that stimulate inflammation, and hence increase nutrient availability to
support P. gingivalis growth (Popadiak et al., 2007). Once P. gingivalis has established an
ecological niche, the increased numbers of P. gingivalis present correlated with the higher levels
of gingipain proteases produced, which may degrade and inactivate complement activity, and
thus help to suppress the immune response (Popadiak et al., 2007). Therefore, the proteolytic
activity of the gingipain proteases enables P. gingivalis to subvert the host immune response by
degrading an array of host immunomodulatory factors.
The gingipain proteases can also manipulate the host immune response by directing immune
signalling crosstalk. For instance, the gingipain proteases can proteolytically process C5 to
44
generate C5a, and thereby activate C5aR on neutrophils and macrophages (Maekawa et al.,
2014; Popadiak et al., 2007; Wang et al., 2010). In neutrophils, signalling crosstalk between
C5aR and TLR2 results in the production of TGF-, which triggers the activation of the E3
ubiquitin ligase SMURF1, and promotes proteasome-mediated degradation of MYD88
(Maekawa et al., 2014). Loss of MYD88 signalling thus suppresses the production of nitric oxide,
which is important for neutrophil-mediated bacterial killing. Concomitantly, the synergistic
activation of MAL-phosphoinositide 3-kinase (PI3K) signalling by C5aR-TLR2 crosstalk
stimulates the expression of pro-inflammatory genes, whilst inhibiting actin polymerisation to
impair phagocytosis. As such, the MYD88-dependent antibacterial response is redirected to a
TLR2-MAL-PI3K signalling response to maintain inflammation. In macrophages, C5aR-TLR2
crosstalk stimulates Gi-dependent intracellular Ca2+ signalling to enhance
P. gingivalis-stimulated cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)
activity (Wang et al., 2010). This results in the inactivation of glycogen synthase kinase-3
(GSK3) and impairs nitric oxide production required for intracellular P. gingivalis killing.
Additionally, P. gingivalis has been demonstrated to regulate the expression of selective
pro-inflammatory cytokines by macrophages to manipulate the host immune response (Liang et
al., 2011). C5aR signalling can synergise with TLR2 to stimulate the expression of TNF, IL-1
and IL-6, and thus stimulate periodontal inflammation and potentially generate nutrients from
tissue breakdown. Concomitantly, C5aR-TLR2 crosstalk leads to ERK1/2-mediated IRF1
suppression, which selectively inhibits the expression of IL-12 and suppresses cell-mediated
immunity. As such, P. gingivalis exploits C5aR-TLR2 crosstalk to subvert host defence, while the
dysregulation of the host immune response via C5aR may generate a microenvironment that
favours microbial dysbiosis. Consistently, C5aR-deficient mice were protected from P. gingivalis-
induced alveolar bone resorption and dysbiosis of the commensal microbiota. Furthermore,
C5aR-deficient mice were able to clear P. gingivalis more efficiently than wildtype mice (Wang
et al., 2010). Therefore, the manipulation of C5aR not only promotes P. gingivalis survival, but
has been proposed to also generate a “bystander” effect that protects otherwise susceptible
bacteria from the host immune system, and thereby contribute to the development of dysbiosis
(Hajishengallis et al., 2011; Liang et al., 2011; Maekawa et al., 2014).
1.7.1.2 Gingipain Proteases and Tissue Destruction
In addition to targeting the immune system, P. gingivalis gingipain proteases can also directly
cause tissue destruction by degrading extracellular matrix proteins, including fibronectin and
laminin (Andrian et al. 2004; Ruggiero et al. 2013). The gingipain proteases also stimulate the
upregulation of host-derived MMPs (e.g. MMP-2), which enhances the degradation of the
extracellular matrix and promotes periodontal ligament detachment (Andrian et al., 2007;
45
Grayson et al., 2003). Moreover, the wound healing response is also compromised as a result of
the gingipain protease-mediated disruption of the coagulation cascade. The gingipain proteases
can proteolytically process and activate kallikrein and bradykinin, which increases vascular
permeability (Hinode et al., 1992; Imamura et al., 1994). Taken together, the effects exerted by
the gingipain proteases allow P. gingivalis to subvert host defences and inflict host tissue
destruction, which ultimately contribute to the pathogenesis of chronic periodontitis.
Fimbriae
P. gingivalis produces two types of fimbriae: FimA (major fimbriae) and Mfa1 (minor fimbriae),
which extend as filamentous protrusions from the surface of the bacterium (Amano et al., 2004).
Genetic variations of FimA are classified as type I, Ib, II, III, IV and V. Patients with chronic
periodontitis have been found to have higher levels of P. gingivalis strains that express type II
FimA, which has been characterised as having the highest adhesive and invasive capacity for
human epithelial cells (Amano et al., 2004; Nakagawa et al., 2002). The fimbriae facilitate
P. gingivalis colonisation by enhancing co-adhesion with other bacteria (e.g. S. gordonii and
T. denticola) (Hashimoto et al., 2003; Park et al., 2005). The adhesive properties of P. gingivalis
fimbriae has also been shown to facilitate host cell invasion of human epithelial cells to subvert
immune detection (Nakagawa et al., 2002; Njoroge et al., 1997). Fimbriae have been shown to
interact with 1 integrins on gingival epithelial cells to promote the initial adherence and
subsequent invasion of P. gingivalis (Yilmaz et al., 2002). This interaction is associated with the
formation of focal adhesions and paxillin phosphorylation, which facilitates cytoskeletal
rearrangement necessary for P. gingivalis invasion (Yilmaz et al., 2002, 2003).
Fimbriae also contribute to the manipulation of the host immune response by inducing
signalling crosstalk between innate immune receptors. For example, P. gingivalis can redirect
TLR2 and CD11b/CD18 receptor signalling in macrophages as a means of immune subversion
(Hajishengallis and Lambris, 2010). Fimbriae-mediated interaction with TLR2 stimulates
MYD88-mediated NF-B activation, enhancing pro-inflammatory gene expression
(Hajishengallis et al., 2009). Concurrently, TLR2 initiates “inside-out” signalling through PI3K to
promote CD11b/CD18 activation (Hajishengallis et al., 2009; Harokopakis and Hajishengallis,
2005). Activated CD11b/CD18 can signal via ERK1/2 to downregulate IL-12 p35/p40
expression, and thus suppress IFN- expression required for the cell-mediated immune
clearance of P. gingivalis (Hajishengallis et al., 2007). Consistently, CD11b/CD18-deficient mice
were demonstrated to clear P. gingivalis infection more efficiently than wildtype mice
(Hajishengallis et al., 2007). TLR2 and CXCR4 signalling in macrophages is also exploited by
P. gingivalis to suppress host defence. P. gingivalis fimbriae engage with CXCR4, which signals
via cAMP-dependent protein kinase A (PKA) to suppress TLR2-mediated NF B activation,
46
resulting in reduced TNF and nitric oxide production (Hajishengallis et al., 2008). Mice treated
with the CXCR4 antagonist, AMD3100, exhibited increased levels of nitric oxide and constrained
P. gingivalis systemic infection more efficiently than untreated mice (Hajishengallis et al., 2008).
As such, P. gingivalis fimbriae not only promote bacterial invasion, but are also involved in
manipulating host immune signalling to impede bacterial clearance.
Atypical Lipopolysaccharides
P. gingivalis has also been shown to antagonise the host immune response by producing atypical
and heterogeneous forms of LPS containing different lipid A moieties (Dixon and Darveau, 2005;
Reife et al., 2006). The post-translational modification to the lipid A of P. gingivalis LPS confers
its ability to act as either a weak TLR4 agonist (in comparison to other Gram-negative species,
such as E. coli) or antagonist to suppress TLR4-mediated immune responses (Domon et al.,
2008; Reife et al., 1995). The acylation and phosphorylation pattern of the lipid A moiety of
P. gingivalis LPS exhibits different PRR-activating capacities, whereby mono/di-phosphorylated,
penta-acylated LPS weakly activates TLR4; non-phosphorylated, tetra-acylated LPS does not
activate TLR4, and mono-phosphorylated, tetra-acylated LPS antagonises TLR4 activation. The
modifications of P. gingivalis LPS is attributable to lipid A 1’-phosphatase (PG1773) and lipid a
4’-phosphatase (PG1587) activity under different haemin conditions (Al-Qutub et al., 2006;
Coats et al., 2009). Under haem-limiting conditions, P. gingivalis utilises lipid A 1’ and 4’-
phosphatase to generate lipid A species that weakly activate TLR4. Conversely, lipid A
1’-phosphatase activity is suppressed under conditions of high haem availability and leads to
the production of TLR4-antagonistic lipid A species. In vitro studies with a P. gingivalis
phosphatase mutant that only express the antagonistic lipid A species revealed that the mutant
inhibited the production of pro-inflammatory mediators (e.g. TNF and IL-6) and suppressed
caspase-11-dependent non-canonical inflammasome activation (Slocum et al., 2014). The
mutant also exhibited increased survival in macrophages. Interestingly,
apolipoprotein-deficient mice orally infected with the same P. gingivalis phosphatase mutant
had no effect on oral inflammation or alveolar bone loss. Instead, the mice infected with the
P. gingivalis phosphatase mutant were found to be more prone to developing vasculature
inflammation associated with increased macrophage infiltration. The phosphatases have also
been shown to be important for bacterial colonisation, in a ligature-induced rabbit model of
periodontitis (Zenobia et al., 2014). Taken together, these studies suggest that the suppression
of TLR4 by P. gingivalis may cause dysbiosis and contribute to the induction of inflammation at
sites distant from the initial infection, and thus potentiate the progression of systemic disease
(e.g. atherosclerosis).
47
SerB Phosphatase
P. gingivalis expresses SerB, a phosphoserine phosphatase that is part of the haloacid
dehydrogenase family of hydrolytic dehydrogenases. P. gingivalis SerB has been shown to be
involved in mediating host cell invasion and immune subversion. Gene expression studies
(e.g. microarray analysis) with gingival epithelial cells suggested that SerB is involved in
regulating the expression of cytoskeletal-associated proteins (e.g. vinculin and paxillin) as well
as MAPK-related proteins (e.g. Cdc42) (Hasegawa et al., 2008). Functional studies revealed that
recombinant SerB can cause the disassembly of the actin by dephosphorylating cofilin, a
regulator of actin stability (Moffatt et al., 2012). The reorganisation of host cytoskeletal proteins
by SerB appears to be important for cellular invasion, as SerB-deficient P. gingivalis exhibited
impaired invasive capabilities and reduced survival in gingival keratinocytes (Hasegawa et al.,
2008; Tribble et al., 2006). In addition, SerB is also involved in modulating the host immune
response. SerB can inhibit IL-8 gene expression in human gingival epithelial cells by
dephosphorylating Ser536 of the p65 subunit of NF-B (Takeuchi et al., 2013). Rats infected
with a SerB-deficient P. gingivalis displayed reduced alveolar bone loss, although the
inflammatory responses were similar between rats infected with SerB-deficient and wildtype
P. gingivalis (Bainbridge et al. 2010). However, greater neutrophil infiltration into the junctional
epithelium in rats infected with SerB-deficient P. gingivalis suggests that SerB impairs innate
immune defences by inhibiting the recruitment of neutrophils (Bainbridge et al. 2010). As such,
SerB supports P. gingivalis immune evasion by mediating cellular invasion and suppressing
NF-B activation.
1.8 Research Objectives
The breakdown of host-microbe (plaque) homeostasis is central to the pathogenesis of chronic
periodontitis. Recent research strongly suggests that P. gingivalis plays a critical role in
promoting the breakdown of homeostasis. Therefore, defining the interactions between host-
and P. gingivalis-derived immunomodulatory factors is crucial for understanding the possible
causes of dysbiosis and inflammation in chronic periodontitis. The overall objective of this
project was to identify and characterise novel host-P. gingivalis interactions. The specific aims of
this project were to:
1. Study the regulation of the chemokine CXCL14 in response to P. gingivalis.
2. Investigate the functions of CXCL14.
3. Identify and characterise potential P. gingivalis TIR domain-containing proteins (Tcp).
4. Study the ability of the P. gingivalis Tcp, PG0382, to modulate the host immune response.
48
Materials and Methods
49
2.1 Materials
Tissue culture reagents
All plasticware was obtained from Falcon (USA). Keratinocyte serum-free medium (K-SFM),
Dulbecco’s Modified Eagle Medium (DMEM), Glutamax-1™, penicillin/streptomycin, bovine
pituitary extract (BPE), human epidermal growth factor (EGF), Gibco® 10X phosphate buffered
saline (PBS), 0.05% Trypsin-EDTA, and foetal bovine serum (FBS) were purchased from
Thermo Fisher (USA).
Bacterial culture reagents
Bacto™ brain heart infusion (BHI) was purchased from BD Biosciences (USA). Blood Agar Base
No. 2 and Agar Bacteriological Agar No. 1 were purchased from Oxoid (United Kingdom).
Defibrinated horse blood was purchased from Equicell (Australia). Menadione, haemin, and
L-cysteine hydrochloride were purchased from Sigma-Aldrich (USA).
General reagents and chemicals
FSL-1 was purchased from Invivogen (USA). ProLongTM Gold Antifade reagent containing
4’6’-diamidino-2-phenylindole (DAPI) stain was purchased from Life Technologies (USA).
Recombinant human and mouse CXCL14 were purchased from R&D Systems (USA). Human
CXCL14 ELISA kit was purchased from Abcam (UK). The following chemicals were purchased
from Sigma-Aldrich (USA): β-mercaptoethanol, isopropanol, calcium chloride, and goat serum.
Molecular biology reagents
The following siRNAs were purchased from Dharmacon (USA): ON-TARGETplus non-targeting
control siRNA, ON-TARGETplus IRF6, ON-TARGET plus IRAK-1, ON-TARGETplus PAR-1,
ON-TARGETplus PAR-2, ON-TARGETplus PAR-3, ON-TARGETplus PAR-4, and ON-TARGETplus
TLR2. Opti-MEM I reduced serum medium, and Lipofectamine RNAiMAX were purchased from
Life Technologies (USA). The Reliaprep™ RNA miniprep system, Reliaprep™ RNA Tissue
Miniprep system, GoScript™ reaction buffer, random primers, nucleotide mix, recombinant
RNasin® ribonuclease inhibitor, GoScript™ reverse transcriptase, GoTaq® probe qPCR master
mix, CXR reference dye, and FuGENE® 6 transfection reagent were purchased from Promega
(USA).
Molecular cloning
PCR primers, SYBR safe DNA gel stain and AxyPrep™ plasmid miniprep kit, and chemically
competent E. coli DH5were purchased from Life Technologies (USA). Pfu DNA polymerase,
and 1 kb DNA ladder were purchased from Promega (USA). The 100 bp DNA ladder was
purchased from Bioline (USA). Restriction enzymes, calf intestinal phosphatase (CIP), and T4
50
DNA Ligase were purchased from New England Biolabs (USA). The DNeasey blood & tissue kit,
MinElute PCR purification kit, and EndoFree® plasmid maxi kit were purchased from Qiagen
(Germany). The UltraClean™ tissue & cells DNA isolation kit and UltraClean™15 DNA
purification kit were purchased from MO BIO laboratories (USA). Ampicillin and erythromycin
were purchased from Sigma-Aldrich (USA).
Quantitative real-time PCR probes
Pre-developed TaqMan assays were purchased from Life Technologies (USA). The following
probe sets for human genes were used: CCL20 (Hs01011368_m1), CXCL14 (Hs01557413_m1),
IL-8 (Hs00174103_m1), IL-36G (Hs00219742_m1), IRAK-1 (Hs01018347_m1), MYD88
(Hs01573837) IRF6 (Hs00196213_m1), PAR-1 (Hs00169258_m1), PAR-2 (Hs00608346_m1),
PAR-3 (Hs00187982_m1), PAR-4 (Hs01006385_g1), TATA box binding protein (TBP)
(Hs00427620_m1), TNF (Hs0113624_g1), and TLR2 (Hs00152932_m1). The following probe
sets for mouse genes were used: CCL2 (Mm00441242_m1), CXCL1 (Mm04207460_m1), IL-6
(Mm00446190_m1), IL-10 (Mm01288386_m1), HPRT (Mm00446968_m1), MAL
(Mm00446502_m1), and TNF (Mm00443258_m1).
SDS-PAGE and Western Blotting
The Bio-Rad protein assay dye reagent was purchased from Bio-Rad (USA). Complete™ EDTA-
free protease inhibitors were purchased from Roche (Switzerland). Bovine serum albumin
(BSA) was purchased from Sigma Aldrich (USA). NuPAGE™ Novex® Bis-Tris precast gels,
NuPAGE™ LDS sample buffer, NuPAGE™ MES buffer, NuPAGE™ MOPS buffer, and Novex® Sharp
pre-stained protein standards were purchased from Life Technologies (USA). Immobilon-P
PVDF Transfer Membrane and Whatman® filter paper were purchased from Millipore (USA).
Enhanced chemiluminescence (ECL) reagent was obtained from GE Healthcare (USA).
Antibodies
The AlexaFluor®-488 conjugated goat anti-rabbit, and AlexaFluor®-594 conjugated goat
anti-mouse antibodies were purchased from Life Technologies (USA). The rabbit monoclonal
anti-FLAG antibody and anti-FLAG M2 affinity gel were from Sigma-Aldrich (USA). The mouse
monoclonal anti-V5 antibody was purchased from Invitrogen (USA). The mouse monoclonal
anti-phospho-ERK1/2, rabbit polyclonal anti-p38, and rabbit polyclonal anti-phospho-p38
antibodies were purchased from Cell Signalling Technology (USA). The rabbit polyclonal
anti-ERK2 antibody was purchased from Santa Cruz (USA). The HRP-conjugated swine
polyclonal anti-rabbit and rabbit polyclonal anti-mouse antibodies were purchased from Dako
(Denmark). The following antibodies were purchased from BD biosciences (USA): mouse
monoclonal anti-HSP90, FITC-conjugated rat monoclonal anti-mouse CD45, PE-conjugated rat
51
monoclonal anti-mouse F4/80, FITC-conjugated rat monoclonal anti-mouse Ly6G, PE-Cy7-
conjugated rat monoclonal anti-mouse CD86, and rat monoclonal anti-mouse CD16/CD32. The
Per-CP-Cy5.5-conjugated rat anti-mouse Ly6C was purchased from Biolegend (USA).
2.2 In vitro methods
Cell culture
2.2.1.1 OKF6/TERT-2 cells
Human telomerase-immortalised OKF6/TERT-2 oral epithelial cells, (were generously provided
by Professor James Rheinwald (Harvard Medical School, Cambridge, MA), and hereafter referred
to as OKF6 cells) (Dickson et al., 2000) were cultured in keratinocyte serum-free medium
(K-SFM) supplemented with 0.4 mM CaCl2, 2 mM GlutaMax, 50 U/ml penicilllin, 50 µg/ml
streptomycin, 25 µg/ml bovine pituitary extract, and 0.2 ng/ml EGF. Cells were cultured at 37 °C
in a humidified atmosphere of 5% CO2 and passaged every 2-3 days, as required.
2.2.1.2 HEK293T cells
HEK293T cells (Graham et al., 1977) were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% (v/v) FBS, 2 mM GlutaMax, and 50 U/ml penicillin and 50
µg/ml streptomycin. The cells were cultured at 37 ˚C in a humidified atmosphere of 5% CO2 and
passaged 2-3 days, as required.
2.2.1.3 RAW 264.7 cells
RAW 264.7 mouse macrophages (Raschke et al., 1978) were cultured in DMEM supplemented
with 10% (v/v) FBS, 2 mM GlutaMax, and 50 U/ml penicillin and 50 µg/ml streptomycin. The
cells were cultured at 37 ˚C in a humidified atmosphere of 5% CO2 and passaged every 2-3 days,
as required.
Bacterial strains and culture conditions
2.2.2.1 P. gingivalis
Freeze-dried cultures of P. gingivalis ATCC 33277 and P. gingivalis W50 were obtained from the
culture collection of the Melbourne Dental School, University of Melbourne. P. gingivalis KDP136
was kindly gifted by Professor Koji Nakayama (Nagasaki University, Graduate School of
Biomedical Sciences). P. gingivalis strains were maintained on 10% defibrinated horse blood
agar base (HBA) supplemented with 5 µg/ml menadione at 37 ˚C in anaerobic conditions (15%
CO2, 5% H2, and 80% N2). The gingipain protease-deficient mutant (KDP136) was created by
homologous recombination, whereby the rgpA and rgpB gene in the P. gingivalis Kgp-deficient
mutant (KDP129) were replaced by a tetracycline-resistance cassette (Shi et al., 1999). Hence,
tetracycline was used to maintain phenotype of the bacterial mutant. Bacteria were passaged
52
weekly on agar plates for a maximum of eight passages, before returning to frozen stocks.
Bacterial colonies were grown in batch culture in Brain Heart Infusion (BHI) broth
supplemented with 5 mg/ml cysteine, 5 µg/ml haemin, and 5 µg/ml menadione.
2.2.2.2 Streptococcus strains
Streptococcus gordonii ATCC 35105 and Streptococcus sp. OT058 were obtained from the
culture collection of the Melbourne Dental School, University of Melbourne. S. gordonii and
S. sp OT058 were cultured on 10% defibrinated HBA, and maintained at 37 ˚C in aerobic and
anaerobic conditions, respectively. Batch cultures were grown in BHI broth supplemented with
5 µg/ml haemin.
2.2.2.3 E. coli
E. coli was cultured on Luria-Bertani (LB) agar, and maintained at 37 °C in aerobic conditions.
Batch cultures were grown in LB broth at 37 ˚C with agitation (200 rpm) on an orbital shaker.
Challenging of OKF6 cells and RAW264.7 cells with P. gingivalis
P. gingivalis cultures were grown to mid to late exponential phase (corresponding to an optical
density of 0.6-0.8 measured at 650 nm) and harvested by centrifugation at 8,000 g for 20 min at
4 °C. The pelleted bacteria were suspended in antibiotic-free K-SFM. The bacteria were diluted
accordingly in K-SFM or DMEM, and added to OKF6 cells or RAW264.7 cells, respectively, to
achieve a bacterium-to-cell ratio of 100:1. Cells challenged with P. gingivalis were incubated in a
humidified atmosphere of 5% CO2 at 37 ˚C for up to 24 h.
RNA interference-mediated gene silencing
A reverse-transfection protocol was used for siRNA transfections. siRNAs were diluted to 120
nM with 100 µl Opti-MEM I reduced serum medium (Life Technologies). The diluted siRNA was
mixed with 100 µl Opti-MEM I reduced serum medium containing 1 µl Lipofectamine RNAiMAX
transfection reagent, and incubated at room temperature for 15 min. The transfection cocktail
was placed in 12-well plates, followed by the addition of 2×105 OKF6 cells. The medium was
replaced 24 h later, and the cells were challenged with P. gingivalis 48 h post-transfection.
RNA purification
Total RNA was purified using the ReliaPrep RNA cell miniprep system. Cells seeded in a 12-well
plates were lysed with 250 μl of BL lysis buffer containing 1-thioglycerol. Genomic DNA was
subsequently sheared by pipetting approximately 10 times. Subsequently, 85 μl of isopropanol
was added into the cell lysate. The cell lysate was applied to the RNA purification column and
centrifuged at 12,000 rpm for 30 sec at room temperature. The column was washed with 500 μl
of RNA Wash solution at 12,000 rpm for 30 sec at room temperature. Next, 30 μl of DNase I
53
Incubation Mix was added to the column for 15 min at room temperature to degrade
contaminating genomic DNA. Subsequently, the column was washed with 200 μl of Column
Wash solution, followed by 500 μl of RNA Wash solution in succession at 12,000 rpm for 30 sec.
Finally, the RNA purification column was washed with 300 μl of RNA Wash solution at 12,000
rpm for 2 min. The RNA was eluted with 20 μl of nuclease-free water by centrifugation at 12,000
rpm for 2 min. RNA concentration and purity were measured with a NanoDrop Lite
Spectrophotometer (Thermo Fisher). The ratio of absorbance at 260 nm and 280 nm was used
to assess the RNA purity, and a ratio of >2.05 was considered acceptable.
Reverse transcription
Total RNA (typically 500 ng per reaction) was incubated with 0.5 µg Oligo(dT)15 and 0.5 µg
random primers at 70 ˚C for 5 min, and then chilled on ice for 5 min. The RNA was reverse
transcribed into cDNA using GoScript Reverse Transcriptase with a BioRad T100™ Thermal
Cycler (BioRad, USA) under the following cycling conditions: annealing at 25 ˚C for 5 min,
extension at 42 ˚C for 45 min, and inactivation at 70 ˚C for 15 min.
Quantitative real-time PCR
Quantitative real-time PCR was performed in duplicate using a QuantStudioTM 7 Flex Real-Time
PCR system under the following cycling conditions: 50 ˚C for 2 min, 95 ˚C for 10 min, followed by
40 cycles of 95 ˚C for 15 s and 60 ˚C for 1 min. Ten ng of cDNA was used per 10 μl reaction. The
data were normalised against the TBP or HPRT gene, and changes in gene expression were
calculated using the ΔΔCt method (Pfaffl, 2001). When appropriate, absolute quantification of
target gene mRNA levels were obtained as 1/(2^ΔCT).
CXCL14 Enzyme-linked immunosorbent assays (ELISA)
CXCL14 protein standards and experimental samples were added to a 96-well micro plate with
anti-CXCL14 capture antibody and incubated at 37 ˚C for 1.5 h. The contents of the wells were
discarded and a biotinylated anti-CXCL14 antibody was added and incubated at 37 ˚C for 1 h.
Thereafter, the wells were washed thrice with PBS, and an avidin-biotin-peroxidase complex
added and the plate was incubated at 37 ˚C for 30 min. The plates were again washed with PBS,
and 3,3’,5,5’-Tetramethylbenzidine (TMD) substrate solution was subsequently added and
incubated in the dark at 37 ˚C for 30 min. Colour development was inhibited by the addition of
the stop solution. Absorbance was measured at 450 nm with a PerkinElmer 1420 Multilabel
Counter VICTOR3™. Background correction was applied by subtracting blank absorbance from
sample readings.
54
In vitro proteolytic degradation of EGF and CXCL14
Purified lysine-specific P. gingivalis gingipain proteinase Kgp was generously provided by
Dr. Laila Huq (Huq et al., 2013). Digestion of EGF and CXCL14 was performed at an enzyme:
substrate ratio of 1:25. Purified Kgp was first activated in 200 mM HEPES (pH 7.6), 4 mM CaCl2,
and 10 mM cysteine for 5 min at 37 ˚C. The substrate (EGF or CXCL14) was then added and
incubated at 37 ˚C. Aliquots of the reaction mixture were removed and Kgp activity was
inhibited by the addition of 2 mM N-Tosyl-L-lysine chlormethyl ketone hydrochloride (TLCK).
The aliquots were then subjected to SDS-PAGE and/or analysis by mass spectrometry.
LTQ Orbitrap Elite mass spectrometry
Kgp-digested recombinant CXCL14 was desalted using C18 zip-tips (Millipore). Prior to sample
loading, the zip-tip was hydrated with 50% (v/v) acetonitrile and 0.1% (v/v) trifluoroacetic
acid, and then thrice washed with 0.1% (v/v) trifluoroacetic acid. The total volume of the
samples was passed through the zip-tips five times, and the zip-tips were then washed five
times with 0.1% (v/v) trifluoracetic acid. Peptides were eluted with 30 % (v/v) acetonitrile
containing 0.1% (v/v) trifluoracetic acid. The eluted samples were frozen with liquid nitrogen
and subsequently vacuum dried in a Digital Series SpeedVac System™ (Thermo Fisher). Samples
were analysed by an LTQ Orbitrap Elite mass spectrometer (Thermo Scientific) with a nanoESI
source interfaced with an Ultimate 3000 RSLC nano-HPLC (Thermo Scientific). The peptides
were loaded onto the enrichment column at an isocratic flow of 5 μl/min in 3% (v/v)
acetonitrile containing 0.1% (v/v) formic acid for 5 min before the enrichment column was
switched in-line with the analytical column. The eluents were used for the LC were 0.1% (v/v)
formic acid (Solvent A) and 100% (v/v) acetonitrile in 0.1% (v/v) formic acid (Solvent B).
Separation was performed with a gradient of 6–80% Solvent B for 53 min. The mass
spectrometer was operated in the data-dependent mode with a nano-ESI spray voltage of 2.0
kV, capillary temperature of 250 ˚C, and S-lens RF value of 55%. All spectra were acquired in
positive mode with full scan MS spectra scanning from m/z 300 – 1650 in the FT mode at
240,000 resolution after accumulating to a target value of 1.0e6. The top 10 most intense
precursor ions were subjected to high energy collision induced dissociation (HCD) with a
normalised collision energy of 35, and activation time of 0.1 ms. Dynamic exclusion with 2
repeat counts over 30 sec, and exclusion for 70 sec was applied. The Mascot MS/MS ions search
was performed using the following settings: Uniprot database, Lys-C, 1 missed cleavage, 10 ppm
peptide tolerance, and 0.2 Da MS/MS tolerance.
55
Antibacterial assays
Mid to late exponential phase bacteria were harvested by centrifugation and washed with PBS.
The bacteria (5×104 ) were suspended in incubation buffer (10 mM Tris-HCl, 5 mM glucose, pH
7.4) and incubated with CXCL14 for 1 h at 37 ˚C. Serial dilutions of P. gingivalis, S. gordonii, and
S. sp. OT058 were plated on HBA, while E. coli was plated on LB agar plates. Bacterial cell
numbers were enumerated and % killing was expressed as: [1 - (number of colonies with
CXCL14 incubation)/ (number of colonies incubated with vehicle control)] × 100.
Wound healing assay
OKF6 cells were seeded into 12-well plates and cultured to 90% confluency. A sterile pipette tip
was used to generate wounds across the cell monolayer. The cells were washed with PBS to
remove detached cells, and replaced with K-SFM containing stimulants. Images were acquired
using an Axio Scope phase contrast microscope. Percentage gap closure was expressed as [1 -
(area of gap measured at specific time)/ (area of gap measured at 0 h)] × 100.
Transfection of HEK293T cells
HEK293T cells were seeded at a density of 1×105 cells/cm2 of culture vessel. The cells were
transfected the next day using FuGENE 6 transfection reagent. Briefly, 1 µg of plasmid DNA was
diluted in 50 µl serum-free DMEM containing 3 µl FuGENE 6 transfection reagent. The
transfection cocktail was incubated at room temperature for 15 min, and then added dropwise
to the cell monolayer. The total amount of plasmid in each transfection was kept constant using
parental empty plasmid. The list of plasmids used are listed in Table 2.1.
Cell lysis
OKF6 and HEK293T cells were washed twice with ice-cold PBS, and then lysed with NP-40 lysis
buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% (v/v) Nonident P-40, 10%
(v/v) glycerol, 1 mM sodium orthovanadate, 10 mM β-glycerol phosphate, and 10 mM sodium
fluoride) supplemented with protease inhibitors on ice for 30 min. The lysates were clarified by
centrifugation at 12,000 rpm for 10 min at 4 ˚C. The protein concentration of the lysates were
determined by the Bradford assay (Bradford, 1976) using the Bio-Rad protein assay dye
reagent. Thereafter, the lysates were either used for co-immunoprecipitation assays, or stored
at -80 ˚C for subsequent analysis by SDS-PAGE.
Co-immunoprecipitation Assay
Lysates were adjusted to a protein concentration of 1 mg/ml in a total volume of 1 ml ice-cold
lysis buffer, and incubated with 10 µl of anti-FLAG beads at 4 ˚C overnight with constant mixing.
The beads were washed four times with ice-cold lysis buffer. Following the final wash, the beads
56
were eluted by heating at 70 ˚C for 10 min in 30 µl of NuPAGE LDS sample buffer containing
50 mM DTT. The eluates were then subjected to SDS-PAGE and Western blotting.
SDS-PAGE
Samples were prepared in NuPAGE LDS sample buffer and heated at 70 ˚C for 10 min.
Approximately 20 µg total protein was loaded per well onto 10% NuPAGE Bis-Tris gels.
Electrophoresis was performed at 100 V for 2 h in 2-(N-morpholino) propanesulfonic acid
(MOPS) (50 mM MOPS, 50 mM Tris (pH 7.7), 0.1 % (w/v) SDS, 1 mM EDTA) or
2-(N-morpholino) ethanesulfonic acid (MES) (50 mM MES, 50 mM Tris (pH 7.3), 0.1% (w/v)
SDS, 1 mM EDTA) running buffer.
Western Blotting
Proteins were transferred to polyvinylidene fluoride (PVDF) membranes at 500 mA for 90 min
in transfer buffer (25 mM Tris-HCl, 200 mM glycine, 20% v/v methanol). Membranes were
rinsed in Tris-buffered saline/Tween-20 (TBST: 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1%
(v/v) Tween-20), and then blocked with 3% (w/v) BSA in TBST for 1 h at room temperature.
Membranes were incubated with primary antibodies (diluted in 1% (w/v) BSA in TBST) at 4 ˚C
overnight. The membranes were washed with TBST over 1 h with several changes of buffer, and
then incubated with the appropriate secondary antibody (diluted in 1% (w/v) BSA in TBST) for
1 h at room temperature. The membranes were again washed with TBST. Immunoreactive
protein bands were visualised with ECL reagent and imaged with a LAS3000 imager (Fujifilm).
Before re-probing membranes with another antibody, bound antibodies were removed by
incubating the membrane in stripping buffer (50 mM Tris-HCl (pH 7.4), 2% (w/v) SDS, 100 mM
β-mercaptoethanol) for 15 min at 55 ˚C. The membrane was washed for 1 h with TBST, and then
blocked with 3% (w/v) BSA in TBST. Densitometric analysis was conducted using the
ImageQuant software.
Immunofluorescence staining and confocal microscopy
Glass coverslips were sterilised with 80% (v/v) ethanol for 10 min. The coverslips were then
transferred to 24-well plates and allowed to air-dry. HEK293T cells were seeded onto the
coverslips at a density of 5×104 cells/well and transfected using FuGENE6 (refer to Section
2.2.13 for further details). Twenty-four h later, the cells were washed with PBS, and fixed with
4% (w/v) paraformaldehyde (PFA) in PBS (pH 7.4) at room temperature for 30 min. The cells
were permeabilised with 0.1% (v/v) Triton X-100 in PBS for 5 min, followed by blocking with
5% (v/v) goat serum in PBS for 1 h at room temperature. Thereafter, the cells were incubated in
the appropriate primary antibody (diluted in 5% (v/v) goat serum in PBS) for 1 h at room
temperature. Following three washes with PBS, the cells were incubated with either an
57
AlexaFluor®-488 conjugated goat anti-rabbit antibody or an AlexaFluor®-594 conjugated goat
anti-mouse antibody for 1 h at room temperature in the dark. The cells were again washed
thrice with PBS, and then mounted onto microscope slides using ProLong™ Gold antifade
reagent with 4’,6-diamidino-2-phenylindole (DAPI) stain. Coverslips were cured overnight in
the dark. Confocal images were acquired with a Leica SP5 microscope (Leica Microsystems).
Construction of PG0382 mammalian expression vectors
2.2.19.1 Polymerase Chain Reaction
P. gingivalis W50 genomic DNA was isolated using a Qiagen DNeasy blood and tissue kit
according to the manufacturer’s instructions. The PG0382 gene was amplified by Polymerase
Chain Reaction (PCR) using Pfu DNA polymerase and custom-designed primers listed in Table
2.2. The thermal cycling conditions were as follow: initial denaturation at 95 ˚C for 4 min,
followed by 35 cycles of denaturation at 95 ˚C for 30 s, annealing at 62 ˚C for 1 min, elongation
at 72 ˚C for 2 min, and a final elongation at 72 ˚C for 10 min. The annealing temperature was
calculated as: Tm – 10 ˚C, where Tm refers to the melting temperature of the primers. PCR
products were purified using a MinElute PCR kit according to the manufacturer’s instructions.
Thereafter, the purified PCR products and the mammalian expression vectors pEF-FLAG and
pEF-V5, were digested with MluI. The linearised vector was dephosphorylated by incubation
with 10 U of calf intestinal phosphatase per µg DNA for 1 h at 37 ˚C. The digested PCR products
and vectors were separated on a 1% agarose gel, excised from the gel, and purified using the
Ultraclean DNA purification kit according to the manufacturer’s instructions. The PCR product
was ligated into the linearised vectors overnight at a 3:1 molar ratio (insert:vector) using T4
DNA ligase.
2.2.19.2 Bacterial transformation
Ten ng of plasmid DNA was first gently mixed with chemically-competent E. coli DH5α (50 μl)
on ice. The bacteria were then subjected to heat-shock at 42 ˚C for 20 sec, followed by
incubation on ice for 2 min. The bacteria were inoculated into pre-warmed LB broth and
incubated at 37 ˚C for 1 h with constant shaking (200 rpm). The bacteria were then spread onto
LB agar plates supplemented with 100 µg/ml ampicillin and incubated overnight at 37 ˚C. Single
colonies were randomly selected and grown in 2 ml LB broth supplemented with 100 µg/ml
ampicillin overnight at 37 ˚C. Miniprep plasmid DNA was purified using the Axygen Plasmid
DNA Purification Miniprep kit. Positive clones were identified by restriction digests of the
purified DNA. Large-scale (maxi prep) plasmid preparations were performed by inoculating 100
µl of the bacterial culture into 200 ml of LB broth supplemented with 100 µg/ml ampicillin and
incubating overnight at 37 ˚C with constant shaking (200 rpm). Plasmid DNA was purified using
58
an EndoFree Maxi kit. DNA sequencing of plasmids was performed at the Centre for
Translational Pathology (The University of Melbourne).
Generation of P. gingivalis PG0382-deficient mutant (ΔPG0382)
2.2.20.1 Splice Overlap Extension Polymerase Chain Reaction (SOE PCR)
A gene deletion cassette, comprised of the erythromycin-resistance gene ermF flanked with 5’
and 3’ sequences from PG0382, was constructed by SOE PCR. Three PCR products, namely,
PG0382_IG5, PG0382_IG3, and ermF_PG0382 were generated using the indicated primer pairs
and thermal cycling conditions (Table 2.3). The PCR products were run on a 1% agarose gel,
excised from the gel, and then purified using the Ultraclean DNA purification kit according to the
manufacturer’s instructions. The first-stage SOE PCR reaction (Table 2.4 - SOE PCR reaction 1),
was undertaken to recombine PG0382_IG5 and ermF_PG0382. Primers PG0382_IG5 Fw and
ermF Rv were added to the reaction at cycle 3 to amplify the recombined PCR products. The SOE
PCR product generated was run on a 1% agarose gel; excised from the gel and purified using the
Ultraclean DNA purification kit. The purified PCR product was used for a subsequent SOE PCR
reaction with PG0382_IG3 (Table 2.4 - SOE PCR reaction 2). Primers PG0382_IG5 Fw and
PG0382_IG3 Rv were added to the reaction at cycle 3 to generate the complete ermF
inactivation cassette. The resulting SOE PCR product was run on a 1% agarose gel; excised from
the gel and purified using the Ultraclean DNA purification kit.
2.2.20.2 Electroporation of P. gingivalis
Two-hundred ml of P. gingivalis in early exponential growth phase (corresponding to an optical
density of 0.5-0.6 measured at 650 nm) were harvested by centrifugation at 8,000 g for 20 min
at 4 ˚C. The pelleted bacteria were washed with 200 ml of ice-cold electroporation buffer (10 %
(v/v) glycerol, 1 mM MgCl2) and re-pelleted by centrifugation at 8,000 g for 20 min at 4 ˚C. The
cell pellet was suspended in 400 µl ice-cold electroporation buffer. Eighty microliters of cells
were aliquoted into Eppendorf tubes containing 200 ng of ermF inactivation cassette. The tubes
were incubated on ice for 5 min before being transferred to ice-cold 0.1-cm gap cuvettes and
electroporated at 1.8 kV, 200 Ω, and 25 µFaradays. Following electroporation, the cells were
suspended in 1 ml of BHI broth supplemented with 0.5 mg/ml cysteine and 5 µg/ml haemin,
and incubated overnight anaerobically at 37 ˚C. The cells were centrifuged at 8,000 g for 5 min.
Most of the supernatant (~900 µl) was discarded and cells were resuspended in the remaining
100 µl of supernatant and plated onto a HBA plate supplemented with 10 µg/ml of
erythromycin for selection. P. gingivalis genomic DNA was purified using a Qiagen DNeasy blood
and tissue kit according to the manufacturer’s instructions, and PG0382 deletion was confirmed
by DNA sequencing performed by the Centre for Translational Pathology (The University of
59
Melbourne), with the following primer pairs: 5’ – TGC ATC AGT GTC TTT CGG TG – 3’ and 5 ‘–
GTA TCC ATG GAT TCG TCT ATC AT – 3’.
Gingipain proteinase assay
Mid exponential phase P. gingivalis were harvested by centrifugation at 8,000 g for 20 min at 4
˚C. The cell pellet was suspended in TC150 buffer (50mM Tris-HCl, pH 7.4, 5 mM CaCl2, and 150
mM NaCl) supplemented with 20 mM cysteine. Kgp- and Rgp-specific activity was assayed using
the chromogenic substrates, N-p-tosyl-Gly-Pro-Lys p-nitroanilide (GPKNA), and N-benzoyl-L-
arginine p-nitroanilide (L-BAPNA) respectively. GPKNA and L-BAPNA cleavage was monitored
at 405 nm for 30 min (at 10 sec intervals) at 37 ˚C using a PerkinElmer 1420 Multilabel Counter
VICTOR3™.
2.3 Bioinformatics methods
Sequence alignments
Multiple sequence alignments were conducted using the ClustalOmega algorithm (with default
parameters) on the EMBL-EBI server with Jalview editing software. Sequence alignments were
coloured using ClustalX colour scheme. Pairwise sequence alignments were conducted using the
EMBOSS NEEDLE algorithm (with default parameters) on the EMBL-EBI server
(https://www.ebi.ac.uk/Tools/psa/emboss_needle/).
Structural modelling
PG0382 amino acid sequence was subjected to FUGUE analysis to identify structural
homologies. FUGUE (http://mizuguchilab.org/fugue/) is an online service that compares query
sequences to a database of structural profiles to identify structural homologs. The program
calculates a level of confidence of resulting matches by utilising an environment-specific
substitution tables and structure-dependent gap penalties to account for the local structural
environment of sequence residues (Shi et al., 2001). A list of sequence-structure compatibility
scores is generated from potential homologues. A 3D structure of PG0382 was modelled based
on structural templates of proteins that were identified in FUGUE using SWISS-MODEL
(https://swissmodel.expasy.org/). The predicted structures were visualised and edited using
PyMOL.
2.4 Mouse studies
Mice
Animal studies were approved by the University of Melbourne Ethics Committee for Animal
Experimentation (Ethics identification number: 1613985 and 1614048). C57BL/6 and BALB/C
60
mice and (6–12 weeks old) were housed at the Bio21 Institute Biological Research Facility. The
mice had access to food and water ad libitum in alternating 12 h periods of light and a dark.
Intragingival injection
C57BL/6 mice (6–12 weeks old) were anaesthetised with a mixture of ketamine (0.1 mg/g) and
xylazine (0.01 mg/g) using a 26G needle. Recombinant mouse CXCL14 (0.5 µg or 2 µg) in PBS
was injected into the gingiva of anaesthetised mice with a 31G needle. Control mice were
injected with the same volume of PBS. Mice were killed at 24 or 48 h-post injection by CO2
gassing. The upper maxilla was dissected from the animal and the gingival tissue was stripped.
Half of the gingival tissue was frozen on dry-ice and stored at -80 ˚C until used for gene
expression analysis.
Mouse gingival RNA extraction
RNA from mouse gingival tissue was purified with the Reliaprep RNA Tissue Miniprep System
(Promega). Excised mouse gingival tissue was crushed in liquid nitrogen with an ice-cold
mortar and pestle. The homogenate was then transferred to 500 µl of LBA lysis buffer
containing 1-thioglycerol. An equal volume of RNA dilution buffer was added, and the lysates
were mixed by vortexing. Subsequently, 85 μl of isopropanol was added into the cell lysate. The
cell lysate was applied to the RNA purification column and centrifuged at 12,000 rpm for 1 min
at room temperature. The column was washed with 500 μl of RNA Wash solution at 12,000 rpm
for 30 sec at room temperature. Next, 30 μl of DNase I Incubation Mix was added to the column
for 15 min at room temperature to degrade contaminating DNA. Subsequently, the column was
washed with 200 μl of Column Wash solution, followed by 500 μl of RNA Wash solution in
succession at 12,000 rpm for 30 sec. Finally, the RNA purification column was washed with 300
μl of RNA Wash solution at 12,000 rpm for 2 min. The RNA was eluted with 25 μl of nuclease-
free water following centrifugation by 12,000 rpm for 1 min. RNA concentration and purity
were measured with a NanoDrop Lite Spectrophotometer (Thermo Fisher). The ratio of
absorbance at 260 nm and 280 nm was used to assess the RNA purity, and a ratio of >2.0 was
considered acceptable.
Intraperitoneal infection
P. gingivalis W50 and P. gingivalis ΔPG0382 cultures were grown to mid to late exponential
phase (corresponding to an optical density of 0.6-0.8 measured at OD650) and harvested by
centrifugation at 8,000 g for 20 min at 4 ˚C and suspended in ice-cold PBS. BALB/c mice (7
weeks old) were injected in the peritoneal cavity with 5×106 or 5×107 bacteria using a 26 G
needle. Control mice were injected with the same volume PBS. Mice were killed 6 or 24 h post-
injection by CO2 gassing. The intraperitoneal cavity was lavaged by injecting 5 ml ice-cold PBS,
61
gently massaging the mouse cavity, and collecting the fluid with a 21 G needle. The peritoneal
cavity fluid was immediately processed for flow cytometric analysis.
Flow cytometric analysis
Mouse peritoneal cavity fluid harvested was centrifuged at 300 g for 10 min at 4 ˚C. The cells
were suspended in PBS and numerated. This was followed by staining with Fixable Viability
Stain 700 (FVS700), used at 1:1000 dilution for 15 min on ice in the dark. The cells were
centrifuged at 300 g for 5 min and washed twice with FACS buffer (2.5 % (v/v) FBS and 2 mM
EDTA in PBS). The cells were then incubated with anti-mouse CD16/CD32 (1:500) on ice for 15
min. Subsequently, 5×105 cells were aliquoted into respective antibody cocktails (Table 2.5) and
incubated on ice for 30 min. Following incubation, the cells were washed twice with FACS
buffer, and then fixed with 2 % (w/v) PFA at 4 ˚C for 15 min. The cells were washed thrice with
PBS and suspended in FACS buffer for analysis using a BD LSRFortessa X-20 (BD bioscience). A
typical forward and side-scatter gate was set to exclude aggregates, and dead cells were
discarded on the basis of the FVS700 staining. Data analysis was conducted using the FlowJo
software (TreeStar Inc).
2.5 Statistical analysis
Data combined from three or more independent experiments are presented as the mean ±
standard error of mean (SEM). Where indicated in the figure legends, n is the number of
biological replicates. Statistical analyses were performed using GraphPad Prism software
version 6.01. Differences between two groups were evaluated using the Student’s t-test. For
multiple comparisons, statistical analysis was performed by ANOVA with Dunnett’s or Sidak’s
post-hoc test. A p-value ˂0.05 was considered to be statistically significant.
62
Table 2.1 List of plasmids used in this study. Plasmid Description Source
pFLAG-MAL Expresses FLAG-tagged mouse MAL. Dr. Ashley Mansell
pFLAG-MYD88 Expresses FLAG-tagged human MYD88. Dr. Ashley Mansell
pEF-V5-PG0382 Expresses V5-tagged P. gingivalis PG0382. This study
pEF-V5-PG0382ΔTD Expresses V5-tagged P. gingivalis PG0382 truncated mutant lacking the TIR domain (Asp2 to Glu342).
This study
pEF-V5-PG0382TD Expresses V5-tagged P. gingivalis PG0382 TIR domain (Lys341 to Gln490).
Ben Huang
63
Table 2.2 PCR primers used for constructing PG0382 mammalian expression vectors.
1 Bolded sequences correspond to MluI restriction sites
Vector PCR primers 1
pEF-V5-PG0382 Fw 5’ – CGA CGC GTG ACT TGG CTG AAG TTT TTG TTA CTG AAG – 3’ Rv 5’ – CGA CGC GTC TAT TGA ACC TTA GAA ATT ATT CTT TC – 3’
pEF-V5-PG0382 ΔTD Fw 5’ – CGA CGC GTG ACT TGG CTG AAG TTT TTG TTA CTG AAG – 3’ Rv 5’ – CGA CGC GTT TCT TTA TAC AAC TTG TTC CAG TCG ATG – 3’
64
Table 2.3 PCR primers used to generate PCR products for constructing ermF cassette.
Product name Genome
position (nt) Primer 1 PCR primers 2,3
Product Description
Thermal Cycling conditions
PG0382_IG5 412991 – 413330
PG0382_IG5 Fw
5’-GAA CAT GGC AAG ATT GCG GT-3’ Contains ermF sequence for SOE PCR and PG0382 5’ intergenic sequences for homologous recombination.
Cycle 1 Denaturation 95 °C 4 min Cycle 2 (4x) Denaturation 95 °C 30 s Annealing 40 °C 1 min Elongation 72 °C 1 min Cycle 3 (25x) Denaturation 95 °C 30 s Annealing 50 °C 1 min Elongation 72 °C 1 min
PG0382_IG5 Rv
5’-GCA ATT TCT TTT TTG TCA TAT GCA GTT AAA-3’
PG0382_IG3 415107 - 415097
PG0382_IG3 Fw
5’-AAA TTT CAT CCT TCG TAG TTA ATT ATA GAA AGG GGG ATT TA-3’
Contains ermF sequence for SOE PCR and PG0382 3’ intergenic sequences for homologous recombination.
Cycle 1 Denaturation 95 °C 4 min Cycle 2 (4x) Denaturation 95 °C 30 s Annealing 42 °C 1 min Elongation 72 °C 1 min Cycle 3 (25x) Denaturation 95 °C 30 s Annealing 51 °C 1 min Elongation 72 °C 1 min
PG0382_IG3 Rv
5’-GAA GCC AAC CGA TAC CGA AT-3’
ermF_PG0382 N/A
ermF Fw 5’-TTT AAC TGC ATA TGA CAA AAA AGA AAT TGC-3’
ermF cassette containing PG0382 overlap sequences for SOE PCR.
Cycle 1 Denaturation 95 °C 4 min Cycle 2 (4x) Denaturation 95 °C 30 s Annealing 40 °C 1 min Elongation 72 °C 1 min Cycle 3 (25x) Denaturation 95 °C 30 s Annealing 48 °C 1 min Elongation 72 °C 1 min
ermF Rv 5’-CCT TTC TAT AAT TAA CTA CGA AGG ATG AAA-3’
65
1 Fw indicates forward primer, and Rv indicates reverse primer. 2 Bolded sequences correspond to overlapping ermF sequence. 3 Underlined sequences correspond to overlapping PG0382 sequence.
66
Table 2.4 SOE PCR reaction thermal cycling conditions.
SOE PCR reaction 1 SOE PCR reaction 2 Cycle 1 Denaturation 95 °C 4 min Cycle 2 (4x) Denaturation 95 °C 30 s Annealing 42.5 °C 1 min Elongation 72 °C 1 min Cycle 3 Hold 12 °C 10min Cycle 4 (25x) Denaturation 95 °C 30 s Annealing 48.8 °C 1 min Elongation 72 °C 1 min
Cycle 1 Denaturation 95 °C 4 min Cycle 2 (4x) Denaturation 95 °C 30 s Annealing 48.8 °C 1 min Elongation 72 °C 1 min Cycle 3 Hold 12 °C 10min Cycle 4 (25x) Denaturation 95 °C 30 s Annealing 51.7 °C 1 min Elongation 72 °C 1 min
67
Table 2.5 Flow cytometric analysis antibody cocktails.
Monocyte and granulocyte antibody cocktail Antigen Conjugate Dilution F4/80 PE 1:400 Ly6G FITC 1:500 CD86 PE-Cy7 1:200 Ly6C Per-CP-Cy5.5 1:200
Lymphocyte antibody cocktail Antigen Conjugate Dilution CD45 FITC 1:100
TCR PE-Cy7 1:50
CD4 APC 1:200 CD8 PE 1:50 CD19 Per-CP-Cy5.5 1:50 CD25 BV421 1:100
68
The regulation of CXCL14 in oral epithelial
cells by Porphyromonas gingivalis
69
3.1 Introduction
The barrier function of the oral epithelium is maintained by cycles of cell proliferation and
differentiation of basal epithelial cells to replace the cells that are constantly lost at the
surface of the epithelium. Mitogens, such as Epidermal Growth Factor (EGF), play
important roles in governing the balance between cell proliferation and differentiation,
and thus contribute to the maintenance of barrier function. In addition, the oral epithelium
also provide host protection by secreting antimicrobial peptides (e.g. -defensins and
cathelicidin) and stimulating host inflammation (Dale and Fredericks, 2005).
Importantly, the oral epithelium also serves as an active participant in host defence by
initiating inflammation and communicating potential threats to immune cells through
PRRs (e.g. TLRs and PARs) (Darveau et al., 2004; Uehara et al., 2005). Once activated, PRRs
can stimulate the expression of chemokines to facilitate the host inflammatory response
by recruiting a range of immune cells (e.g. neutrophils and macrophages) to the site of
infection. Interestingly, EGF receptor (EGFR) signalling appears to play an important role
in the differential regulation of chemokine expression by epithelial cells. For instance,
EGFR signalling was shown to regulate the expression of IL-8, CCL2 and CXCL10 in
epidermal keratinocytes (Mascia et al., 2003). The coordinated response mediated by
chemokines through multiple regulatory pathways is essential for maintaining tissue
homeostasis and preventing chronic inflammation. However, in the case of chronic
periodontitis, the continual stimulation of the host immune response by a dysbiotic
microbial community can inflict damaging effects on host tissues (Darveau, 2010).
P. gingivalis drives this process by expressing virulence factors (e.g. gingipain proteases)
to dysregulate the host immune response and promote microbial dysbiosis (Hajishengallis
et al., 2012). For example, P. gingivalis virulence factors can antagonise the host immune
response by dysregulating the expression of chemokines. Accordingly, disordered
chemokine expression may stimulate a sustained inflammatory response and turn a
normally host-protective response into one that causes pathology.
Our laboratory had previously undertaken a preliminary study to identify P. gingivalis-
inducible genes in human oral epithelial cells (e.g. OKF6 cells). Specifically, an Open-Array
human inflammation panel, which allows the mRNA levels of 586 inflammatory genes to
be measured, was used to screen for genes whose expression is regulated by P. gingivalis.
The gene encoding the orphan chemokine, CXCL14, was identified as one such gene.
Therefore, the aim of this Chapter was to examine the regulation of CXCL14 in oral
epithelial cells by P. gingivalis.
70
3.2 Results
P. gingivalis stimulates CXCL14 expression in human oral epithelial
cells in a TLR2-independent manner
OKF6 cells, which are a non-transformed, telomerase-immortalised human oral epithelial
cell line (Dickson et al., 2000), have been used in a number of studies to investigate the
response of oral epithelial cells to bacteria (Giacaman et al., 2009; Macpherson et al.,
2014). Therefore, OKF6 cells were considered an appropriate in vitro cell system to
investigate the inflammatory response of human oral epithelial cells to P. gingivalis.
To validate the identification of CXCL14 as a P. gingivalis-inducible gene, OKF6 cells were
challenged with P. gingivalis over a 24 h time-course and CXCL14 mRNA levels were then
measured by Real-Time PCR. CXCL14 gene expression was found to be strongly
upregulated 24 h-post challenge with P. gingivalis (Fig. 3.1A). The levels of CXCL14 protein
in the cell culture supernatants were measured by ELISA to determine whether the
upregulation of CXCL14 mRNA resulted in a corresponding increase in CXCL14 protein.
Although CXCL14 protein was detected in the cell culture supernatants from cells that had
not been challenged with P. gingivalis, CXCL14 protein levels were not increased by
P. gingivalis challenge (Fig. 3.1B). Other studies have also reported difficulties in
measuring cytokine/chemokine levels in cell culture supernatants due to their proteolysis
by P. gingivalis gingipain proteases (Darveau et al. 1998; Stathopoulou et al. 2009;
Zhang et al. 1999).
Our laboratory has recently demonstrated that TLR2, IRAK-1 and IRF6 function as a
signalling axis to differentially regulate cytokine and chemokine expression in oral
epithelial cells (Huynh et al., 2016; Kwa et al., 2014). Therefore, a role for these proteins in
regulating P. gingivalis-inducible CXCL14 expression was investigated using a
siRNA-mediated gene knockdown approach. The transfection of OKF6 cells with the TLR2
siRNA reduced TLR2 mRNA levels by >80% relative to cells transfected with the
non-targeting control siRNA (Fig. 3.1C). The effect of TLR2 knockdown on the induction of
CXCL14 expression by P. gingivalis was next evaluated. Notably, knockdown of TLR2 did
not inhibit the upregulation of CXCL14 (Fig. 3.1D), suggesting that the regulation of
CXCL14 gene expression in response to P. gingivalis was not mediated by the TLR2
signalling pathway. A complementary approach was also taken, whereby OKF6 cells were
stimulated with the TLR2 agonist FSL-1. Consistent with the findings from the TLR2 gene
silencing experiment, FSL-1 did not stimulate CXCL14 gene expression (Fig. 3.1E). To
confirm that OKF6 cells are responsive to stimulation by FSL-1, the expression levels of
the chemokine, CCL20, which is known to be a target of TLR2 signalling in human
keratinocytes (Niebuhr et al., 2010), were measured. As shown in Figure 3.1F, FSL-1
71
strongly stimulated CCL20 gene expression. Together, these data suggest that
P. gingivalis-inducible CXCL14 gene expression is regulated in a TLR2-independent
manner.
Figure 3.1 P. gingivalis stimulates CXCL14 gene expression in a TLR2-independent manner. (A-B) OKF6 cells were challenged with P. gingivalis at 100 MOI for the times indicated. CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mocked-challenged cells (n=3). (B) CXCL14 protein levels in the cell culture supernatants were measured by ELISA (n=3). (C-D) OKF6 cells were transfected with a non-targeting (-) or a TLR2 (+) siRNA for 48 h, and subsequently challenged with P. gingivalis at 100 MOI for 24 h. (C) TLR2 mRNA levels were measured by Real-Time PCR, and levels in cells transfected with the non-targeting siRNA control were arbitrarily assigned a value of 100% (n=3). (D) CXCL14 mRNA levels were measured by Real-Time PCR, and shown as a fold change relative to mock-challenged cells (n=3). (E-F) OKF6 cells were stimulated with FSL-1 (100 ng/ml) for the times indicated. (E) CXCL14 and (F) CCL20 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n=3). All data are presented as the mean ± SEM (* = p <0.05, *** = p <0.001).
A
C D
E F
B
72
Despite the above finding, roles for IRAK-1 and IRF6 in regulating CXCL14 gene expression
were also investigated. Transfection of OKF6 cells with the IRAK-1 siRNA, which reduced
IRAK-1 gene expression by >90% (Fig. 3.2A), did not inhibit the stimulation of CXCL14
gene expression by P. gingivalis (Fig. 3.2B). Similarly, gene silencing of IRF6 (Fig. 3.2C) did
not inhibit P. gingivalis-inducible CXCL14 gene expression (Fig. 3.2D). Collectively, these
data suggest that P. gingivalis-inducible CXCL14 gene expression in oral epithelial cells
(e.g. OKF6 cells) is regulated in a TLR2-, IRAK-1-, and IRF6-independent manner.
Figure 3.2 P. gingivalis stimulates CXCL14 gene expression in an IRAK-1 and IRF6-independent manner. (A-B) OKF6 cells were transfected with a non-targeting (-) or a IRAK-1 (+) siRNA for 48 h and subsequently challenged with P. gingivalis at 100 MOI for 24 h. (A) IRAK-1 mRNA levels were measured by Real-Time PCR, and levels in cells transfected with the non-targeting siRNA control was arbitrarily assigned a value of 100% (n=3). (B) CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-challenged cells (n=3). (C-D) OKF6 cells were transfected with a non-targeting (-) or an IRF6 (+) siRNA for 48 h and subsequently challenged with P. gingivalis at 100 MOI for 24 h. (C) IRF6 mRNA levels were measured by Real-Time PCR, and levels in cells transfected with the non-targeting siRNA control was arbitrarily assigned a value of 100% (n=3). (D) CXCL14 mRNA levels were measured by Real-Time PCR and shown as a fold change relative to mock-challenged cells (n=3). All data are presented as the mean ± SEM (** = p < 0.01, *** = p < 0.001).
A B
C D
73
Gingipain proteases mediate the stimulation of CXCL14 expression by
P. gingivalis
The gingipain proteases are central to the ability of P. gingivalis to antagonise the host
immune response (O’ Brien-Simpson et al., 2003). They not only stimulate the expression
of cytokines and chemokines in oral epithelial cells, but can also degrade them once
secreted (Darveau et al., 1998; Dommisch et al., 2007; Lourbakos et al., 2001), and thereby
contribute to the dysregulation of the host immune response. Therefore, a role for the
gingipain proteases in the stimulation of CXCL14 gene expression by P. gingivalis was
investigated. OKF6 cells were challenged with either wildtype P. gingivalis or P. gingivalis
KDP136, an isogenic Kgp/Rgp-deficient mutant (Shi et al., 1999), and CXCL14 gene
expression was then measured. In contrast to wildtype P. gingivalis, the gingipain
protease-deficient mutant did not stimulate CXCL14 gene expression (Fig. 3.3A). To
confirm the importance of the gingipain proteases for P. gingivalis-inducible CXCL14
expression, OKF6 cells were challenged with P. gingivalis that had first been treated with
the irreversible serine protease chemical inhibitor, N--Toysl-L-Lysine chloromethyl
ketone hydrochloride (TLCK), to inhibit gingipain protease activity. Consistent with the
data presented in Figure 3.3A, inhibition of gingipain protease activity with TLCK
prevented the stimulation of CXCL14 gene expression by P. gingivalis (Fig. 3.3B). These
data therefore suggest that the gingipain proteases are necessary for the stimulation of
CXCL14 gene expression in OKF6 cells by P. gingivalis.
In addition to being attached to the cell-surface of P. gingivalis, the gingipain proteases are
also found on P. gingivalis outer membrane vesicles (OMVs) (Gui et al., 2015). To
determine whether OMV-associated gingipain proteases can likewise stimulate CXCL14
gene expression, OKF6 cells were treated with either cell-free culture supernatants
derived from wildtype P. gingivalis or P. gingivalis KDP136. Kgp- and Rgp-specific activity
in the cell-free culture supernatants were first determined by measuring the hydrolysis of
chromogenic substrates, N-p-Tosyl-Gly-Pro-Lys 4-nitroanilide (GPKNA) and
N-Benzoyl-L-arginine 4-nitroanilide hydrochloride (L-BAPNA), respectively. As shown in
Figure 3.3C, supernatants derived from wildtype P. gingivalis exhibited Kgp activity, whilst
Kgp activity was not detected in supernatants derived from P. gingivalis KDP136.
Similarly, Rgp activity was only detected in supernatants derived from wildtype P.
gingivalis and not P. gingivalis KDP136 (Fig. 3.3D). The same supernatants were
subsequently used to stimulate OKF6 cells. As seen in Figure 3.3E, CXCL14 gene
expression was strongly upregulated when stimulated with the wildtype P. gingivalis cell-
free culture supernatants. In comparison, the P. gingivalis KDP136 cell-free culture
supernatants stimulated a significantly weaker CXCL14 response (Fig 3.3E). In summary,
74
these data suggest that cell-surface and OMV-associated gingipain proteases are involved
in the stimulation of CXCL14 gene expression in OKF6 cells by P. gingivalis.
Figure 3.3 P. gingivalis gingipain proteases stimulate CXCL14 expression. (A) OKF6 cells were challenged with wildtype P. gingivalis or P. gingivalis KDP136 at 100 MOI for 24 h. CXCL14 mRNA levels were measured by Real-Time PCR and shown as a fold change relative to mock-challenged cells (n=3). (B) OKF6 cells were challenged with P. gingivalis that had been pre-treated with TLCK, and CXCL14 mRNA levels were measured by Real-Time PCR and shown as a fold change relative to mock-challenged cells (n=3). (C-D) Proteolytic activity of (C) Kgp and (D) RgpA/B in cell-free culture supernatant from wildtype P. gingivalis and P. gingivalis KDP136 were measured (n=3). (E) OKF6 cells were stimulated with brain heart infusion broth (BHI) or cell-free culture supernatant from wildtype P. gingivalis and P. gingivalis KDP136 for 24 h. CXCL14 mRNA levels were measured by Real-Time PCR and shown as a fold change relative to BHI-stimulated cells (n=3). All data are presented as the mean ± SEM (** = p < 0.01, *** = p < 0.001).
A B
C D E
75
PAR-3-dependent regulation of P. gingivalis-stimulated CXCL14 gene
expression
PARs can mediate the recognition of host as well as microbial proteases (Soh et al., 2010).
P. gingivalis gingipain proteases have been shown to stimulate cytokine responses in
KB cells and human keratinocytes via PAR-1 and PAR-2 (Dommisch et al., 2007; Giacaman
et al., 2009; Lourbakos et al., 2001). Given the involvement of the gingipain proteases in
the stimulation of CXCL14 gene expression by P. gingivalis, a role for the PARs in
regulating CXCL14 response was investigated. The basal gene expression levels of PARs in
OKF6 cells were first measured. PAR-2 was found to be most highly expressed, whilst
PAR-1 and PAR-3 were expressed at lower levels, and PAR-4 mRNA was not detected
(Fig. 3.4A). The effects of P. gingivalis on the expression levels of the PARs were also
investigated. P. gingivalis was found to weakly induce the expression of PAR-2 (Fig. 3.4B),
whilst PAR-1 and PAR-3 expression remained unchanged (Fig. 3.4C-D).
Figure 3.4 P. gingivalis stimulates PAR-2 gene expression in oral epithelial cells. (A) Basal PAR-1, PAR-2, PAR-3 and PAR-4 mRNA levels in OKF6 cells were measured by Real-Time PCR and are shown as relative to TBP (endogenous control gene) (n=3). (B-D) OKF6 cells were challenged with P. gingivalis at 100 MOI for 24 h. (B) PAR-2, (C) PAR-1, and (D) PAR-3 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-challenged cells (n=3). All data are presented as the mean ± SEM (ND = not detected, *= p < 0.05).
C
A
D
B
76
The involvement of PAR-1, PAR-2, and PAR-3 in regulating P. gingivalis-inducible CXCL14
expression was subsequently investigated by siRNA-mediated gene silencing. The
knockdown of PAR-1, PAR-2, and PAR-3 gene expression in OKF6 cells was confirmed by
Real-Time PCR (Fig. 3.5A, C and E). As shown in Figure 3.5B, knockdown of PAR-1 did not
affect the stimulation of CXCL14 gene expression by P. gingivalis. Likewise,
P. gingivalis-inducible CXCL14 gene expression was not affected by PAR-2 knockdown
(Fig. 3.5D). In contrast, P. gingivalis-inducible CXCL14 gene expression was significantly
inhibited by knockdown of PAR-3 (Fig. 3.5F). Taken together, these data indicate that
P. gingivalis stimulates CXCL14 expression in OKF6 cells in a PAR-3-dependent manner.
77
Figure 3.5 PAR-3-dependent regulation of P. gingivalis-inducible CXCL14 expression. OKF6 cells were transfected with a non-targeting (-) siRNA, or a (A-B) PAR-1, (C -D) PAR-2, or (E-F) PAR-3 (+) siRNA for 48 h, and subsequently challenged with P. gingivalis at 100 MOI for 24 h. (A) PAR-1, (C) PAR-2, and (E) PAR-3 mRNA levels were measured by Real-Time PCR, and levels in cells transfected with the non-targeting siRNA control were arbitrarily assigned a value of 100%. (B, D, and F) CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-challenged cells (n=3). All data are presented as the mean ± SEM (ns = non-significant, * = p < 0.05, *** = p < 0.001).
D
B
F
C
A
E
78
EGFR signalling negatively regulates CXCL14 transcription in a
MEK-dependent manner
Although chemokines are essential for host defence, their expression must be tightly
regulated to prevent the development of pathological immune responses. EGFR signalling
is associated with epithelial proliferation and differentiation required for maintaining
tissue homeostasis. Studies have also demonstrated a role for EGFR in regulating skin
inflammation by modulating chemokine expression (Lichtenberger et al., 2013; Mascia et
al., 2003; Pastore et al., 2008). EGFR signalling was recently reported to inhibit CXCL14
gene expression in epidermal keratinocytes (Lichtenberger et al., 2013). Therefore, the
ability of EGF to regulate CXCL14 gene expression in OKF6 cells was investigated. EGF
stimulation was found to strongly suppress CXCL14 gene expression (Fig. 3.6A).
Time-course experiments also demonstrated that CXCL14 was rapidly downregulated
upon EGF stimulation (Fig. 3.6B). The protein levels of CXCL14 in cell culture supernatants
of OKF6 cells were also measured. Consistent with the reduction of CXCL14 mRNA levels
in response to EGF stimulation, CXCL14 protein levels were also reduced, although the
reduction was not statistically significant (Fig 3.6C). EGF has also been shown to modulate
the expression of other chemokines (e.g. CXCL1 and CCL5) (Mascia et al., 2003; Pastore et
al., 2005). Therefore, the effects of EGF on the expression of other cytokines/chemokines
in OKF6 cells were also investigated. EGF strongly stimulated the expression of the
neutrophil chemokine, CXCL1 (Fig. 3.6D). In contrast, EGF suppressed TNF gene
expression (Fig. 3.6E). IL-8 and CCL20 gene expression was unaffected by EGF stimulation
(Fig. 3.6F-G). Thus, EGF can differentially regulate the expression of specific cytokines and
chemokines in OKF6 cells.
79
Figure 3.6 EGF differentially regulates cytokine expression. (A) OKF6 cells were stimulated with the indicated concentration of EGF for 24 h. CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n=3). (B) OKF6 cells were stimulated with EGF (5 ng/ml) for the times indicated. CXCL14 mRNA levels were then measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n=3). (C) OKF6 cells were stimulated with EGF (20 ng/ml) for 24 h, and CXCL14 protein levels in the cell culture supernatants were measured by ELISA. (n=3). (D-G) OKF6 cells were stimulated with the indicated concentration of EGF for 24 h. (D) CXCL1, (E) TNF, (F) IL-8, and (G) CCL20 mRNA levels were measured by Real-Time PCR, and shown as a fold change relative to mock-stimulated cells (n=3). All data are presented as the mean ± SEM (* = < 0.05, ** = p < 0.01, *** = p < 0.001).
D
A
E
B
F G
C
80
Ligand-induced activation of EGFR induces a series of signalling cascades mediated by
mitogen-activated protein kinases (MAPKs), including ERK1/2 and p38 MAPK (Avraham
and Yarden, 2011). The ability of EGF to stimulate ERK1/2 and p38 MAPK activation in
OKF6 cells were first investigated. Specifically, phospho-specific antibodies were used to
assess EGF-mediated activation of ERK1/2 and p38 MAPK. As shown in Figure 3.7A-B,
ERK1/2 and p38 MAPK were rapidly activated in response to EGF stimulation.
Subsequently, the involvement of ERK1/2 and p38 MAPK in the regulation of CXCL14 by
EGFR signalling was investigated with pharmacological inhibitors. The blockade of
ERK1/2 signalling with the MEK inhibitor, U0126 (Duncia et al., 1998), prevented the
suppression of CXCL14 expression by EGF (Fig. 3.7C). By contrast, the p38 MAPK inhibitor,
SB203580 (Cuenda et al., 1995), did not prevent the suppression of CXCL14 (Fig. 3.7C).
This suggested that EGF suppresses CXCL14 expression by activating the MEK-ERK1/2
pathway. The EGF-MEK-ERK1/2-dependent downregulation of CXCL14 expression could
potentially be attributable to transcriptional repression or decreased mRNA transcript
stability. The mode of regulation was investigated by inhibiting gene transcription with
the RNA polymerase inhibitor, Actinomycin D. Blocking transcription inhibited the
upregulation of CXCL14 caused by the MEK inhibitor (Fig. 3.7D). This suggests that the
suppression of CXCL14 gene expression by EGF-induced MEK-ERK1/2 signalling is a
transcription-dependent event. The rate of CXCL14 mRNA decay in the presence and
absence of EGF was also investigated. CXCL14 mRNA stability was found to be unaffected
by EGF-MEK-ERK1/2 signalling (Fig. 3.7E). Taken together, these data suggest that EGF
signals through the MEK-ERK pathway to inhibit the transcription of CXCL14.
81
Figure 3.7 EGF suppresses CXCL14 transcription via MEK signalling. (A-B) OKF6 cells were stimulated with EGF (20 ng/ml) for the indicated times. The cell lysates were then subjected to Western blotting with (A) anti-phospho-ERK1/2 and anti-ERK2 antibodies, and (B) anti-phospho-p38 MAP kinase and anti-p38 MAP kinase antibodies. The positions of molecular mass standards (in kDa) are as indicated. The data are representative of two independent experiments. (C) OKF6 cells were stimulated with EGF (20 ng/ml) for 24 h and treated with the MEK inhibitor U0126 (10 µM) or the p38 inhibitor SB203580 (5 µM) for 8 h. CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to cells stimulated with EGF (n=3). (D) OKF6 cells were stimulated with EGF (20 ng/ml) for 24 h and treated with the MEK inhibitor U0126 (10 µM) and Actinomycin D (1 µg/ml) for 8 h. CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to cells stimulated with EGF (n=3). (E) OKF6 cells were treated with Actinomycin D (1 µg/ml) for 1 h, and stimulated with EGF (20 ng/ml) for the indicated times. CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n=3). All data are presented as the mean ± SEM (ns = non-significant, ** = p < 0.01).
C D
p-ERK1/2
0 5 15 30
ERK2
EGF (min)
p-p38
p38
0 5 15 30 EGF (min) A B
E
40
40
40
40
82
P. gingivalis gingipain proteases antagonise the negative regulation
of CXCL14 gene expression by EGF
P. gingivalis is known to antagonise immune signalling networks as a means of subverting
host defence (Hajishengallis, 2009, 2014), and has been shown to dampen EGFR signalling
in epidermal fibroblasts by inactivating EGF (Pyrc et al., 2013). Thus, there was a
possibility that EGF may modulate the stimulation of CXCL14 gene expression by
P. gingivalis. To investigate this possibility, OKF6 cells were cultured in the presence or
absence of EGF, and subsequently challenged with P. gingivalis. EGF was found to
significantly reduce, by around 50%, the stimulation of CXCL14 gene expression by
P. gingivalis (Fig. 3.8A). As expected, the P. gingivalis gingipain-deficient mutant
(P. gingivalis KDP136) did not stimulate CXCL14 gene expression, and nor did it relieve
EGF-mediated suppression of CXCL14 transcription (Fig. 3.8A). For comparison, the effect
of EGF on the stimulation of CXCL1 gene expression by P. gingivalis was also examined.
Although P. gingivalis and EGF both stimulated CXCL1 gene expression, they did not exert
an additive effect (Fig. 3.8B). Notably, the P. gingivalis gingipain-deficient mutant and EGF
appeared to exert additive effects on the stimulation of CXCL1 expression (Fig. 3.8B).
Taken together, these data suggest that P. gingivalis can antagonise the EGF-mediated
negative and positive regulation of CXCL14 and CXCL1 expression, respectively.
The gingipain proteases expressed by P. gingivalis have been demonstrated to be capable
of degrading host immunomodulatory factors (e.g. C3 complement factor and IL-8) (Zhang
et al., 1999; Stathopoulou et al. 2009; Potempa et al., 2009). Therefore, the ability of the
gingipain proteases to degrade EGF was tested. Specifically, EGF was incubated with
P. gingivalis for up to 3 h, and proteolysis was then assessed by SDS-PAGE. For
comparison, the ratio of P. gingivalis to EGF in Figure 3.8A-B was 2×106 P. gingivalis per ng
EGF. The incubation of 500 ng EGF with 1×106 P. gingivalis (i.e. 2×103 P. gingivalis per ng
EGF) resulted in significant EGF degradation within 60 min (Fig. 3.8C). Complete EGF
degradation was observed within 30 min when incubated with 1×108 P. gingivalis
(Fig. 3.8C). Conversely, EGF was not degraded when incubated with 1×106 P. gingivalis
KDP136 (Fig. 3.8C); significant degradation was only apparent when EGF was incubated
with 1×108 P. gingivalis KDP136 for 3 h (Fig. 3.8C), which was most likely attributable to
the activity of other P. gingivalis proteases (e.g. PrtT and Tpr) (Otogoto and Kuramitsu,
1993; Park and McBride, 1993). The ability of purified Kgp to degrade EGF was also
investigated. Consistently, EGF was rapidly degraded by Kgp (Fig. 3.8D). Collectively, these
data suggest that P. gingivalis gingipain protease-mediated degradation of EGF may
dysregulate host expression of CXCL14.
83
Figure 3.8 P. gingivalis antagonises the regulation of CXCL14 by EGF. (A-B) OKF6 cells were stimulated with EGF (20 ng/ml) for 24 h, and then challenged with P. gingivalis or P. gingivalis KDP136 at 100 MOI for 24 h. (A) CXCL14 and (B) CXCL1 mRNA levels then measured by Real-Time PCR, and are shown as a fold change relative to mock-challenged and unstimulated cells (n=3). (C) EGF (500 ng) was incubated with 1×106, 1×107, or 1×108 P. gingivalis or P. gingivalis KDP136 for the indicated times, and then subjected to SDS-PAGE and silver staining. The positions of molecular mass standards (in kDa) are as indicated. The data are representative of three independent experiments. (D) EGF was incubated with purified Kgp for the indicated times, and aliquots of the incubation mixtures were then subjected to SDS-PAGE and silver staining. The positions of molecular mass standards (in kDa) are as indicated. The data are representative of two independent experiments. All data are presented as the mean ± SEM (** = p < 0.01, * = p <0.05).
A
B
Kgp
EGF
1 0.5 2 3
10
3.5
Kgp 50
Time (h) 0
D
EGF
Time (h)
P. gingivalis KDP136 P. gingivalis
0 1 0.5 2 3
3.5
1x106
0 1 0.5 2 3
EGF
Time (h)
P. gingivalis KDP136 P. gingivalis
0 1 0.5 2 3
3.5
1x107
0 1 0.5 2 3
EGF
Time (h)
P. gingivalis KDP136 P. gingivalis
0 1 0.5 2 3
3.5
1x108
0 1 0.5 2 3
C
84
3.3 Discussion
Chemokines are essential immunomodulatory regulators of host defence. For instance,
they are important for directing the migration of both innate and adaptive immune cells to
sites of infection. In chronic periodontitis, P. gingivalis antagonises and dysregulates the
host immune response to create a favourable niche to sustain its growth. Oral epithelial
cells lining the surface of the oral mucosa express PRRs, which are essential for
recognising and mounting an immune response against oral pathogens. TLR2 is a critical
mediator of the inflammatory response to P. gingivalis, whilst other PRRs, such as PAR-1
and PAR-2, can also stimulate inflammation when proteolytically activated by P. gingivalis
gingipain proteases (Burns et al., 2010; Dommisch et al., 2007; Giacaman et al., 2009).
However, P. gingivalis can hijack and manipulate the host immune response to cause its
dysregulation (Hajishengallis, 2014; Hajishengallis et al., 2009). In this Chapter, the
expression of the orphan chemokine, CXCL14, in human oral epithelial cells (e.g. OKF6
cells) was identified to be strongly upregulated in response to P. gingivalis. The regulation
(and function) of the orphan chemokine CXCL14 has yet to be fully elucidated, therefore
the regulation of CXCL14 in oral epithelial cells was investigated.
P. gingivalis gingipain proteases (e.g. Kgp and RgpA/B) are critical virulence factors
because of their ability to antagonise and dysregulate host immunity (Imamura, 2003).
The results presented in this Chapter showed that the gingipain proteases play a central
role in the P. gingivalis-inducible stimulation of CXCL14 expression in OKF6 cells.
P. gingivalis has also been demonstrated to induce CXCL14 expression in primary gingival
epithelial cells (Chung and Dale, 2008), therefore the stimulation of CXCL14 by
P. gingivalis is not limited to OKF6 cells. Epithelial cells typically express cytokines
relatively rapidly in response to bacterial challenge, and thus the somewhat delayed
induction of CXCL14 expression when OKF6 cells were challenged with P. gingivalis
suggests that additional regulatory mechanisms, such as autocrine factors, may be
required for the optimal stimulation of CXCL14 expression. Further studies will therefore
be required to determine whether other factors induced by P. gingivalis might also
contribute, in an autocrine manner, to the regulation of CXCL14 expression.
CXCL14 protein levels in the cell culture supernatant were not significantly increased by
P. gingivalis. This was not entirely surprising, because other studies have encountered
difficulty in measuring cytokines and chemokines in cell culture supernatants due to their
proteolysis by P. gingivalis proteases (Darveau et al. 1998; Zhang et al. 1999; Stathopoulou
et al. 2009). In addition to degrading host cytokines and chemokines, the gingipain
proteases are also required for the efficient processing of P. gingivalis fimbriae (Nakayama
et al., 1996; Kadowaki et al., 1998). Fimbriae not only facilitate the adherence and invasion
85
of oral epithelial cells by P. gingivalis (Weinberg et al., 1997; Yilmaz et al. 2002), they also
stimulate the expression of pro-inflammatory genes (Hajishengallis et al., 2009). However,
the fact that treating wildtype P. gingivalis with the protease inhibitor TLCK also
prevented the stimulation of CXCL14 largely excludes the possibility that impaired
fimbriae processing at the cell surface might have been responsible for the lack of CXCL14
stimulation by the isogenic P. gingivalis gingipain protease-deficient mutant, P. gingivalis
KDP136. Nonetheless, additional studies will be needed to definitively exclude a role for
fimbriae in the stimulation of CXCL14 expression by P. gingivalis, including by mediating
epithelial cell adherence and invasion.
In addition to being attached to the outer membrane of P. gingivalis, the gingipain
proteases are also associated with OMVs, which are released into the surrounding
environment (O’ Brien-Simpson et al., 2003; Potempa et al., 1995). P. gingivalis cell-free
culture supernatants containing gingipain proteases have been shown to stimulate
chemokine expression (e.g. CCL20) in oral epithelial cells (Dommisch et al., 2007). CXCL14
gene expression in OKF6 cells was similarly upregulated when treated with cell-free
culture supernatants derived from wildtype P. gingivalis. Cell-free culture supernatants
derived from the P. gingivalis gingipain protease-deficient mutant also weakly induced
CXCL14 expression. These findings suggest that, although the stimulation of CXCL14 gene
expression in oral epithelial cells in response to P. gingivalis is predominantly mediated by
the gingipain proteases, there are also other factors present in the cell-free culture
supernatants that can also stimulate CXCL14 gene expression. Further studies will be
required to identify and determine the roles of other P. gingivalis-secreted factors in
stimulating CXCL14 gene expression in oral epithelial cells.
P. gingivalis-stimulated CXCL14 gene expression was found to be regulated in a
PAR-3-dependent manner. Previous studies using P. gingivalis cell-free culture
supernatants and purified gingipain proteases have demonstrated roles for PAR-1 and
PAR-2 in the induction of inflammatory gene expression (e.g. IL-6 and CCL20) in gingival
epithelial cells (Chung et al., 2004; Dommisch et al., 2007; Lourbakos et al., 2001). This is
the first study to provide evidence for PAR-3 in regulating chemokine expression in oral
epithelial cells in response to P. gingivalis. The function of PAR-3 is poorly characterised.
PAR-3 has been shown to act as a co-factor, by dimerising and potentiating PAR-1
activation in endothelial cells (Mclaughlin et al., 2007). Interestingly, the tethered ligand
produced from canonical PAR-3 cleavage at Lys38 by thrombin has been shown to also
directly activate PAR-1 and PAR-2 (Hansen et al., 2004). Although there is still little
evidence to suggest that PAR-3 can signal autonomously, it has recently been reported
that thrombin cleavage of endogenous PAR-3 regulated IL-8 gene expression in human
86
lung epithelial cells and astrocytoma cells (Ostrowska and Reiser, 2008). PAR-3 has also
been shown to be activated, by non-canonical cleavage at Arg41, in endothelial cells by the
coagulation enzyme, activated protease C, to promote endothelial barrier protection
(Burnier and Mosnier, 2013). The consequence of differential cleavage of canonical Lys38
and non-canonical Arg41 cleavage in inflammation has yet to be explored, and thus would
be an avenue for further investigation. Given that PAR-3 can potentially be activated by
both host- and pathogen-produced proteases, the complexity of PAR-3 activation and
signalling may therefore depend on the milieu of proteases present. Additionally, PRRs
have been shown to act in synergy to regulate cytokine and chemokine expression. For
example, the stimulation of THP-1 monocytes with different combinations of PRR agonists,
including PAR activating peptides, TLR agonists (e.g. FSL-1 and PolyI:C), NOD agonists
(e.g. FK156 and MDP), resulted in additive IL-8 response (Uehara et al., 2008). Therefore,
the possibility of PARs cooperating with alternative PRRs may also add to the complexity
of PAR signalling.
EGF is an important regulator of epithelial cell proliferation and differentiation, and hence
a critical mediator of epithelial tissue homeostasis (Parkar et al., 2001). EGF has been
reported to act as an important inflammatory mediator, by regulating cytokine and
chemokine expression (Lichtenberger et al., 2013; Mascia et al., 2003; Pastore et al., 2008).
In this study, EGF was found to differentially regulate cytokine/chemokine responses
differentially in oral epithelial cells (e.g. OKF6 cells). Significantly, EGF strongly inhibited
CXCL14 transcription in a MEK-ERK1/2-dependent manner. However, given that
Actinomycin D acts as a global transcriptional inhibitor, further studies will be required to
determine whether other signalling modulators (e.g. protease or kinase/phosphatase)
might also contribute to the regulation of CXCL14 expression by EGF. TNF gene expression
was similarly suppressed, whilst CXCL1 gene expression was upregulated with EGF
stimulation. Consistently, EGF has also been reported to regulate CXCL14, TNF, and CXCL1
in a similar manner in epidermal keratinocytes (Lichtenberger et al., 2013; Mascia et al.,
2003). Although EGF is constitutively present in saliva, gingival tissues, and gingival
crevicular fluid, the role of EGF in periodontitis is unclear (Chang et al., 1996; Mogi et al.,
1999). EGF has been shown to be important for the proliferation of human periodontal
ligament fibroblasts and human gingival fibroblasts (Matsuda et al., 1992). Additionally,
during periodontitis suggest that EGFR signalling is likely important for the wound healing
response in periodontitis (Chang et al., 1996). Critically, the loss of EGFR signalling in the
epidermis has been demonstrated to increase cytokine and chemokine levels and immune
cell infiltration (e.g. macrophages and neutrophils), resulting in skin inflammation
(Lichtenberger et al., 2013; Pastore et al., 2005). Thus, EGFR signalling in the oral
87
epithelium may also be important for coordinating the recruitment of immune cells during
the transition between the inflammatory and the wound healing response.
The findings presented in this Chapter revealed that P. gingivalis can negate EGF-mediated
regulation of CXCL14 (and CXCL1) through gingipain-mediated degradation of EGF.
Interestingly, in addition to EGF, other growth factors, including keratinocyte growth
factor (KGF) and insulin-like growth factor 1 (IGF-1), which likewise activate
MEK-ERK1/2, are also upregulated in the oral mucosa during inflammation and wound
healing (Li et al., 2005; Werner and Katz, 2004). Therefore, P. gingivalis gingipain
protease-mediated degradation of various host factors that can activate the MEK-ERK1/2
pathway may potentially contribute to the optimal stimulation of CXCL14 expression.
P. gingivalis can also antagonise EGF signalling through other mechanisms. For instance,
P. gingivalis expresses a peptidylarginine deiminase (PAD) that inhibits EGF-induced EGFR
signalling by catalysing the citrullination of the C-terminal arginine residue in EGF (Pyrc et
al., 2013). Furthermore, P. gingivalis LPS has been shown to attenuate EGFR-regulated
signalling, including ERK1/2 activation (Elkaim et al., 2017; Quinchia-Rios et al., 2008).
Accordingly, P. gingivalis gingipain proteases likely stimulate CXCL14 through
PAR-3-dependent signalling, and by antagonising signalling growth factors (e.g. EGF) that
activate the MEK-ERK1/2 pathway.
This Chapter has defined a regulatory pathway for P. gingivalis-stimulated CXCL14 gene
expression in oral epithelial cells. Specifically, the upregulation of CXCL14 gene expression
by P. gingivalis in OKF6 cells was shown to be dependent on the host protease-activated
receptor, PAR-3. Concomitantly, P. gingivalis can potentiate CXCL14 gene expression by
degrading EGF to relieve its transcriptional suppression of CXCL14 (Fig. 3.9). Given that
P. gingivalis dysregulates the host immune response to promote oral dysbiosis and
inflammation, host immunomodulatory factors can participate in the progression of
chronic periodontitis. Therefore, further studies will be required to determine whether
CXCL14 has a host-protective or destructive role in chronic periodontitis.
88
Figure 3.9 A proposed model for the regulation of CXCL14 gene expression in oral epithelial cells by P. gingivalis. The outer membrane-associated gingipain proteinases of P. gingivalis (Kgp/Rgp) stimulate the expression of CXCL14 in a PAR3-dependent manner. Maximal CXCL14 expression requires the gingipain-mediated degradation of EGF, which relieves the transcriptional repression of the CXCL14 gene by the ERK1/2 pathway.
CXCL14
P. gingivalis
EGF
MEK-ERK1/2
PAR-3
Kgp/Rgp
89
The work presented in this Chapter has given rise to the following publication: Aw, J., Scholz, G.M., Huq, N.L., Huynh, J., O’Brien‐Simpson, N.M., Reynolds, E.C. (2018),
“Interplay between Porphyromonas gingivalis and EGF signalling in the regulation of
CXCL14”, Cellular Microbiology, e12837
90
Function of CXCL14
91
4.1 Introduction
Chemokines have pleiotropic immunomodulatory functions in the host immune response
to infection. For example, they play important roles in the regulation of immune
surveillance and inflammation by directing the migration of immune cells to sites of tissue
injury and/or infection. CXCL14, which is an orphan member of the CXC family of
chemokines, was initially identified in breast and kidney tissues, and was later found to
also be expressed in other epithelia, including the oral epithelium (Hromas et al., 1999;
Meuter and Moser, 2008). The conservation of the amino acid sequence of CXCL14
between vertebrates suggests that it likely possesses indispensable function(s). However,
our understanding of the physiological function of CXCL14 is still relatively limited due to
its orphan state.
CXCL14 has been reported to possess chemotactic activity towards several immune cell
populations. For example, in vitro studies have demonstrated the ability of CXCL14 to
induce the migration of natural killer cells, dendritic cells, monocytes and neutrophils (Cao
et al., 2000; Kurth et al., 2001; Shellenberger et al., 2004; Shurin et al., 2005; Starnes et al.,
2006). However, the normal immune phenotype of CXCL14-deficient mice suggests that
compensatory mechanisms for CXCL14-regulated chemotaxis likely exist (Meuter et al.,
2007). In addition to chemotactic activity for immune cells, CXCL14 has also been reported
to regulate angiogenesis by inhibiting the migration of endothelial cells (Shellenberger et
al., 2004). Thus, further studies will be required to define the chemotactic activity of
CXCL14.
In addition to regulating immune cell chemotaxis, a number of chemokines, including
CCL20 and CCL28, have been shown to possess direct antibacterial activity (Hieshima et
al., 2003; Yang et al., 2003). The antibacterial activity of the chemokines is likely to be
attributed to the formation of a positively-charged surface patch, similar to antimicrobial
peptides (e.g. -defensins). CXCL14 has recently been demonstrated to exhibit bactericidal
activity; for instance, CXCL14 was shown to kill Streptococcus pyogenes and Pseudomonas
pneumoniae by causing cell membrane depolarisation (Dai et al., 2015; Frick et al., 2011).
Notably, the bactericidal activity has been mapped to a peptide encompassing Ser1-Arg13
at the N-terminus of CXCL14 (Dai et al., 2015). Importantly, it has been proposed that the
constitutive expression of CXCL14 in the epidermis may be an immediate defence
mechanism to provide protection against bacterial pathogens upon cutaneous injury
(Maerki et al., 2009).
The results presented in Chapter 3 suggest that P. gingivalis may dysregulate CXCL14 gene
expression in oral epithelial cells by activating PAR-3-dependent signalling whilst
92
impairing the EGF-mediated transcriptional repression of CXCL14. The function of CXCL14
in the inflammatory response to infection is still unclear. Therefore, this Chapter will
examine the potential regulatory role of CXCL14 in inflammation as well as its ability to
exert bactericidal activity against oral bacteria.
4.2 Results
Effects of CXCL14 on inflammatory gene expression in macrophages
CXCL14 is constitutively expressed in the oral epithelium (Hromas et al., 1999; Meuter and
Moser, 2008), and the results presented in Chapter 3 demonstrate that its expression by
oral epithelial cells is further upregulated in response to P. gingivalis. As briefly described
above, CXCL14 has been shown to regulate the migration of immune cells, including
monocytes (Kurth et al., 2001). Therefore, the ability of oral epithelial cell-derived CXCL14
to potentially act in a paracrine manner to regulate inflammatory gene expression in
macrophages was investigated. Specifically, the mouse macrophage-like cell line,
RAW 264.7, was stimulated over a 24 h time-course with recombinant murine CXCL14,
and changes in the mRNA expression levels of selected cytokines and chemokines were
then measured. Macrophages can produce chemokines that stimulate the recruitment of
monocytes to facilitate the clearance of infection (Murray and Wynn, 2012). Therefore, the
effect of CXCL14 on CCL2 (aka monocyte chemotactic protein-1 (MCP-1)) gene expression
was determined. As shown in Figure 4.1A, CCL2 expression was not modulated by CXCL14
stimulation. In addition to the recruitment of monocytes, macrophages can reinforce the
host immune response by producing CCL5 (aka regulated on activation, normal T cell
expressed and secreted (RANTES)), which is chemotactic for an array of immune cells,
including dendritic cells and T lymphocytes (Marques et al., 2013). However, CXCL14
stimulation did not affect the gene expression of CCL5 (Fig. 4.1B). In addition to
chemokines, macrophages also produce pro-inflammatory and anti-inflammatory
cytokines to regulate the host inflammatory response (Murray and Wynn, 2012).
Therefore, the ability of CXCL14 to regulate the expression of the pro-inflammatory
cytokine, TNF, and anti-inflammatory cytokine, IL-10, was also investigated. TNF and IL-
10 gene expression were not affected by CXCL14 stimulation (Fig. 4.1C-D). Collectively,
these findings suggest that CXCL14 does not regulate the expression of key inflammatory
genes in macrophages (e.g. RAW 264.7 cells). However, given the limited number of genes
examined, further studies will be required to establish whether CXCL14 may regulate the
inflammatory properties of macrophages.
93
Figure 4.1 CXCL14 stimulation of mouse macrophage RAW 264.7 cells. RAW 264.7 cells were stimulated with CXCL14 (100 ng/ml) for the indicated times. (A) CCL2, (B) CCL5, (C) TNF, and (D) IL-10 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n=3). All data are presented as the mean ± SEM.
Effects of CXCL14 on inflammatory gene expression in oral epithelial
cells
The ability of CXCL14 to act in an autocrine manner to regulate inflammatory gene
expression in oral epithelial cells was also investigated. Specifically, OKF6 cells were
stimulated over a 24 h time-course with recombinant human CXCL14, and changes in the
mRNA expression levels of selected cytokines and chemokines were then measured.
Neutrophils are instrumental to the host immune response to infection, and constitute a
large proportion of the inflammatory infiltrate in the junctional epithelium in periodontitis
(Scott and Krauss, 2013). In addition to mediating direct antimicrobial responses,
neutrophils can also act in conjunction with Th17 lymphocytes to sustain the
inflammatory response (Hajishengallis, 2014; Pelletier et al., 2010). Accordingly, the
ability of CXCL14 to regulate the expression of the neutrophil chemoattractant, IL-8, and
Th17 chemoattractant, CCL20, were examined. As shown in Figure 4.2A, IL-8 gene
expression was not significantly affected by CXCL14 stimulation. Similarly, CCL20 gene
expression was also unaffected by CXCL14 stimulation (Fig. 4.2B). The family of colony
stimulating factors: CSF-1 (M-CSF), CSF-2 (GM-CSF) and CSF-3 (G-CSF), are potent
A B
C D
94
haematopoietic growth factors and important for the development and functions of
macrophages and neutrophils (Hamilton, 2008; Metcalf, 2008), and as described in
Chapter 1, macrophages and neutrophils are important in the pathogenesis of chronic
periodontitis. Therefore, the effects of CXCL14 stimulation on these factors were
examined. CSF-1 gene expression was unchanged with CXCL14 stimulation (Fig. 4.2C), and
whilst CSF-2 expression appeared to be weakly upregulated, the increase was not
statistically significant at either the 2 h or 8 h time-point (Fig. 4.2D). Likewise, CSF-3 gene
expression was not significantly regulated by CXCL14 (Fig. 4.2E). CXCL14 gene expression
was also measured to determine whether CXCL14 may regulate its own expression,
potentially as part of a feedback mechanism; however, its expression was unchanged with
CXCL14 stimulation (Fig. 4.2F). Taken together, these results suggest that CXCL14 does not
regulate the expression of genes involved in the control of immune cells in oral epithelial
cells (e.g. OKF6 cells), though this conclusion is based on examination of a limited number
of genes.
Figure 4.2 CXCL14 stimulation of OKF6 cells. OKF6 cells were stimulated with CXCL14 (100 ng/ml) for the indicated times. (A) IL-8, (B) CCL20, (C) CSF-1, (D) CSF-2, (E) CSF-3, and (F) CXCL14 mRNA levels were measured by Real-Time PCR, and are shown as a fold change relative to mock-stimulated cells (n≥3). All data are presented as the mean ± SEM.
A B C
D E F
95
Effects of CXCL14 on inflammatory gene expression in the mouse
gingiva
In vitro studies are limited in that they cannot entirely reflect what occurs in vivo. Thus, a
pilot study was undertaken to investigate the ability of CXCL14 to regulate inflammatory
gene expression in vivo. Briefly, recombinant murine CXCL14 (either 0.5 µg or 2 µg) was
injected into the gingiva of BALB/c mice. Mice were killed either 24 h or 48 h post-
injection, and gingival tissues were harvested for analysis of inflammatory gene
expression. The effect of CXCL14 administration on the expression of the neutrophil
chemoattractant CXCL1 was first assessed. There was no difference in CXCL1 mRNA levels
24 h or 48 h post-intragingival injection of either 0.5 μg or 2.0 μg CXCL14, when compared
to time-matched, PBS-injected control mice (Fig. 4.3A). The effects of CXCL14 on the
mRNA expression levels of the pro-inflammatory cytokines, TNF and IL-6, were also
investigated. TNF mRNA levels in mice injected with either 0.5 μg or 2.0 μg CXCL14 were
comparable to PBS-injected, control mice at 24 or 48 h-post injection (Fig. 4.3B). Similarly,
intragingival injection of CXCL14 did not affect IL-6 mRNA levels (Fig. 4.3C). Consistent
with previous findings (Meuter and Moser, 2008), CXCL14 mRNA was found to be highly
expressed in the gingiva of mice (Fig. 4.3D); however, they were not further modulated in
response to intragingival injection of CXCL14 (Fig. 4.3D). Because the mRNA levels for the
genes measured were highly variable between mice within the same treatment group, no
conclusive results were obtained from this pilot study. In addition, the time-points at
which inflammatory gene expression were measured may also have not been optimal.
96
Figure 4.3 Effects of CXCL14 on inflammatory gene expression in the mouse gingiva. Murine CXCL14 (0.5 µg or 2 µg) was injected into the gingiva of BALB/c mice for 24 and 48 h. (A) CXCL1, (B) TNF, (C) IL-6, and (D) CXCL14 mRNA levels in the gingiva were measured by Real-Time PCR and are shown as relative to HPRT (endogenous control gene) (n=3). All data are presented as the mean ± SEM.
A B
C D
97
Effect of CXCL14 on oral epithelial cell migration
Wound healing is a highly dynamic process, involving reepithelisation and accompanied
by angiogenesis. In addition to regulating immune cell chemotaxis, CXCL14 has been
reported to antagonise IL-8-mediated endothelial cell migration and thereby inhibit
angiogenesis (Shellenberger et al., 2004). CXCL14 gene expression has also been found to
be dysregulated in various cancers (e.g. pancreatic and breast cancer) and associated with
enhanced cancer cell invasion (Pelicano et al., 2009; Wente et al., 2008). Therefore, a role
for CXCL14 in regulating oral epithelial cell migration was investigated with the well-
established “scratch wound” assay. The assay is a relatively simple method that allows
assessment of cell migration based on the rate of gap closure (Liang et al., 2007). Briefly,
OKF6 cells were cultured to confluence, and a “scratch” (wound) was then created in the
cell monolayer with a sterile pipette tip. The cells were washed with warm culture
medium to remove detached cells, and then cultured in medium (without EGF and BPE) in
the presence and absence of added CXCL14 for 6 h. Photographic images were taken at 2 h
intervals to enable assessment of gap (wound) closure. The images taken at the 2 h time-
point showed the uniform migration of OKF6 cells at the leading edge of the scratch, and
by 6 h the wound area was visibly reduced (Fig. 4.4A). The rate of gap closure was
quantified by measuring the area of the gap at each time point and expressing the value as
a percentage relative to the gap at time = 0 h. Gap closure increased by approximately 10%
within 2 h, and 60% after 6 h (Fig. 4.5B). However, CXCL14 did not appear to affect the
rate of gap closure (Fig. 4.4B). Therefore, these results suggest that CXCL14 does not
regulate oral epithelial cell (e.g. OKF6 cells) migration.
98
PBS CXCL14
0 h
2 h
4 h
6 h
B
A
Figure 4.4 Effect of CXCL14 on OKF6 cell migration. (A) OKF6 cells were subjected to a scratch-wound assay, and stimulated with PBS or CXCL14 (100 ng/ml). The data presented is representative of three independent experiments. Scale bar: 100 µm. (B) Gap closure was quantified by measuring the area of the gap at each time point, and expressed as % gap closure relative to 0 h. The data is presented as the mean ± SEM.
99
Bactericidal activity of CXCL14 against oral bacteria
In addition to being inflammatory mediators, some chemokines (e.g. CCL20) have also
been demonstrated to exert direct bactericidal activity. CXCL14 was recently reported to
possess bactericidal activity against bacteria, including Streptococcus pneumoniae, which
is associated with respiratory tract infection (Dai et al., 2015; Frick et al., 2011; Yang et al.,
2003). Therefore, the bactericidal activity of CXCL14 against oral bacteria was
investigated. Because CXCL14 has previously been shown to kill E. coli (Dai et al., 2015;
Maerki et al., 2009), the bactericidal activity of recombinant CXCL14 against E. coli was
used as a positive control in this study. Consistent with those previous studies, E. coli was
found to be highly susceptible to killing by CXCL14. Approximately 50% killing was
achieved at the lowest CXCL14 concentration tested (e.g. 0.25 µM), and 100% killing
achieved at ≥1 µM CXCL14 (Fig. 4.5A). The bactericidal activity of CXCL14 against oral
bacteria was next investigated. Notably, P. gingivalis appeared to be resistant to killing by
CXCL14, even when treated with 2 μM CXCL14 (Fig. 4.5B). Contrastingly, Streptococcus
gordonii, a Gram-positive bacterium and early coloniser of the oral biofilm, was highly
susceptible to killing by CXCL14 (Fig. 4.5C). For example, >95% killing of S. gordonii was
achieved at 1 μM CXCL14. Streptococcus sp. OT058, which is frequently associated with
periodontal health, was likewise highly susceptible to CXCL14-mediated killing (Fig. 4.5D).
These data demonstrate significant differences in the ability of CXCL14 to kill oral bacteria.
Moreover, they show that P. gingivalis is resistant to killing by CXCL14.
100
Figure 4.5 Bactericidal activity of CXCL14. (A) Escherichia coli, (B) P. gingivalis, (C) Streptococcus gordonii, and (D) Streptococcus sp. OT058 were incubated with the indicated concentration of recombinant CXCL14 for 1 h, and killing then measured by CFU assay (n=3). All data are presented as the mean ± SEM (* = p < 0.05, ** = p < 0.01, *** = p < 0.001).
A B
C D
101
Gingipain protease-dependent degradation of CXCL14 by P. gingivalis
The mature human CXCL14 protein contains fourteen lysine and seven arginine residues
(Fig. 4.6A), thus potentially making CXCL14 susceptible to degradation by the P. gingivalis
gingipain proteases, Kgp and RgpA/B. This raised the possibility that P. gingivalis may
resist CXCL14-mediated killing by proteolytically degrading CXCL14. Therefore, the ability
of the gingipain proteases to degrade CXCL14 was investigated. Briefly, CXCL14 was
incubated with P. gingivalis for up to 1 h, and proteolysis of CXCL14 was then assessed by
SDS-PAGE. The incubation of 500 ng CXCL14 with 1×104 P. gingivalis, which was
equivalent to the ratio of CXCL14 to P. gingivalis in the antimicrobial assays in Figure 4.5,
resulted in significant proteolysis of CXCL14 (Fig. 4.6B). CXCL14 was completely degraded
when incubated with greater numbers of P. gingivalis (e.g. 1×106 P. gingivalis) (Fig. 4.6B).
In contrast, when CXCL14 was incubated with the isogenic P. gingivalis gingipain
protease-deficient mutant, P. gingivalis KDP136, CXCL14 degradation was only evident
when incubated with 1×108 bacteria (Fig 4.6B). The ability of purified Kgp to degrade
CXCL14 was also tested. As shown in Figure 4.6C, CXCL14 was rapidly degraded by Kgp.
These findings indicate that the gingipain proteases can directly degrade CXCL14.
Figure 4.6 Gingipain protease-mediated degradation of CXCL14. (A) Amino acid sequence of mature human CXCL14. (B) CXCL14 (500 ng) was incubated with 1×104, 1×106, or 1×108 P. gingivalis or P. gingivalis KDP136 for the time indicated, and then subjected to SDS-PAGE and silver staining. (C) CXCL14 was incubated with purified Kgp for the indicated times, and then subjected to SDS-PAGE and silver staining. The positions of molecular mass standards (in kDa) are as indicated. The data in (B) and (C) are representative of two independent experiments.
SKCKCSRKGPKIRYSDVKKLEMKPKYPHCEEKMVIITTKSVSRYRGQEHCLHPKLQSTKRFIKWYNAWNEKRRVYEE
10 20 30 40 50 60 70 A
Kgp
0 1 0.5 2 3
CXCL14
10
3.5
Kgp 50
Time (h)
B
C
10
10
0 1 0.5
CXCL14
Time (h) 0 1 0.5 0 1 0.5
0 1 0.5
P. gingivalis
Time (h) 0 1 0.5 0 1 0.5
1x106 1x10
4 1x10
8
CXCL14
P. gingivalis KDP136
1x106 1x10
4 1x10
8
102
Identification of CXCL14 peptides resulting from Kgp digestion
A recent report revealed that the first thirteen amino acids of the mature CXCL14 protein
(i.e. Ser1‒Arg13) largely mediate its bactericidal activity, whilst Tyr14-Lys54 was shown
to stimulate chemotaxis of THP-1 monocytes (Dai et al., 2015). Thus, aliquots of the
digestion reactions shown in Fig. 4.6C were subjected to analysis by mass spectrometry to
identify the CXCL14-derived peptides generated by Kgp digestion. Several peptides from
the N-terminal half of CXCL14 were identified; however, none contained Ser1-Arg13 or
Tyr14-Lys54 (Table 4.1). This was not particularly surprising given that the amino acid
sequence spanning Ser1‒Arg13 contains four lysine residues, and Tyr14-Lys54 contains
six lysine residues (Fig. 4.6A). Several relatively short peptides from the C-terminal half of
CXCL14 were also identified (Table 4.1). Taken together, these data indicate that the
gingipain proteases of P. gingivalis can degrade CXCL14, and thereby likely inhibit its
bactericidal activity.
Table 4.1 CXCL14 peptides identified by MS from Kgp digestion.
Peptides Identified Ion Score 12
IRYSDVKK19 10
19KLEMKPK
25 34 20
LEMKPKYPHCEEK32 57
26YPHCEEK
32 17 33
MVIITTK39 53
64WYNAWNEK
71 36 64
WYNAWNEKRRVYEE77 9
103
4.3 Discussion
The host immune system relies on the tightly coordinated actions of immunomodulatory
factors to clear infection by pathogens. Chemokines are instrumental in coordinating the
immune response as they regulate the recruitment of both innate and adaptive immune
cells to sites of infection. Although the inflammatory response is typically host-protective,
P. gingivalis can manipulate and subvert the host immune response for its own benefit.
The dysregulation of cytokine and chemokine expression by P. gingivalis can lead to the
excessive recruitment of immune cells with suboptimal functions, and thus cause chronic
inflammation that mediates host tissue breakdown to increase nutrient availability
(e.g. haem). The data presented in Chapter 3 demonstrated that P. gingivalis can
potentially dysregulate CXCL14 gene expression by stimulating its expression via PAR-3,
while concurrently inhibiting EGF-mediated transcriptional repression of CXCL14. This
Chapter explored some of the potential immunomodulatory functions of CXCL14. A
gingipain protease-mediated mechanism that may enable P. gingivalis to further
dysregulate CXCL14 function was also identified.
The amino acid sequence of CXCL14 is highly conserved in vertebrates, and its constitutive
expression in various epithelia suggests that CXCL14 may have important inflammatory
and homeostatic functions. The participation of CXCL14 in modulating the inflammatory
response was examined in this study. It was found that CXCL14 did not regulate the
expression of the inflammatory genes examined in either human oral epithelial cells
(e.g. OKF6 cells) or mouse macrophages (e.g. RAW 264.7 cells). However, the potential to
identify CXCL14-regulated inflammatory genes was limited, as only a small number of
genes were investigated in this study. A more comprehensive, and unbiased approach,
such as RNA-seq, would likely provide greater insight into the potential role of CXCL14 as
a regulator of inflammatory genes in oral epithelial cells and macrophages. However, given
that the receptor for CXCL14 is still unknown, there is also the possibility that neither of
these cell types express the receptor for CXCL14.
Recent studies suggest that CXCL14 can interact with CXCR4, the receptor for CXCL12 (aka
stromal cell-derived factor-1 (SDF-1)) (Collins et al., 2017; Hara and Tanegashima, 2014;
Tanegashima et al., 2013). However, CXCL14 does not stimulate CXCR4 receptor signalling,
instead it functions as an allosteric modulator to prime and sensitise CXCL12-mediated
CXCR4 signalling. This regulatory function for CXCL14 was demonstrated in natural killer
cells, T lymphocytes and B lymphocytes (Collins et al. 2017). Interestingly, CXCR4 has
been shown to participate in signalling crosstalk with TLR2 and TLR4 to dampen the host
inflammatory response (Hajishengallis et al., 2008; Kishore et al., 2005). For example,
P. gingivalis fimbriae can stimulate the co-association of CXCR4 and TLR2 in lipid rafts in
104
macrophages to promote signalling crosstalk. Importantly, this inhibits TLR2-mediated
NF-B activation and nitric oxide production, which is required for the intracellular killing
of P. gingivalis (Hajishengallis et al., 2008). In a similar vein, CXCL12-mediated activation
of CXCR4 signalling has been shown to inhibit TLR4 activation at low LPS concentrations,
potentially preventing inappropriate inflammatory responses to the normal microbiota
(Kishore et al., 2005). Accordingly, the stimulation of CXCL14 expression by P. gingivalis
might potentiate the aforementioned signalling crosstalk by enhancing the potency of
CXCR4 ligands. It is therefore tempting to speculate that the dysregulation of CXCL14
expression by P. gingivalis might potentiate CXCR4 crosstalk and contribute to microbial
dysbiosis by not only suppressing macrophage antimicrobial activity, but also increasing
the activation threshold of TLR4 and thereby alter the host inflammatory response to the
oral biofilm. Thus, the contribution of CXCL14 to CXCR4/TLR signalling crosstalk warrants
further investigation.
A mouse intragingival injection model was established to enable in vivo investigation of
CXCL14 function. Consistent with a previous study (Meuter and Moser, 2008), CXCL14
mRNA was found to be highly expressed in the gingival tissues of mice. However, due to
the variability in gene expression between mice, it is unclear whether CXCL14 may
potentially play a role in regulating inflammatory gene expression in the gingiva.
Furthermore, the time-points selected may not have been optimal to identify changes in
the expression of CXCL14-target genes. The mouse model undertaken here was a pilot
study, with the aim of gaining data to justify a follow-up study involving larger numbers of
mice. Although in vivo mouse models allow biological studies to be conducted in a more
physiologically relevant environment, responses can be highly variable between mice.
Relatively large numbers of mice are therefore often required to provide experiments with
sufficient statistical power.
Nevertheless, the ability of recombinant CXCL14 to promote leukocyte recruitment when
injected into the footpads of BALB/c nude mice (Sleeman et al., 2000) suggests that
CXCL14 is capable of regulating inflammation in vivo. In the study by Sleeman et al.,
CXCL14 induced a similar inflammatory response when injected into C3H/HeJ mice,
indicating that the response was specific to CXCL14, rather than a consequence of
potential endotoxin (LPS) contamination (Sleeman et al., 2000). Furthermore, CXCL14 has
been found to be strongly upregulated in inflamed joints in autoimmune arthritis
(Lindberg et al., 2006). The transgenic expression of CXCL14 in mice exacerbates
collagen-induced arthritis, and was found to be associated with an increased T lymphocyte
response (Chen, Guo, et al., 2012). Moreover, in vitro studies have demonstrated that
CXCL14 possesses pleiotropic chemotactic activity towards monocytes, B lymphocytes,
105
neutrophils, immature dendritic cells and natural killer cells (Dai et al., 2015;
Shellenberger et al., 2004; Sleeman et al., 2000; Starnes et al., 2006). CXCL14-deficient
mice appear to have a normal immune phenotype, although the mice weigh less than their
wildtype littermate counterparts (Meuter et al., 2007). Therefore, the possibility of
functional redundancy that compensates for the chemotactic activity of CXCL14 cannot be
excluded. Intriguingly, CXCL14 knockout mice are born at a lower than expected
Mendelian frequency, suggesting a potential role for CXCL14 in embryogenesis (Meuter et
al., 2007).
CXC chemokines that lack the ELR (glutamic acid-leucine-arginine) motif (e.g. CXCL9 and
CXCL10) have been shown to be potent angiostatic factors (Strieter et al., 1995, 2005). As
an ELR-negative chemokine, CXCL14 has been demonstrated to inhibit angiogenesis by
interacting with angiogenic ligands (e.g. IL-8 and basic fibroblast growth factor (bFGF)) to
inhibit endothelial cell migration (Shellenberger et al., 2004). The ability of CXCL14 to
direct oral epithelial cell migration was assessed in this study by the “scratch assay”. The
findings obtained suggest that CXCL14 does not regulate the rate of oral epithelial cell
migration (e.g. OKF6 cells). However, CXCL14 has been shown to regulate the migration of
other cell types, such as osteosarcoma cells and mammary epithelial cells (Lu et al., 2015;
Pelicano et al., 2009). The heterogenous expression of CXCL14 in various cancers (e.g.
breast and pancreatic cancer) suggests that CXCL14 may be involved in mediating cancer
progression (Pelicano et al., 2009; Wente et al., 2008). It is uncertain whether CXCL14
might be acting as a tumour suppressor or oncogene, because CXCL14 gene expression has
been reported to be increased in some cancers (e.g. pancreatic and prostate cancer) and
decreased in others (e.g. lung cancer) (Augsten et al., 2009; Tessema et al., 2010; Wente et
al., 2008). Various studies have provided evidence for the involvement of CXCL14 in
promoting cancer metastasis (Augsten et al., 2009; Pelicano et al., 2009), whilst others
have shown that the expression of CXCL14 suppressed tumour growth (Ozawa et al., 2006;
Tessema et al., 2010). Therefore, CXCL14 tumour-suppressing and tumour-promoting
activities are possibly dependent on the tumour type. Nevertheless, the functional
characteristics of CXCL14 in cancer studies could potentially provide inferences about the
role of CXCL14 in the context of infection, inflammation, and wound healing.
CXCL14 has recently been shown to possess bactericidal activity against some skin and
respiratory pathogens, including Pseudomonas aeruginosa and Staphylococcus aureus (Dai
et al., 2015). Here, CXCL14 was shown to kill oral Streptococcus species, namely S. gordonii
and S. sp. OT058. S. gordonii is a primary coloniser of the tooth-accreted oral biofilm and
promotes P. gingivalis colonisation (Park et al., 2005). Accordingly, S. gordonii has been
described as being an accessory pathogen in chronic periodontitis (Lamont et al., 1993;
106
Lamont and Hajishengallis, 2015). Conversely, the abundance of S. sp. OT058 in the biofilm
has been reported to be associated with periodontal health (Hong et al., 2015). CXCL14
has also been demonstrated to kill Streptococcus mitis, one of the most abundant
Streptococcus species in the biofilm (Dai et al., 2015). Significantly, P. gingivalis was shown
to be resistant to CXCL14-mediated killing. This is potentially attributable to the ability of
the gingipain proteases produced by P. gingivalis to proteolytically degrade CXCL14. The
results presented in this Chapter suggest that CXCL14 may also be susceptible to
degradation by other P. gingivalis proteases (e.g. PrtT and Tpr). In addition to proteolytic
degradation of antimicrobial peptides, unique modifications of P. gingivalis LPS have also
been proposed to protect P. gingivalis from cationic antimicrobial peptides (e.g. LL-37 and
polymyxin B) (Bachrach et al., 2008; Coats et al., 2009). Specifically, phosphorylation of
the lipid A moiety of LPS is proposed to contribute resistance to antimicrobial peptides by
imparting P. gingivalis with a negative surface-charge. Therefore, the P. gingivalis outer
membrane LPS composition may also affect the ability of CXCL14 to kill P. gingivalis.
Human CXCL14 is translated as an 111-amino acid pro-protein, and is cleaved C-terminal
of Gly-34 to yield the mature 77-amino acid CXCL14 protein (Cao et al., 2000). The
antimicrobial activity of the mature CXCL14 protein has been demonstrated to lie within a
short sequence at the N-terminus (i.e. Ser1-Arg13), while Tyr14-Lys54 has been shown to
be sufficient for CXCL14 to promote chemotaxis of human monocytes (e.g. THP-1 cells)
(Dai et al., 2015). Peptides arising from in vitro Kgp digestion of CXCL14 were analysed by
mass spectrometry to determine if they contain Ser1-Arg13 or Tyr14-Lys54. Although this
analysis identified several peptides from the N-terminal half of CXCL14, Ser1-Arg13 and
Tyr14-Lys54 peptides were not detected. This suggests that the gingipain protease-
mediated degradation of CXCL14 likely eliminates CXCL14 bactericidal and chemotactic
activity, and hence protects P. gingivalis from CXCL14-mediated host defence. A similar
protease-mediated protective mechanism has been proposed to explain the resistance of
Finegoldia magna, an opportunistic pathogen, to CXCL14 (Frick et al., 2011). Importantly,
the degradation of CXCL14 by P. gingivalis might also potentially protect otherwise
susceptible bacteria, including accessory pathogens (e.g. S. gordonii), from killing by
CXCL14, and thereby play a role in promoting dysbiosis. In addition, loss of CXCL14-
mediated chemotactic activity might also contribute to the proliferation of a dysbiotic
biofilm. Additional studies will be required to determine whether the CXCL14 peptides
generated from gingipain protease digestion have bactericidal activity against other
bacteria, as this could potentially also contribute to the development of dysbiosis.
This study has explored several potential functions of CXCL14 relevant to the oral mucosa.
Due to the orphan nature of CXCL14, it is unclear whether CXCL14 can regulate the
107
inflammatory response of oral epithelial cells or macrophages. The findings from this
study also suggest that CXCL14 does not regulate the migration of oral epithelial cells.
However, this study did demonstrate that CXCL14 can exert differential bactericidal
activity against oral bacteria. Significantly, the dysregulation of CXCL14 by P. gingivalis
may potentially destabilise the proportions of bacterial species in the biofilm and thereby
promote biofilm dysbiosis. Thus, although the expression of CXCL14 by oral epithelial cells
is a host-initiated response, its role in host defence might need to be reconsidered in the
context of infection by pathogens that rely on dysbiosis to fulfil their nutritional
requirements.
108
The work presented in this Chapter has given rise to the following publication: Aw, J., Scholz, G.M., Huq, N.L., Huynh, J., O’Brien‐Simpson, N.M., Reynolds, E.C. (2018),
“Interplay between Porphyromonas gingivalis and EGF signalling in the regulation of
CXCL14”, Cellular Microbiology, e12837
109
Identification and characterisation of
P. gingivalis TIR domain-containing proteins
110
5.1 Introduction
The dysregulation of the host immune response by P. gingivalis can induce biofilm
dysbiosis and chronic inflammation (Darveau, 2010; Hajishengallis, 2014). Pattern-
recognition receptors, such as TLRs, are important for recognising and mounting immune
responses against potential pathogens. The TIR domain of TLRs is an essential signalling
module in innate immunity. As described in Chapter 1, homotypic interactions between
the TIR domain of TLRs provides a platform for the assembly of adaptor proteins, which is
necessary for the activation of downstream signal transduction. Importantly, TLR
signalling can be hijacked by P. gingivalis as a means of immune subversion
(Hajishengallis, 2014). Other bacterial species have been shown to express TIR domain-
containing proteins (Tcps) as a means of immune subversion. Newman et al. were the first
to show that TlpA from S. enterica was not only capable of suppressing TLR4-mediated
NF-B activation in vitro, but was also required for S. enterica virulence in vivo (Newman
et al., 2006). This led to the subsequent identification of additional bacterial Tcps
(e.g. TcpC from E. coli CFT037 and TcpB from B. melitensis), and the proposal of the
“subversion hypothesis”, whereby pathogens adopt molecular mimicry to inhibit the host
immune response (Cirl and Miethke, 2010; Radhakrishnan et al., 2009; Sengupta et al.,
2010). Although P. gingivalis can interfere with TLR signalling through multiple virulence
factors (refer to Section 1.7 for specific details), the existence of P. gingivalis Tcp(s) has not
been reported. Therefore, this Chapter will characterise putative P. gingivalis Tcps, using a
combination of bioinformatics and biochemical approaches.
5.2 Results
Identification of putative P. gingivalis TIR domain-containing
proteins
A search for TIR domains in the Interpro database was conducted to identify putative
P. gingivalis Tcps. The search revealed that there are over 9,000 bacterial proteins
annotated with TIR domain signatures, and P. gingivalis is annotated to have twelve Tcps
in nine different strains (Table 5.1). A phylogenetic tree was subsequently constructed,
using the BLOSUM62 score matrix (Henikoff and Henikoff, 1992), to infer the evolutionary
relationships between the Tcps. P. gingivalis W83 and W50 share highly related, if not
nearly identical genomes, and thus are denoted as P. gingivalis W83/W50 (Chen et al.,
2004). Phylogenetic analysis suggested that the P. gingivalis Tcps can be divided into two
groups, which, for simplicity, are denoted as Group A and Group B (Fig. 5.1). The Group A
proteins Q7MX37 (PG0382) from P. gingivalis W83/W50 and U2JPW4
(HMPREF1989_02328) from P. gingivalis F0566 differ by only two amino acids (Met169
and Gly231 in Q7MX37, and Arg169 and Glu231 in U2JPW4), and thus are the same
111
protein. The three unique Tcps in Group A are <500 amino acids in length, and share
between 13 and 20% overall amino acid sequence identity (Table 5.2). The putative TIR
domains of Group A proteins share amino acid sequence identities between 15 to 32%
(Table 5.3.). Tcps from Group B comprise proteins >800 amino acids in length, and share
>60% overall amino acid sequence identity (Table 5.2). Notably, the putative TIR domains
in Tcps within Group B have amino acid sequence identities >70% (Table 5.3).
Furthermore, the TIR domains in W1R842 and F5X7L6, and T2N8U1 and A0A0K2J754, are
identical (Table 5.3).
Table 5.1 Annotated TIR domain-containing proteins of P. gingivalis strains.
P. gingivalis strain
Accession number
Gene Protein length
ATCC 33277 B2RLS0 PGN1796 1125 W83/W50 Q7MTS7 PG1864 1266 W83/W50 Q7MX37 PG0382 490 AJW4 A0A0K2J754 PGJ00016810 1109 F0566 U2JFL6 HMPREF1989_01737 875 F0566 U2JPW4 HMPREF1989_02328 490 F0566 U2JRV0 HMPREF1989_00095 401 F0570 A0A0E2LT79 HMPREF1555_00114 881 JCVI SC001 T2N8U1 A343_0215 814 JCVI SC001 T2NDV9 A343_1154 440 SJD2 W1R842 SJDPG2_06385 1145 TDC60 F5X7L6 PGTD600128 1384
112
Group A
Group B
Figure 5.1 Phylogenetic tree of P. gingivalis TIR domain-containing proteins.
113
Table 5.2 Amino acid sequence identity (%) between P. gingivalis TIR domain-containing proteins.
Strain W83/W50 F5066 F0566 SC001 ATCC33277 F0566 SJD2 TDC60 F0570 W83 SC001 SC001
Strain Protein Q7MX37 U2JRV0 U2JPW4 T2NDV9 B2RLS0 U2JFL6 W1R842 F5X7L6 A0A0E2LT79 Q7MTS7 T2N8U1 A0A0K2J754 W83/W50 Q7MX37 - 12.8 99.6 20.1 10.1 12.0 9.4 8.6 12.6 9.8 14.1 9.5
F0566 U2JRV0 12.8
13.0 13.9 5.9 7.3 5.7 4.9 7.9 5.9 7.9 5.9 F0566 U2JPW4 99.6 13.0 - 20.1 10.0 11.9 9.3 8.8 12.5 9.8 14.6 9.4 SC001 T2NDV9 20.1 13.9 20.1 - 2.7 3.3 2.5 2.1 4.0 2.6 3.7 2.9
ATCC33277 B2RLS0 10.1 5.9 10.0 2.7 - 71.5 81.5 62.4 67.9 71.5 79.9 77.9 F0566 U2JFL6 12.0 7.3 11.9 3.3 71.5 - 61.4 51.3 80.5 57.5 79.7 66.2 SJD2 W1R842 9.4 5.7 9.3 2.5 81.5 61.4 - 68.1 67.5 70.9 59.5 76.4
TDC60 F5X7L6 8.6 4.9 8.8 2.1 62.4 51.3 68.1 - 53.3 71.5 52.5 64.4 F0570 A0A0E2LT79 12.6 7.9 12.5 4.0 67.9 80.5 67.5 53.3 - 62.0 84.4 72.2 W83 Q7MTS7 9.8 5.9 9.8 2.6 71.5 57.5 70.9 71.5 62.0 - 59.6 76.1
SC001 T2N8U1 14.1 7.9 14.6 3.7 79.9 79.7 59.5 52.5 84.4 59.6 - 69.3 AJW4 A0A0K2J754 9.5 5.9 9.4 2.9 77.9 66.2 76.4 64.4 72.2 76.1 69.3 -
114
Table 5.3 Amino acid sequence identity (%) between P. gingivalis TIR domain-containing proteins TIR domains.
Strain W83/W50 F5066 F0566 SC001 ATCC33277 F0566 SJD2 TDC60 F0570 W83 SC001 SC001 Strain Protein Q7MX37 U2JRV0 U2JPW4 T2NDV9 B2RLS0 U2JFL6 W1R842 F5X7L6 A0A0E2LT79 Q7MTS7 T2N8U1 A0A0K2J754
W83/W50 Q7MX37 - 15.2 100.0 32.1 18.6 18.6 22.7 22.7 17.4 17.4 17.4 17.4 F5066 U2JRV0 15.2 - 15.2 19.5 22.0 22.0 19.5 19.5 19.5 19.5 19.5 19.5 F0566 U2JPW4 100.0 15.2 - 32.1 18.6 18.6 22.7 22.7 17.4 17.4 17.4 17.4 SC001 T2NDV9 32.1 19.5 32.1 - 23.5 23.5 23.7 23.7 24.5 24.5 25.5 25.5
ATCC33277 B2RLS0 18.6 22.0 18.6 23.5 - 98.7 74.7 74.7 98.7 87.5 81.6 81.6 F0566 U2JFL6 18.6 22.0 18.6 23.5 98.7 - 75.8 75.8 88.2 87.5 73.7 82.9 SJD2 W1R842 22.7 19.5 22.7 23.7 74.7 75.8 - 100.0 73.7 73.7 73.7 73.7
TDC60 F5X7L6 22.7 19.5 22.7 23.7 74.7 75.8 100.0 - 73.7 73.7 73.7 73.7 F0570 A0A0E2LT79 17.4 19.5 17.4 24.5 98.7 88.2 73.7 73.7 - 98.0 94.7 94.7 W83 Q7MTS7 17.4 19.5 17.4 24.5 87.5 87.5 73.7 73.7 98.0 - 92.8 92.8
SC001 T2N8U1 17.4 19.5 17.4 25.5 81.6 73.7 73.7 73.7 94.7 92.8 - 100.0 AJW4 A0A0K2J754 17.4 19.5 17.4 25.5 81.6 82.9 73.7 73.7 94.7 92.8 100.0 -
115
A NCBI Conserved Domain Search (www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) was
performed to establish whether the proteins contain other annotated domains. A nucleoside-
triphosphatase (NTPase) domain, which can catalyse the hydrolysis of nucleoside triphosphates
to nucleotides (Vetter and Wittinghofer, 1999), was predicted to be present in several Tcps,
including U2JPW4/Q7MX37 (PG0382) (Fig. 5.2). A leucine-rich repeat (LRR) domain, which is
involved in mediating protein-protein interactions (Kobe and Kajava, 2001), was predicted to
be present in all Group B Tcps, except T2N8U1. Interestingly, T2NDV9 was predicted to contain
a SIR2 domain, which are involved in transcriptional regulation (North and Verdin, 2004).
Therefore, in addition to a TIR domain, putative P. gingivalis Tcps may possess other functional
domains. Based on its predicted structural domains, Q7MX37 (referred to as, PG0382,
hereafter) from P. gingivalis W83/W50 was selected for further investigation.
Figure 5.2 Predicted protein domains of annotated P. gingivalis TIR domain-containing proteins.
B2RLS0
Q7MTS7
Q7MX37/ U2JPW4
F5X7L6
A0A0K2J754
U2JFL6
U2JRV0
A0A0E2LT79
T2N8U1
T2NDV9
W1R842
Domain:
LRR
NTPase
SIR2
TIR
116
PG0382 is predicted to contain a coiled-coil motif
Some bacterial Tcps have been identified to contain coiled-coil motifs comprised of heptad
sequence repeats, which may facilitate dimer formation and thereby potentially enhance
inhibition of TLR signalling (Low et al., 2007; Rana et al., 2011; Fekonja et al. 2012). Therefore,
the presence of putative coiled-coil motifs in PG0382 was investigated, using the COILS
prediction server (https://embnet.vital-it.ch/software/COILS_form.html) (Lupas et al., 1991).
The COILS prediction server compares input sequences with a database of known two-stranded
parallel coiled-coils to derive a similarity score, and then generates a probability score that
reflects the likelihood of the input protein sequence having a coiled-coil motif (Lupas et al.,
1991). Based on the COILS output, PG0382 was predicted to form coiled-coil motifs, in a
14-residue window, between: Gln200-Lys250, Gly350-Glu400, and Thr450-Gln490 (Fig. 5.3).
PG0382 is also predicted to form coiled-coil motifs at the same positions when calculated by
applying a 21-residue window, albeit with lower probability. Therefore, PG0382 likely contains
coiled-coil motifs within the N-terminal half of the protein.
Figure 5.3 COILS analysis output for PG0382
1
0.8
0.6
0.4
0.2
0
0 50 100 150 200 250 300 350 400 450 500
Co
iled
-co
il fo
rmin
g p
rob
abili
ty
Residue number
117
Comparison of PG0382 with mammalian TLRs and adaptor proteins
PG0382 is comprised of 490 amino acids and predicted to contain an NTPase domain between
Val8 and Leu52, and a TIR domain between Lys341 and Gln490 (Fig. 5.4). Pairwise sequence
alignments were conducted with EMBOSS Needle (www.ebi.ac.uk/Tools/psa/emboss_needle/)
to determine the amino acid sequence identity between the PG0382 TIR domain and TLR TIR
domains. The accession numbers of the mammalian TLRs are presented in Appendix Table A1.
The alignment indicated that the PG0382 TIR domain shares highest sequence identity with
TLR1 (17.8%), and lowest identity with TLR4 (3.8%) (Table 5.4). In comparison, TLR TIR
domains have sequence identities between 87% (TLR1 and TLR6) and 6% (TLR1 and TLR9).
TLR1 and TLR6 are highly homologous, most likely because they heterodimerise with TLR2.
However, most TLR TIR domains have sequence identities around 20-30%.
Figure 5.4 Amino acid sequence of PG0382. The putative NTPase domain is shown in orange text, and the TIR domain is shown in purple text.
10 20 30 40 50 MDLAEVFVTE GFPHLTYVEP LNYYEILIDV KSKKKPVIIE GQTGTGKTST 60 70 80 90 100 ILKILSDIKE EIHFEYLSAR NIDETTKINQ LINSNFEEGG NFVIDDFHRL 110 120 130 140 150 VDHLKLRLSN IAKLAADNVA NPKYPKLVII GINQTGRELL KLSPDIAKRF 160 170 180 190 200 GVHKIQPATE ENVKSIIEMG EKLLNIKFLR HKPIYSESKG DYWLTQHICQ 210 220 230 240 250 TICTQNGVIN TQEETKQIKL NIKEARRKII GRLEYIYNDI VKEFCRGKRF 260 270 280 290 300 RPSNDAYIKF LESVSKMDDF PIDLNELIGN VDDHHRIAIS SIKGHRLDVL 310 320 330 340 350 IKEKIDLRNN FYYSKDTKLF NIEDPALQYY IKHIDWNKLY KECGFKKNNG 360 370 380 390 400 SYKYDIAISF AGEKRELAEE IADQLQRSDY EVFYDRLYED NYLGMSLSDE 410 420 430 440 450 FERIFTSESK FVVCLLDKNH KKKIWPTFER DCFLEKVQTN EVIPIFLDDT 460 470 480 490 KFPGIPNDIA CIRYEEKQED KKRSERVQRE IIERIISKVQ
118
Table 5.4 Amino acid sequence identity (%) between PG0382 TIR domain and TLR TIR domains.
PG0382 TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR7 TLR8 TLR9
TLR1 17.8 - 51.0 27.7 38.9 25.3 86.9 29.6 33.1 28.9 TLR2 14.0 51.0 - 27.0 40.5 28.2 49.7 38.1 39.1 28.9 TLR3 17.6 27.7 27.0 - 25.2 27.3 27.0 29.9 32.5 28.0 TLR4 3.8 38.9 40.5 25.2 - 28.7 35.1 25.9 28.2 30.9 TLR5 12.6 25.3 28.2 27.3 28.7 - 22.7 24.3 28.3 25.8 TLR6 17.0 86.9 49.7 27.0 35.1 22.7 - 30.5 32.5 27.6 TLR7 14.0 29.6 38.1 29.9 25.9 24.3 30.5 - 58.8 42.4 TLR8 8.9 33.1 39.1 32.5 28.2 28.3 32.5 58.8 - 43.3 TLR9 6.0 28.9 28.9 28.0 30.9 25.8 27.6 42.4 43.3 -
The TIR domain contains conserved sequence motifs in box 1, box 2 and box 3 (Watters et al.,
2007). Therefore, a multiple sequence alignment analysis was conducted using ClustalOmega to
identify sequence similarities in the conserved TIR domain box 1, box 2 and box 3 sequence
motifs of PG0382 and human TLRs (Fig. 5.5). PG0382 contains multiple amino acid residues
corresponding to the TIR domain box 1 consensus sequence: F/YDAFISY, including Tyr354,
Asp355, Ile358, and Ser359. The sequence alignment also identified differences between
PG0382 and TLRs. For example, all TLRs contain the consensus alanine residue, which is
replaced by an isoleucine (Ile356) in PG0382. In addition, the phenylalanine residue is replaced
by an alanine (Ala357) in PG0382. At the same position, TLRs either contain the conserved
phenylalanine or is replaced by tyrosine. Like TLR5 and TLR9, the final consensus tyrosine
residue is replaced by phenylalanine (Phe360) in PG0382. The BB loop in box 2 is characterised
by an RDxxPG motif, where x denotes a hydrophobic amino acid. Although PG0382 does not
contain the consensus arginine residue, it does contain the consensus aspartic acid residue
(Asp390) and glycine residue (Gly394). The consensus proline residue, which is critical for
TLR4 signalling (Poltorak et al., 1998), and present in all TLRs (except TLR3), is replaced by a
leucine (Leu393) in PG0382. Box 3 is defined by an FW motif, and like TLR3 and TLR5, box 3 is
absent in PG0382.
119
Figure 5.5 Multiple sequence alignment of PG0382 TIR domain with TLR TIR domains. The consensus sequence for box 1, box 2, and box 3 are as indicated. The x in box 2 denotes any hydrophobic amino acid.
Pairwise amino acid sequence alignments were also performed to compare the TIR domain of
PG0382 with those of mammalian TLR adaptor proteins (e.g. MYD88, MAL, TRAM, TRIF, and
SARM). The accession numbers of the mammalian TLRs are presented in Appendix Table A1.
The sequence alignments revealed that the PG0382 TIR domain shares highest sequence
identity with MAL (15.2%), and lowest identity with TRIF (9.7%) (Table 5.5). The TIR domains
of TLR adaptor proteins have sequence identities between 25.5% (MYD88 and MAL) and 7.8%
(MYD88 and TRIF). A multiple sequence alignment was also conducted to compare the TIR
domain with adaptor protein TIR domains (Fig. 5.6). PG0382 box 1 shares some conserved
amino acid residues with the TLR adaptor proteins. Like MAL and MYD88, PG0382 contains the
conserved aspartate residue (Asp355). The amino acid residues following Asp355 in PG0382
are less well conserved with the adaptor proteins; however, the substituted amino acids share
similar chemical properties. For instance, PG0382 Iso356, MAL Val88 and MYD88 Ala163 are
aliphatic, hydrophobic amino acids. PG0382 and TLR adaptor proteins (except MAL), contain
the conserved isoleucine residue in the box 1 sequence motif. The box 2 sequence motif of
F/YDAFISY box 1
RDxxPG box 2
FW box 3
120
PG0382 was more similar to MAL and MYD88, than to TRAM, TRIF and SARM. Like MAL and
MYD88, PG0382 contains the consensus aspartate (Asp390) and glycine (Gly394) residue. By
contrast, the box 2 sequence motif in TRAM, TRIF and SARM are less well conserved. TRAM and
TRIF only share the conserved glycine (Gly118 and Gly435, respectively) with PG0382. SARM
lacks both the proline and glycine residues important for mediating TIR-TIR interactions. Like
PG0382, the box 3 sequence motif in TLR adaptor proteins is also poorly defined. In conclusion,
the PG0382 TIR domain contains a number of amino acid residues that are identical to the TIR
domain consensus sequence in box 1 and box 2. In addition, the TIR domain of PG0382 was also
found to be more similar to the TIR domain of TLR adaptor proteins than TLRs.
Table 5.5 Amino acid sequence identity (%) between PG0382 TIR domain and TIR domains of TLR protein adaptors.
PG0382 MYD88 MAL TRIF TRAM SARM
MYD88 10.9 - 25.5 7.8 12.6 14.0 MAL 15.2 25.5 - 12.8 22.9 12.7 TRIF 9.7 7.8 12.8 - 20.9 10.4
TRAM 15.0 12.6 22.9 20.9 - 9.2 SARM 14.8 14.0 12.7 10.4 9.2 -
Figure 5.6 Multiple sequence alignment of PG0382 TIR domain with TLR adaptor proteins TIR domains. The consensus sequence for box 1 and box 2 are as indicated. The x in box 2 denotes any hydrophobic amino acid.
F/YDAFISY box 1
RDxxPG box 2
121
Comparison of PG0382 with bacterial Tcps
The TIR domain of PG0382 was also compared with the TIR domain of previously characterised
bacterial Tcps (Table 5.6). The accession numbers of the bacterial Tcps are presented in
Appendix Table A2. Pairwise sequence alignment revealed that PG0382 TIR domain shares
highest sequence identity with the TIR domain of P. dentrificans PdTIR (25.3%), and lowest
sequence identity with E. coli CFT037 TcpC (15.1%). The bacterial Tcps have sequence
identities between 51.9% (PdTIR and TcpB) and 12.4 % (TcpC and YpTdp). Multiple sequence
alignment of bacterial Tcp TIR domains showed that box 1 of bacterial Tcps were relatively
similar between one another, and contained amino acid residues equivalent to the consensus
sequence (Fig. 5.7). The bacterial Tcps all shared the conserved isoleucine (except TirS, which is
substituted with leucine) and serine residues in box 1. The bacterial Tcps also have a histidine
residue in box 1, while PG0382 contains a phenylalanine (Phe360) at that position. The
conserved box 2 sequence motif of bacterial Tcps differs from the mammalian TIR domain
consensus sequence. Like PG0382, the conserved proline residue in box 2 is absent in TcpB,
TcpC and TirS. The conserved glycine residue in PG0382 is also present in the bacterial Tcps,
except YpTdp. Box 3 is not present in the bacterial Tcps. Taken together, these analyses indicate
that the PG0382 TIR domain has features similar to the TIR domain of other bacterial Tcps.
Table 5.6 Amino acid sequence identity (%) between PG0382 TIR domain and bacterial Tcp TIR domains.
PG0382 TcpB TcpC PdTIR YpTdp TirS
TcpB 19.2 - 51.1 51.9 17.3 29.3 TcpC 15.1 51.1 - 39.5 12.4 29.5 PdTIR 25.3 51.9 39.5 - 12.8 28.2 YpTdp 18.0 17.3 12.4 12.8 - 14.6
TirS 17.5 29.3 29.5 28.2 14.6 -
122
Figure 5.7 Multiple sequence alignment of PG0382 TIR domain with TIR domains of known bacterial TIR domain-containing proteins. The consensus sequence for box 1 and box 2 are as indicated. The x in box 2 denotes any hydrophobic amino acid.
F/YDAFISY box 1
RDxxPG box 2
123
Structural prediction of PG0382
Mammalian TIR domains are structurally defined by a flavodoxin-fold comprised of five central
-sheets and five -helices (Xu et al., 2000). In silico analysis was performed to determine
whether the putative PG0382 TIR domain might adopt a similar structural conformation.
FUGUE analysis (http://mizuguchilab.org/fugue/prfsearch.html), which aligns sequence-
structure pairs to generate a list of structural homologs (Shi et al., 2001), was performed to first
identify potential PG0382 structural homologs for tertiary structure modelling. This analysis
revealed that PG0382 shares greatest structural homology with the TIR domain of PdTIR
(Table 5.7). The TIR domains of human TLR1, TLR2, and MAL were also identified to be PG0382
structural homologs (Table 5.7). Interestingly, TIR domains in plants (e.g. Linum usitatissimum
and Arabidopsis thaliana) were also identified as potential structural homologs of the PG0382
TIR domain (Table 5.7).
Table 5.7 Identification of PG0382 TIR domain structural homologs using FUGUE.
Protein PDB code Species Z-score
PdTIR 3H16 Paracoccus dentrificans 11.81
TLR1 TIR domain 1FYV Homo sapiens 9.85
MAL variant TIR domain 3UB4 Homo sapiens 8.43
L6 TIR domain 3OZI Linum usitatissimum 7.41
TLR2 variant TIR domain 1FYX Homo sapiens 7.07
RRS1 TIR domain 4C6S Arabidopsis thaliana 5.21
Nuclear receptor coactivator 5 binding domain 1V95 Homo sapiens 4.81
IL-17R SEFIR domain 3VBC Mus musculus 4.74
Homology modelling with SWISS-MODEL (https://swissmodel.expasy.org/) was performed
next to produce a structural model based on protein sequence and model protein structures
(Schwede et al., 2003). Given that PdTIR (PDB 3H16) was identified as a potential structural
homolog for the TIR domain of PG0382 (Table 5.7), it was used as the template structure for
modelling (Fig. 5.8A). As shown in Figure 5.8C, the model for PG0382 was predicted to consist of
four -helices surrounding three -sheets. PG0382 was also modelled using the TIR domain of
TLR1 (PDB 1FYV) as the template, for comparison (Fig. 5.8B). The resulting PG0382 homology
124
model generated suggested that the PG0382 TIR domain may form a similar / conformation,
as when PdTIR was used as the structural template (Fig. 5.8D). The PdTIR TIR domain contains
-helices that are more compact when compared to TLR1 TIR domain, but the overall structure
of the PdTIR and TLR1 TIR domains are similar (Fig. 5.8A-B). In summary, the homology models
suggest that the annotated TIR domain of PG0382 may adopt a TIR domain-like conformation.
Figure 5.8 PG0382 structural prediction. Three dimensional models of the TIR domains of (A) PdTIR, (B) PG0382 modelled using PdTIR as a template, (C) TLR1, and (D) PG0382 modelled using TLR1 as a template. Box 1, box 2 and box 3 are as indicated by the yellow, green and pink colouring, respectively.
A
PdTIR TIR domain
PG0382 (TLR1 TIR domain template)
D
TLR1 TIR domain
B
PG0382 (PdTIR template)
C
125
Expression of PG0382 in HEK293T cells
To better understand the biochemical properties of PG0382, mammalian expression plasmids
encoding PG0382 and a mutant thereof lacking the TIR-like domain (i.e. PG0382ΔTD) were
constructed (Fig. 5.9A). An expression plasmid encoding the TIR-like domain of PG0382
(i.e. PG0382TD) was created by another student (Ben Huang) in the laboratory. Briefly, the
coding sequences for PG0382 and PG0382ΔTD were amplified by PCR, using P. gingivalis W50
genomic DNA as the template. The PCR products generated were subsequently cloned into the
mammalian expression vector, pEF-V5, such that the expressed proteins contain an N-terminal
V5 epitope tag. Following transformation into E. coli DH5α bacteria, positive transformants
were identified by restriction endonuclease digestion (Fig. 5.9B-C). The absence of mutations in
the cloned cDNA sequences was confirmed by DNA sequencing (performed at The Centre for
Translational Pathology, The University of Melbourne).
126
Figure 5.9 Construction of pEF-V5-PG0382 and pEF-V5-PG0382ΔTD expression vectors. (A) Schematic representation of PG0382, PG0382ΔTD and PG0382TD. (B-C) Restriction endonuclease (HindIII) analyses of (B) pEF-V5-PG0382 and (C) pEF-V5-PG0382ΔTD. the bolded clones contain plasmid inserts with the correct orientation. The positions of DNA standards are as indicated.
PG0382ΔTD
N-terminus (coiled-coil motif)
342
TIR domain
490
PG0382
PG0382TD
N-terminus (coiled-coil motif)
342
342
TIR domain
490 p
EF-V
5
Size (kb)
3.0
10
1.5
2.0
4.0
pEF
-V5
3.0
1.0
2.0
8.0
Size (kb)
A
B C
0.2
1 k
b N
EB D
NA
lad
der
1 k
b B
iolin
e D
NA
lad
der
NTPase
NTPase
8 52
8 52
Clo
ne
1
Clo
ne
2
Clo
ne
3
Clo
ne
4
Clo
ne
5
Clo
ne
1
Clo
ne
2
Clo
ne
3
Clo
ne
4
127
The expression of the proteins (PG0382, PG0382ΔTD, and PG0382TD) was tested by transient
transfection of HEK293T cells, and subsequently subjecting lysates of the cells to Western blot
analysis with an anti-V5 antibody. Other studies have demonstrated a propensity for bacterial
Tcps to form dimers (Rana et al., 2011; Fekonja et al., 2012). Coiled-coil domains can drive
oligomerisation of proteins (Low et al., 2007), and thus can affect protein solubility. PG0382 is
predicted to contain a coiled-coil motif in the N-terminal half of the protein, and therefore both
the detergent-soluble and detergent-insoluble fractions of the cells were analysed. Briefly, the
cells were lysed with buffer containing 1% NP-40 (non-ionic detergent) and centrifuged to
pellet insoluble material. The supernatant (representing the detergent-soluble fraction) was
retained, whilst the pelleted material was solubilised with LDS sample buffer (representing the
detergent-insoluble fraction). As shown in Figure 5.10, PG0382 and PG0382ΔTD were detected
in both the detergent-soluble and detergent-insoluble fractions. Higher molecular weight bands
were also detected for PG0382ΔTD (Fig. 5.10). Contrastingly, PG0382TD was found exclusively
in the detergent-soluble fraction (Fig. 5.10), suggesting that the N-terminus of PG0382 may
strongly influence its solubility under these experimental conditions. Full-length PG0382 was
expressed at lower levels in comparison to PG0382ΔTD and PG0382TD (Fig. 5.10).
128
Figure 5.10 Ectopic expression of V5-PG0382, V5-PG0382ΔTD, and V5-PG0382TD in HEK293T cells. HEK293T cells were transiently transfected with vectors expressing the indicated proteins, and lysed 24 h post-transfection. The detergent-soluble and detergent-insoluble fractions were subjected to Western blotting with an anti-V5 and anti-HSP90 (loading control) antibodies. The positions of molecular mass standards (in kDa) are as indicated. The data presented are representative of three independent experiments.
Detergent-soluble Detergent-insoluble
Anti-HSP90
60
50
40
30
20
15
Anti-V5
80
129
Given the differences in their detergent-solubilities, PG0382, PG0382ΔTD, and PG0382TD were
also investigated by immunofluorescence confocal microscopy; this also enabled examination of
their subcellular localisation. PG0382 and PG0382ΔTD were detected in the cytoplasm, and
appeared to form localised punctate bodies (Fig. 5.11). By contrast, PG0382TD exhibited
dispersed cytoplasmic staining (Fig. 5.11). Collectively, the findings presented in Fig. 5.10 and
Fig. 5.11 suggest that the structural elements in the N-terminal half of PG0382 may be
important in dictating the solubility and subcellular localisation of PG0382.
Figure 5.11 Subcellular localisation of ectopically expressed PG0382, PG0382ΔTD, and PG0382TD in HEK293T cells. HEK293T cells were transiently transfected with vectors expressing the indicated proteins. The cells were fixed 24 h-post transfection, and stained with an anti-V5 antibody (red staining); nuclei were counterstained with DAPI (blue staining). The data presented are representative of three independent experiments. Scale bar = 10 µm.
Merged Anti-V5 DAPI
V5-PG0382
V5-PG0382TD
V5-PG0382ΔTD
130
PG0382 expression causes the loss of MAL and MYD88 in HEK293T cells
Bacterial Tcps have previously been shown to inhibit TLR signalling by interacting with MAL
and MYD88 (Sengupta et al., 2010). Therefore, the effects of PG0382 on MAL and MYD88 were
investigated. FLAG-tagged murine MAL and FLAG-tagged human MYD88 were transiently
co-expressed with PG0382, PG0382ΔTD, or PG0382TD, in HEK293T cells. MAL was exclusively
detected in the detergent-soluble fraction (Fig. 5.12A). However, MAL was not detected, in
either the detergent-soluble or detergent-insoluble fraction, when co-expressed with PG0382
(Fig. 5.12A-B). MAL was not similarly affected when co-expressed with either PG0382ΔTD or
PG0382TD (Fig. 5.12A-B). The effect of PG0382 on MYD88 was also investigated. Notably,
MYD88 was exclusively detected in the detergent-insoluble fraction (Fig. 5.12C). MYD88 levels
were greatly reduced when co-expressed with PG0382, whilst PG0382ΔTD and PG0382TD did
not affect MYD88 levels (Fig. 5.12C-D). The mRNA expression levels of MAL and MYD88 were
also measured by Real-time PCR to determine whether PG0382 might have affected the
transcription of the MAL and MYD88 expression plasmids. Because murine MAL was used in
this study, the level of overexpressed MAL was not directly comparable with endogenous MAL
(Fig. 5.13A). MYD88 mRNA levels were increased by ~100-fold when transfected with the
MYD88 expression plasmid (Fig. 5.13B). As shown in Figure 5.13A-B, co-expression with
PG0382 did not cause a reduction in the mRNA levels of ectopically expressed MAL or MYD88.
These data suggest that PG0382 causes the loss of MAL and MYD88 protein when co-expressed
in HEK293T cells.
131
Figure 5.12 Effects of PG0382 on MAL and MYD88 expression. HEK293T cells were transiently transfected vectors expressing the indicated proteins, and were lysed 24 h-post transfection. (A and C) The detergent-soluble and detergent-insoluble fractions were subjected to Western blotting. The positions of molecular mass standards (in kDa) are as indicated. The data presented are representative of three independent experiments. (B and D). The protein levels of MAL from (A) and MYD88 from (C) were quantified by densitometric analysis. (B) MAL and (D) MYD88 transfected alone were given an arbitrary value of 100%. All data are presented as the mean ± SEM (*=p<0.05, ***=p<0.001).
Anti-V5
Anti-HSP90
Anti-FLAG
FLAG-MYD88 + + + + - + + + + -
- + - - - - + - - - - - + - - - - + - -
- - - + - - - - + -
Anti-HSP90
Anti-V5
Anti-FLAG
FLAG-MAL
V5-PG0382
V5-PG0382 ΔTD
V5-PG0382 TD
Detergent-Soluble Detergent-Insoluble
V5-PG0382
V5-PG0382 ΔTD
V5-PG0382 TD
50
40
30
20
50
40
30
20
A
C
B
D
+ + + + - + + + + -
- + - - - - + - - - - - + - - - - + - -
- - - + - - - - + -
80
80
30
30
Detergent-Soluble Detergent-Insoluble
132
Figure 5.13 Effects of PG0382 on MAL and MYD88 mRNA levels in HEK293T cells. HEK293T cells were transiently transfected with the vectors expressing the indicated proteins for 24 h. (A) MAL and (B) MYD88 mRNA levels were measured by Real-Time PCR, and are shown as relative to HPRT (endogenous control gene) (n=3). All data are presented as the mean ± SEM (ND=not detected, *=p<0.05).
Given the findings above, the effects of PG0382 on MAL and MYD88 were further investigated in
titration experiments, where increasing amounts of the PG0382 expression plasmid were
co-transfected with the MAL or MYD88 expression plasmids. MAL protein was reduced by
approximately 90%, even at the lowest PG0382 plasmid concentration tested (Fig. 5.14A-B).
MYD88 was less susceptible to the effects of PG0382, in comparison to MAL. MYD88 protein
expression was reduced by approximately 50% when co-transfected with the lowest amount of
PG0382 plasmid, and was reduced by approximately 90% at the highest amount of PG0382
plasmid transfected (Fig. 5.14C-D). Taken together, these findings suggest that the MAL is more
susceptible to the effects of PG0382 than MYD88.
A B
ND
133
Figure 5.14 Concentration-dependent effects of PG0382 on MAL and MYD88 protein expression. HEK293T cells were transiently transfected with vectors expressing the indicated proteins, and were lysed 24 h-post transfection. (A and C) The detergent-soluble and detergent-insoluble fractions were subjected to Western blotting. The detergent-soluble fraction was probed for MAL and HSP90, whilst the detergent-insoluble fraction was probed for MYD88 and PG0382. The positions of molecular mass standards (in kDa) are as indicated. The data presented are representative of three independent experiments. (B and D) The protein levels of MAL from (A) and MYD88 from (C) were quantified by densitometric analysis. (B) MAL and (D) MYD88 transfected alone were given an arbitrary value of 100%. The data are presented as the mean ± SEM (***=p<0.001).
MYD88 (0.1 µg)
PG0382 (µg)
MYD88
1
+
0
+
0.1
+
0.4
+
0
-
PG0382
HSP90
MAL (0.1 µg)
PG0382 (µg) 1
+
0
+
0.1
+
0.4
+
0
-
MAL
PG0382
HSP90
A
C
B
D
30
30
50
50
80
80
134
Lack of complex formation between PG0382TD and MAL
The ability of PG0382 to form a stable complex with MAL was next investigated. Its ability to
likewise interact with MYD88 was not investigated because denaturing conditions were
required to recover MYD88 from cell lysates. Additionally, because PG0382 causes the loss of
MAL protein, PG0382TD was used to determine whether the TIR domain of PG0382 can interact
with MAL. Briefly, FLAG-tagged murine MAL was co-expressed with V5-PG0382 in HEK293T
cells, and lysates of the cells were subsequently subjected to immunoprecipitation with an
anti-FLAG antibody. As shown in Figure 5.15, FLAG-MAL was successfully immunoprecipitated
from the detergent-soluble cell lysates. However, V5-PG0382TD was not detected in the
anti-FLAG immunoprecipitates. PG0382TD was detected in the detergent-soluble cell lysates
(Fig. 5.15), indicating that the protein had been successfully expressed. This suggests that the
absence of complex formation between the PG0382TD and MAL is unlikely to be due to poor
expression of PG0382TD. Therefore, it was concluded that the TIR domain of PG0382 does not
form a stable complex with MAL.
Figure 5.15 Analysis of PG0382TD and MAL interaction by co-immunoprecipitation. HEK293T cells were transiently transfected with vectors expressing the indicated proteins, and lysed 24 h-post transfection. FLAG-MAL was immunoprecipitated from the cell lysates with anti-FLAG antibodies, followed by Western blotting with anti-V5 and anti-FLAG antibodies. The lysates (input) were subjected to Western blotting with an anti-V5 antibody. The positions of molecular mass standards (in kDa) are as indicated. The data is representative of three independent experiments.
Anti-V5
Input
Anti-FLAG
Anti-V5
V5-PG0382TD
FLAG-MAL - + +
FLAG-IP
- - +
20
20
30
135
Subcellular localisation of PG0382TD, MAL and MYD88
Complex formation by proteins might not be detectable by immunoprecipitation assay if the
proteins form weak or transient interactions. Therefore, immunofluorescence confocal
microscopy was also performed to investigate whether PG0382TD might co-localise or affects
the subcellular localisation of MAL and/or MYD88. When expressed in HEK293T cells, MAL
appeared to be largely localised towards the cell periphery (Fig. 5.16A). When co-expressed
with PG0382TD, MAL remained localised around the cell periphery, and did not co-localise with
PG0382TD (Fig. 5.16A). In contrast to MAL, MYD88 was observed to form distinct foci
throughout the cytoplasm (Fig. 5.16B), potentially consistent with MYD88 forming detergent-
resistant homo-oligomers. The subcellular localisation of MYD88 was not affected by the
co-expression of PG0382TD, and nor did MYD88 co-localise with PG0382TD (Fig. 5.15B). Taken
together, these data suggest that PG0382 does not co-localise with MAL or MYD88, or affect
their subcellular localisation.
136
Figure 5.16 Co-localisation of PG0382TD with MAL and MYD88 by immunofluorescence. HEK293T cells were transiently transfected with vectors expressing the indicated proteins. The cells were fixed 24h-post transfection, and were stained with an anti-FLAG antibody (green staining), and an anti-V5 antibody (red staining); nuclei were counterstained with DAPI (blue staining). The data presented are representative of three independent experiments. Scale bar = 10 µm.
A Merged Anti-V5 Anti-FLAG
FLAG-MAL
FLAG-MAL and
V5-PG0382TD
B
FLAG-MYD88
FLAG-MYD88 and
V5-PG0382TD
Merged Anti-V5 Anti-FLAG
137
5.3 Discussion
TLRs are integral to the host innate immune system where they play important roles in the
recognition of pathogens and initiating the ensuing immune response. Upon recognition of their
cognate ligand, TLRs trigger a series of intracellular signalling cascades that culminate in the
stimulation of host inflammation. Notably, the intracellular TIR domains of TLRs are critical for
facilitating downstream signalling because they recruit TIR domain-containing signalling
adaptor proteins, such as MAL and MYD88, into the receptor complex. Given the critical roles of
TLRs, pathogens have evolved mechanisms to suppress TLR signalling. Bacterial Tcps have been
proposed to be a subversion mechanism to block the interaction of TLRs with downstream
adaptor proteins. Indeed, in vivo studies have demonstrated important roles for Tcps in
bacterial virulence and dissemination (Cirl et al., 2008; Newman et al., 2006; Radhakrishnan et
al., 2009). Tcp(s) have yet to be defined in P. gingivalis. Thus, potential P. gingivalis Tcps were
identified and characterised in this Chapter.
The InterPro database integrates amino acid “signatures” representing protein domains from
multiple source databases, such as Pfam and Prosite, into a single consortium (Hunter et al.,
2009). The database can therefore provide a global overview of identified and annotated
protein domains across various species. This approach led to the identification of eleven
putative Tcps, across nine different strains, in P. gingivalis. The putative Tcps were classified
into two distinct groups, which were denoted, Group A and Group B. Proteins in Group A had
low overall amino acid sequence conservation (5-20%), and shared approximately 20%
sequence identity between TIR domains. For comparison, mammalian TIR domains typically
share around 20-30% sequence conservation (Xu et al., 2000). Therefore, it was not surprising
that the TIR domains of Group A Tcps exhibited relatively low sequence conservation. The low
degree of sequence conservation in TIR domains of TLRs and adaptor proteins is proposed to
provide the domains with sufficient structural diversity to enable specific TIR-TIR interactions
(Xu et al., 2000). In contrast to the Tcps in Group A, Group B Tcps shared a higher level of amino
acid sequence conservation (60-80%), and the TIR domains have sequence identities greater
than 70%. In addition, NCBI Conserved Domain searches revealed that Group B Tcps also have
similar domain arrangements, suggesting that they may have similar functions. Cluster analysis
suggests that the distribution of TIR domains in bacteria occurred through horizontal gene
transfer (Zhang et al., 2011), however the relationship between the two groups of P. gingivalis
Tcps is unclear. Further studies will be required to determine whether the proteins from the
two groups are involved in similar biological functions.
Despite having low overall amino acid sequence conservation, TIR domains contain conserved
sequence motifs, namely box 1, box 2 and box 3, which have been shown to be important for
138
TLR signalling. Multiple sequence alignment analysis revealed that PG0382 shares conserved
amino acid residues with TLRs, TLR adaptor proteins, and bacterial Tcps in box 1. Box 2 of
PG0382 was found to be more similar to TLR adaptor proteins (e.g. MAL and MYD88) and
bacterial Tcps than TLRs. PG0382 lacks box 3, which is also absent in TLR adaptor proteins and
bacterial Tcps. The role of box 2 is best understood and has been shown to be important for
mediating heterotypic TIR-TIR interaction (Poltorak et al., 1998; Slack et al., 2000). PG0382
lacks the invariant proline residue found in box 2. Mutation of the proline residue rendered
TLR4 hyporesponsive to LPS stimulation (Poltorak et al., 1998). However, the mutation did not
prevent TLR4 from interacting with MAL or MYD88 (Dunne et al., 2003). This contrasts with the
effect of mutating the corresponding proline residue in TLR2, which inhibited MYD88 binding
(Xu et al., 2000). This implies that the conserved proline residue may be important for surface
properties (e.g. surface charge) required for some TIR-TIR interactions, but not others. PG0382
contains the consensus box 2 glycine residue conserved in TLRs, adaptor proteins and bacterial
Tcps. Mutation of the conserved glycine residue (Gly158 to Ala) in TcpB impaired the inhibition
of TLR2-mediated NF-B activation (Alaidarous et al., 2014; Radhakrishnan et al., 2009).
Similarly, the conserved glycine was also found to be important for SARM-mediated immune
suppression (Carlsson et al., 2016). Similar mutagenesis studies will be required to determine
the importance of the conserved box 2 residues (e.g. Gly394) for PG0382 function. Nonetheless,
PG0382 contains TIR domain sequence features similar to TLR adaptor proteins and previously
characterised bacterial Tcps.
Structurally, TIR domains are comprised of five central -sheets and five -helices, described as
a flavadoxin fold (Xu et al., 2000). Structural homology modelling suggested that the annotated
TIR domain in PG0382 might adopt a similar flavadoxin fold, with central -sheets surrounded
by -helices. The prediction of coiled-coil motifs within the PG0382 TIR domain
(Gly350-Glu400, and Thr450-Gln490) is consistent with the ability of PG0382 TIR domain to
form -helices, because coiled-coil motifs, like -helices, form secondary helical structures. In
addition, box 1 and box 2 in PG0382 were predicted to adopt similar secondary conformations
compared to resolved TIR domain crystal structures. However, the generation of a reliable
protein structure model with known structural templates typically requires >30% amino acid
sequence identity (Sánchez and Šali, 1997). The relatively low sequence conservation between
the TIR domain of PG0382 and structural homologs identified (e.g. PdTIR TIR domain and TLR1
TIR domain) thus posed as a restriction on generating an accurate structural prediction of
PG0382. Moreover, the models are limited by the fact that they are built around existing
structures and hence are restricted by computational algorithms. Therefore, laboratory-based
139
experimental approaches (e.g. circular dichroism and x-ray crystallography) with purified
PG0382 will likely be required to provide further insight into the structure of PG0382.
The COILS prediction analysis suggested that PG0382 may contain a coiled-coil motif within the
N-terminal half (Gln200-Lys250) of the protein. Other bacterial Tcps that have been studied for
their ability to suppress TLR-mediated immune responses, including TcpC, TcpB, and YpTdp,
also possess coiled-coil motifs (Alaidarous et al., 2014; Cirl et al., 2008; Rana et al., 2011). The
primary role of a coiled-coil domain is to mediate protein oligomerisation (Burkhard et al.,
2001). For example, purified full-length PdTIR, as well as its isolated N-terminal coiled-coil
domain, have been shown to exist in monomers and oligomers (Low et al., 2007). Therefore, it is
tempting to speculate that the predicted coiled-coil motif in PG0382 may likewise promote its
oligomerisation. Notably, the detection of PG0382 and PG0382ΔTD in the detergent-insoluble
fraction of transfected HEK293T cells, and their subcellular distribution as punctate bodies, is
potentially consistent with PG0382 forming dimers/oligomers. TirS from S. aureus has also been
shown to be highly insoluble (Patot et al., 2017). Interestingly, the addition of an artificial
coiled-coil motif to the MYD88 TIR domain was shown to inhibit TLR4 activation in vitro
(Fekonja et al., 2012). The addition of a coiled-coil motif was proposed to not only promote
MYD88 TIR domain dimerisation, and thereby provide a larger interface to interact with
dimerised TLRs, but also to sterically hinder the recruitment of downstream TLR adaptor
proteins (Fekonja et al., 2012). Therefore, bacterial Tcps may have adopted coiled-coil motifs to
potentiate the inhibition of TLR signalling. Further biochemical studies, such as size exclusion
chromatography and sedimentation equilibrium analysis, will be required to gain direct insight
into the ability of PG0382 to form dimers/oligomers.
Other studies have demonstrated the ability of bacterial Tcps to interact with MAL and MYD88
as a mechanism to inhibit NF-B signalling (Alaidarous et al., 2014; Askarian et al., 2014; Cirl et
al., 2008; Newman et al., 2006; Sengupta et al., 2010). The results presented in this study
revealed that co-expression with PG0382 in HEK293T cells significantly reduced the protein
expression of MAL and MYD88. This effect likely occurred at the protein level, because PG0382
did not similarly affect MAL or MYD88 mRNA expression levels. The interaction of TcpB with
MAL was shown to promote MAL ubiquitination, leading to its rapid proteasomal degradation
(Sengupta et al., 2010). The mechanism whereby PG0382 causes MAL and MYD88 protein levels
to be markedly reduced is unclear at this time. Co-immunoprecipitation and
immunofluorescence confocal microscopy experiments suggested that the PG0382 TIR domain
does not interact with MAL or MYD88. Chemical cross-linking reagents (e.g. disuccinimidyl
suberate) can be used in co-immunoprecipitation assays to “capture” weak/transient
interactions, and thus could be used to further investigate the ability of PG0382 to bind MAL or
140
MYD88. However, it is important to appreciate that cross-linking reagents can potentially result
in the “capturing” of artefactual protein complexes that do not form under native conditions. It
is also possible that other regions/domains of PG0382 might be required for its interaction with
MAL and MYD88. Therefore, further studies will be required to elucidate the effects of PG0382
on MAL and MYD88.
In summary, the bioinformatics analyses performed in this Chapter identified eleven putative
P. gingivalis Tcps, which can be divided into two groups with distinctive characteristics. Further
bioinformatics analysis of P. gingivalis PG0382 revealed that its putative TIR domain contains
sequence features similar to TLR adaptor proteins and bacterial Tcps. In addition, homology
modelling suggests that PG0382 may adopt a TIR-like structure. Further characterisation by
transient expression in HEK293T cells and analysis by Western blotting and
immunofluorescence confocal microscopy showed that the N-terminus of PG0382 exhibits
properties consistent with it promoting oligomerisation. Significantly, the co-expression of
PG0382 reduced MAL (and MYD88) protein levels, but the PG0382 TIR domain does not appear
to directly interact with MAL. Therefore, further studies will be required to better understand
the effects of PG0382 on MAL and MYD88.
141
Investigation of PG0382 and host inflammation
142
6.1 Introduction
The oral epithelium expresses TLRs (e.g. TLR2) that enable the detection of potential pathogens
and stimulation of host inflammation. The activation of TLR signalling in oral epithelial cells
leads to the production of inflammatory mediators, including cytokines and chemokines, which
promote the recruitment of immune cells. Neutrophils and inflammatory monocytes are
typically the first immune cells that respond to infection by pathogens. Collectively, neutrophils
and inflammatory monocytes mount a robust antimicrobial response. They also activate and
direct adaptive immune cells (e.g. lymphocytes) to generate an appropriately tailored immune
response. Together, the two arms of the immune system collaborate to efficiently eliminate
potential pathogens.
Bacterial TIR domain-containing proteins (Tcps) have been proposed to subvert the host
immune response by suppressing TLR signalling. Several in vitro studies have demonstrated the
ability of bacterial Tcps to inhibit the activation of the transcription factor NF-B (Cirl et al.,
2008; Newman et al., 2006; Radhakrishnan et al., 2009). Importantly, blockade of NF-B activity
potentially translates to reduced inflammatory cytokine production. Consistently, macrophages
mounted enhanced cytokine responses when infected with a TcpC-deficient E. coli CFT037
mutant, in comparison to infection with wildtype E. coli CFT037(Cirl et al., 2008). Moreover,
in vivo studies have demonstrated a role for bacterial Tcps in virulence. For example, mice
infected with a B. melitensis TcpB-deficient mutant had prolonged survival, when compared to
mice infected with wildtype B. melitensis (Salcedo et al., 2013). TcpC was also shown to be
important for the survival of E. coli CFT037 in mice, and induction of kidney pathology in a
mouse model of urinary tract infection (Cirl et al., 2008). In this Chapter, in vitro and in vivo
systems were used to investigate a role for the putative P. gingivalis Tcp, PG0382, in modulating
the host inflammatory response.
6.2 Results
Generation of an isogenic P. gingivalis PG0382-deficient mutant
To study the function of PG0382, an isogenic P. gingivalis PG0382-deficient mutant (ΔPG0382)
was created (Fig. 6.1). Purified genomic DNA from P. gingivalis W50 was used as the template to
generate PCR products from the 5’ and 3’ intergenic regions of the PG0382 gene (Fig. 6.2A).
Similarly, PCR was also used to amplify the erythromycin-resistance gene (ermF) from the
pAL30 plasmid (Dashper et al., 2009). The PCR reactions were conducted with primers
containing complementary sequence overhangs to subsequently enable a single, linear PCR
product to be generated by splice-overlap extension PCR (SOE PCR) (Fig. 6.1). The resulting SOE
PCR product was comprised of the ermF gene, flanked by 5’ and 3’ intergenic regions of the
143
PG0382 gene (Fig. 6.2B). The SOE PCR product was then introduced into P. gingivalis W50 by
electroporation. Successful integration of the SOE PCR product into the P. gingivalis genome
occurs through homologous recombination with the 5’ and 3’ intergenic regions of the PG0382
gene. Transformed P. gingivalis were selected on blood agar plates supplemented with
erythromycin. Genomic DNA from positive transformants was then purified, and PCR primers
specific for the 5’ and 3’ intergenic regions of the PG0382 gene were used to amplify the
corresponding region of the P. gingivalis genome for DNA sequencing to confirm that the
PG0382 gene had been replaced with the ermF gene (Fig. 6.2C).
144
Figure 6.1 Strategy for the generation of an isogenic P. gingivalis PG0382-deficient mutant.
PG0382 5’ intergenic
region
3’ intergenic region ermF
ermF
ermF 5’ intergenic
region 3’ intergenic
region
PG0382
PCR products
Splice Overlap Extension PCR
5’ intergenic region
3’ intergenic region
ermF 5’ intergenic
region 3’ intergenic
region
Electroporation
PG0382_IG5 PG0382_IG3 ermF_PG0382
Selection
Homologous recombination
P. gingivalis W50 genomic DNA pAL30
145
Figure 6.2 Gel electrophoresis analysis of PCR products. (A-B) Gel electrophoresis analysis of the indicated PCR products. (C) Genomic DNA from positive P. gingivalis transformants were purified and used as a template for PCR amplification. The PCR products were then analysed by gel electrophoresis. Bolded clones contain ermF integrated into the positive P. gingivalis transformants. The positions of DNA standards are as indicated.
1 k
b la
dd
er
PG
038
2 S
OE
pro
du
ct
500
300
1000
2000
1000
250/253
A B
Wild
typ
e
500
1000
2000
Size (bp)
4000
Clo
ne
1
Clo
ne
3
Clo
ne
4
Clo
ne
2
1 k
b la
dd
er
C
500
4000
Size (bp) Size (bp) 10
0 b
p la
dd
er
PG
038
2_I
G5
erm
F_P
G0
38
2
PG
03
82
_IG
3
250/253
146
Phenotypic characterisation of P. gingivalis ΔPG0382
P. gingivalis ΔPG0382 growth properties were first characterised to ensure that the deletion of
PG0382 does not affect P. gingivalis phenotype. The mean generation time of P. gingivalis
ΔPG0382 was determined to assess whether the absence of PG0382 might affect the growth of
the bacterium. The mean generation time for P. gingivalis ΔPG0382 was 3.3±0.1 h, which was
comparable to the mean generation time of 3.0±0.2 h for wildtype P. gingivalis (Fig. 6.3). The
latter finding is consistent with published studies, which also established that wildtype
P. gingivalis has a mean generation time of approximately 3 h (Aruni et al., 2011; Fletcher et al.,
1995). Therefore, the deletion of the PG0382 gene does not affect the growth rate of P. gingivalis,
at least under the in vitro conditions used.
Figure 6.3 Growth rates of wildtype P. gingivalis and P. gingivalis ΔPG0382. The mean generation time was calculated based on the rate of change of optical density (OD650) (n=3). The data are presented as the mean ± SEM.
P. gingivalis forms black-pigmented colonies when cultured on horse blood agar plates due to
the degradation of haemoglobin by the gingipain proteases (Smalley et al., 1998; Zambon et al.,
1981). The gingipain proteases are also major components of the electron-dense surface layer
(EDSL) of the outer membrane of P. gingivalis (Chen et al., 2011). The effects of deleting the
PG0382 gene on colony pigmentation and the EDSL were therefore examined. Like wildtype
P. gingivalis (Fig. 6.4A), P. gingivalis ΔPG0382 also grew as black-pigmented colonies when
cultured on horse blood agar plates (Fig. 6.4B). Cryo-electron microscopy was performed to
determine if the PG0382 gene is required for EDSL formation. Like wildtype P. gingivalis
(Fig. 6.4C), P. gingivalis ΔPG0382 had a distinct EDSL associated with the outer membrane
P. gingivalis Mean generation time (h) Wildtype 3.0±0.2 ΔPG0382 3.3±0.1
147
(Fig. 6.4D). Collectively, these results suggest that the absence of PG0382 does not affect these
defining characteristics of P. gingivalis.
Figure 6.4 Phenotypic characterisation of P. gingivalis ΔPG0382. Images of (A) wildtype P. gingivalis and (B) P. gingivalis ΔPG0382 cultured on 10% defibrinated horse blood agar plates for 7 days at 37°C in anaerobic conditions. (C-D) EDSL of (C) wildtype P. gingivalis and (D) P. gingivalis ΔPG0382 were examined by cryo-EM. EDSL = Electron surface dense layer, OM = outer membrane, and IM = inner membrane. Scale bar = 200 nm.
EDSL
OM IM
EDSL OM
IM
A B
C D
Wildtype P. gingivalis P. gingivalis ΔPG0382
Wildtype P. gingivalis P. gingivalis ΔPG0382
148
Gingipain protease activity of P. gingivalis ΔPG0382
The findings above suggested that deletion of the PG0382 gene does not affect the expression
and processing of the gingipain proteases. Nonetheless, this was also directly assessed by
measuring the Kgp and RgpA/B proteolytic activity of P. gingivalis ΔPG0382. P. gingivalis
ΔPG0382 was cultured to exponential growth phase, and both whole-cells and cell-free culture
supernatants were then harvested to assay Kgp and RgpA/B activity. The whole-cell Kgp activity
of P. gingivalis ΔPG0382 was comparable to wildtype P. gingivalis (Fig. 6.5A). Similarly, the
cell-free culture supernatants derived from P. gingivalis ΔPG0382 and wildtype P. gingivalis
exhibited comparable Kgp activity (Fig. 6.5B). Deletion of the PG0382 gene also did not affect
whole-cell RgpA/B activity (Fig. 6.5C) or RgpA/B activity in cell-free supernatants (Fig. 6.5D).
Therefore, it can be concluded that deletion of the PG0382 gene does not affect the cell-surface
attachment or secretion of Kgp or RgpA/B.
Figure 6.5 P. gingivalis gingipain protease activity. (A-B) Kgp and (C-D) RgpA/B proteolytic activity in (A and C) whole-cells and (B and D) cell-free culture supernatants from wildtype P. gingivalis and P. gingivalis ΔPG0382 were measured (n=3). All data are presented as the mean ± SEM.
A B
C D
149
Stimulation of inflammatory gene responses in oral epithelial cells by
P. gingivalis ΔPG0382
The bioinformatics and functional biochemical analyses in Chapter 5 suggest that PG0382
contains TIR-like properties comparable to bacterial Tcps that have been shown to suppress
TLR-mediated inflammatory cytokine responses. Therefore, a role for PG0382 in modulating
inflammatory cytokine responses in oral epithelial cells was investigated. OKF6 cells were
challenged with either wildtype P. gingivalis or P. gingivalis ΔPG0382, and changes in the mRNA
expression levels of inflammatory cytokines were then assessed. Fimbriae from P. gingivalis
have previously been shown to stimulate IL-8 expression in a TLR2-dependent manner in
human gingival epithelial cells (Asai et al., 2001; Eskan et al. 2007). Thus, the stimulation of IL-8
expression in OKF6 cells by P. gingivalis ΔPG0382 was investigated. As shown in Figure 6.6A, no
difference was found between P. gingivalis ΔPG0382 and wildtype P. gingivalis in the
stimulation of IL-8 gene expression. Recent work by our laboratory has demonstrated that TLR2
signalling in OKF6 cells also regulates the expression of IL-36G in response to P. gingivalis
(Huynh et al., 2016). Therefore, the ability of P. gingivalis ΔPG0382 to stimulate IL-36G
expression was investigated. The stimulation of IL-36G gene expression by P. gingivalis
ΔPG0382 was comparable to that by wildtype P. gingivalis (Fig 6.6B). Tumour necrosis factor
alpha-induced protein 3 (TNFAIP3) expression is also induced via TLR signalling, and functions
to negatively regulate NF-B signalling (Boone et al., 2004). Both P. gingivalis ΔPG0382 and
wildtype P. gingivalis were found to stimulate comparable TNFAIP3 responses (Fig. 6.6C). Taken
together, these data suggest that PG0382 does not suppress the stimulation of inflammatory
gene expression in oral epithelial cells (e.g. OKF6 cells) by P. gingivalis, although only a small
number of genes were examined.
Figure 6.6 Effects of P. gingivalis ΔPG0382 on inflammatory responses of oral epithelial cells. OKF6 cells were challenged with wildtype P. gingivalis or P. gingivalis ΔPG0382 at 100 MOI for 24 h. (A) IL-8, (B) IL-36G, and (C) TNFAIP3 mRNA levels were then measured by Real-Time PCR, and are shown as a fold change relative to mock-challenged cells (n=3). All data are presented as the mean ± SEM (* = p <0.05).
A B C
150
Stimulation of inflammatory gene responses in macrophages by
P. gingivalis ΔPG0382
Macrophages are critical mediators of host inflammation, and TLR2 and TLR4 have been shown
to be important regulators of the inflammatory responses of macrophages to P. gingivalis
(Holden et al., 2014; Papadopoulos et al., 2013). Therefore, the effect of PG0382 deletion on
P. gingivalis-inducible macrophage inflammatory responses was investigated with the murine
macrophage cell line, RAW 264.7 (Raschke et al., 1978). TNF and IL-6 levels closely correlate
with disease progression in chronic periodontitis, and are induced by P. gingivalis (Graves, 2008;
Graves and Cochran, 2003), therefore the stimulation of TNF and IL-6 by P. gingivalis were
examined. Wildtype P. gingivalis stimulated strong TNF gene expression (>10-fold) in
RAW 264.7 cells (Fig. 6.7A). P. gingivalis ΔPG0382 stimulated similar upregulation of TNF gene
expression, and accordingly, there was no difference in the stimulation of TNF expression
between wildtype P. gingivalis and P. gingivalis ΔPG0382 (Fig. 6.7A). As shown in Figure 6.7B,
wildtype P. gingivalis stimulated robust IL-6 gene expression (>150-fold) in RAW 264.7 cells,
albeit more slowly than TNF (Fig. 6.7A). P. gingivalis ΔPG0382 also stimulated strong IL-6 gene
expression (Fig. 6.7B). Although the IL-6 responses stimulated by wildtype P. gingivalis and
P. gingivalis ΔPG0382 were not statistically different, the trend for all experiments was the same,
whereby P. gingivalis ΔPG0382 stimulated weaker IL-6 responses. The expression of the
monocyte/macrophage chemokine, CCL2, was also found to be strongly induced by both
wildtype P. gingivalis and P. gingivalis ΔPG0382, with no statistically significant differences
between the responses (Fig. 6.7C). However, the trend for all experiments revealed a weaker
CCL2 response when the cells were challenged with P. gingivalis ΔPG0382 (Fig. 6.7C). The
anti-inflammatory cytokine, IL-10, plays an important role in limiting inflammatory responses
(Couper et al., 2008). IL-10 gene expression was induced (5 to 10-fold) by both wildtype
P. gingivalis and P. gingivalis ΔPG0382 (Fig. 6.7D). Notably, the IL-10 response 24 h-post
challenge was weaker with P. gingivalis ΔPG0382 (Fig. 6.7D). Overall, these data suggest that
PG0382 may affect the inflammatory responses elicited in macrophages by P. gingivalis.
151
Figure 6.7 Effects of P. gingivalis ΔPG0382 on inflammatory cytokine responses of macrophages. RAW 264.7 cells were challenged with wildtype P. gingivalis or P. gingivalis ΔPG0382 at 100 MOI for up to 24 h. (A) TNF, (B) IL-6, (C) CCL2, and (D) IL-10 mRNA levels were then measured by Real-Time PCR, and are shown as a fold change relative to time-matched and mock-challenged cells (n≥3). All data are presented as the mean ± SEM (* = p<0.05, ** = p<0.01, *** = p<0.001).
Innate immune response to P. gingivalis ΔPG0382 in mice
Given the limitations of in vitro systems for studying immunological responses, an in vivo
approach was also undertaken to investigate the potential role of PG0382 in modulating the
host immune response to P. gingivalis. Due to time limitations towards the end of my PhD
candidature, a pilot study was undertaken to determine whether PG0382 may affect the
recruitment of host immune cells in response to P. gingivalis. Briefly, wildtype P. gingivalis and
P. gingivalis ΔPG0382 were injected into the peritoneal cavity of BALB/c mice, and
intraperitoneal fluid was then harvested 6 or 24 h-post infection. The cells recovered in the
peritoneal lavage fluid were stained with fluorochrome-conjugated antibodies, and immune cell
populations of interest (e.g. macrophages, inflammatory monocytes, and neutrophils) were then
quantified by fluorescence-activated cell sorting (FACS) analysis. A representative FACS-gating
strategy is shown in Figure 6.8. Debris, cell doublets and aggregates were excluded from the
analyses. Fixable viability dye 700 (FVS700) is a cell-impermeable dye that reacts with free
A B
C D
152
amines in the cytoplasm of dead cells (Perfetto et al., 2006), and hence was used to exclude dead
cells from the analysis.
Figure 6.8 FACs Gating strategy for identifying innate immune cells. Single cells were analysed by excluding debris (top left panel) and cell aggregates (top centre panel). Live/dead discrimination was determined using Fixable Viability Dye 700 (top right panel). Cells were stained with fluorochrome conjugated mouse antibodies to identify F4/80+ CD86+ activated macrophages and F4/80lo Ly6C+
inflammatory monocytes. Ly6G+ Ly6Clo neutrophils were
identified by excluding macrophages and monocytes from the analyses.
FSC-A
SSC
-A
FSC-A
FSC
-H
FSC
-H
FVS700
F4/8
0
Ly6C
Ly6
G
Ly6C
Single cells Live cells
F4/8
0
CD86
Cells
Activated macrophages
(F4/80+ CD86
+)
Inflammatory monocytes
(F4/80lo
Ly6C+)
Neutrophils
(Ly6G+ Ly6C
lo)
153
Tissue-resident macrophages are critical immune sentinels; therefore, the proportion of
macrophages that were activated in response to intraperitoneal infection by P. gingivalis was
measured. F4/80 is a unique, mouse macrophage cell-surface marker (Austyn and Gordon, 1981)
and routinely used for the identification of macrophage populations by FACS analysis. Activated
macrophages can be identified based on increased expression levels of CD86, a costimulatory
molecule involved in T lymphocyte activation (Mosser and Edwards, 2008). Thus, cells that
were double-positive for F4/80 and CD86 (i.e. F4/80+, CD86+) were defined as activated
macrophages. The analysis showed that the numbers of activated macrophages from mice
infected with 5×106 wildtype P. gingivalis or P. gingivalis ΔPG0382 were similar to
sham-infected mice, at either 6 or 24 h post-infection (Fig. 6.9A-B). The infection of mice with a
larger inoculum of P. gingivalis (e.g. 5×107) did not appear to affect the numbers of activated
macrophages (Fig. 6.9A-B).
154
B
Figure 6.9 Activation of macrophages in response to P. gingivalis. BALB/c mice were infected intraperitoneally with wildtype P. gingivalis or P. gingivalis ΔPG0382. Mouse peritoneal fluid was harvested 24 h or 48 h-post injection and subjected to flow cytometric analysis. (A) F4/80+ CD86+ FACS plots from individual mice. NB: Due to the variation in response between mice, the FACs plots presented do not necessarily represent the “average” response. (B) Quantification of F4/80+ CD86+ activated macrophages. Each data point represents an individual mouse (n=5).
F4/8
0
F4/8
0
F4/8
0
F4/8
0
F4/8
0
CD86 CD86 CD86 CD86 CD86
F4/8
0
F4/8
0
F4/8
0
F4/8
0
F4/8
0
CD86 CD86 CD86 CD86 CD86
6 h
24 h
Sham
P. gingivalis 5×106
P. gingivalis 5×107
P. gingivalis ΔPG0382 5×107
P. gingivalis ΔPG0382 5×106
A
155
In addition to tissue-resident macrophages, inflammatory monocytes are recruited to sites of
infection to reinforce the host immune response. Inflammatory monocytes express low levels of
F4/80 but high levels of Ly6C (i.e. F4/80lo, Ly6C+) (Geissmann et al., 2003, 2008) and were
identified on that basis. For most mice, infection with wildtype P. gingivalis or P. gingivalis
ΔPG0382 stimulated an increase in the numbers of inflammatory monocytes by 6 h-post
infection (Fig. 6.10A-B). However, the increases in inflammatory monocytes were not
statistically significant due to the variation in responses between mice, specifically, the
responses by mice infected with 5×106 wildtype P. gingivalis. (The increases are statistically
significant if the two mice that did not respond to infection with 5×106 wildtype P. gingivalis are
excluded from the analysis). Infection with 5×107 wildtype P. gingivalis or P. gingivalis ΔPG0382
did not stimulate a further increase in the numbers of inflammatory monocytes (Fig. 6.10B),
suggesting that infection with 5×106 P. gingivalis induces a maximal response in this model.
Notably, the numbers of inflammatory monocytes recruited in response to P. gingivalis was not
affected by deletion of the PG0382 gene (Fig. 6.10B).
156
Figure 6.10 Recruitment of inflammatory monocytes in response to P. gingivalis. BALB/c mice were infected intraperitoneally with wildtype P. gingivalis or P. gingivalis ΔPG0382. Mouse peritoneal fluid was harvested 24 h or 48 h-post injection and subjected to flow cytometric analysis. (A) F4/80lo Ly6C+
FACS plots from individual mice. NB: Due to the variation in response between mice, the FACs plots presented do not necessarily represent the “average” response. (B) Quantification of F4/80lo Ly6C+ inflammatory monocytes. Each data point represents an individual mouse (n=5).
F4/8
0
Ly6C
F4/8
0
Ly6C
F4/8
0
F4/8
0
Ly6C
F4/8
0
Ly6C
Sham
6 h
24 h
Ly6C
F4/8
0
Ly6C
F4/8
0
Ly6C
F4/8
0
F4/8
0
Ly6C
F4/8
0
Ly6C Ly6C
A P. gingivalis
5×106 P. gingivalis
5×107 P. gingivalis ΔPG0382
5×107 P. gingivalis ΔPG0382
5×106
B
157
Neutrophils are typically the major responders during the early stages of infection, and mediate
bacterial clearance by producing reactive oxygen species and releasing granules containing
antimicrobial proteins (e.g. -defensins) (Faurschou and Borregaard, 2003; Rice et al., 1987).
Thus, the recruitment of neutrophils in response to P. gingivalis was also examined. Neutrophils
express high levels of Ly6G and low levels of Ly6C, and do not express F4/80 (i.e. Ly6G+, Ly6Clo,
F4/80-) (Lee et al., 2013). Neutrophil numbers were found to have significantly increased
6 h-post infection with either wildtype P. gingivalis or P. gingivalis ΔPG0382 (Fig. 6.11A-B). As
for inflammatory monocytes, infection with 5×107 wildtype P. gingivalis or P. gingivalis
ΔPG0382 did not stimulate a further increase in neutrophil numbers (Fig. 6.11). In contrast to
inflammatory monocytes, however, the neutrophil response was greatly reduced by 24 h-post
infection (Fig. 6.11A-B). Collectively, the results presented in Figure 6.10 and Figure 6.11
suggest that expression of PG0382 by P. gingivalis does not affect the recruitment of
inflammatory monocytes and neutrophils.
158
Figure 6.11 Recruitment of neutrophils in response to P. gingivalis. BALB/c mice were infected intraperitoneally with wildtype P. gingivalis or P. gingivalis ΔPG0382. Mouse peritoneal fluid was harvested 24 h or 48 h-post injection and subjected to flow cytometric analysis. (A) Ly6G+ Ly6Clo FACS plot from individual mice. NB: Due to the variation in response between mice, the FACs plots presented do not necessarily represent the “average” response. (B) Quantification of Ly6G+ Ly6Clo neutrophils. Each data point represents an individual mouse (n=5).
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
Sham
6 h
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
Ly6
G
Ly6C
24 h
A P. gingivalis
5×106
P. gingivalis
5×107
P. gingivalis ΔPG0382
5×107
P. gingivalis ΔPG0382
5×106
B
159
6.3 Discussion
The TIR domain plays a central role in TLR signalling by acting as a modular, interactive
domain to enable the recruitment of specific downstream signalling adaptor proteins (e.g.
MYD88 and MAL). Signal transduction initiated by TLRs results in the expression of
cytokines and chemokines, which stimulate the activation and recruitment of immune
cells to combat infection. P. gingivalis can subvert the host immune response by
suppressing TLR activation. For instance, P. gingivalis produces heterogeneous forms of
LPS with different acylation patterns that inhibit or weakly activate TLR4 signalling (Dixon
and Darveau, 2005; Reife et al., 2006). By contrast, bacterial Tcps are proposed to interact
with TLR adaptor proteins to attenuate signalling (Patterson and Werling, 2013; Rana et
al., 2013). The bioinformatics analyses and functional biochemical experiments performed
in Chapter 5 suggested that P. gingivalis PG0382 possesses characteristics of a bacterial
Tcp. Therefore, this Chapter examined a potential immunomodulatory role for PG0382.
An isogenic P. gingivalis PG0382-deficient mutant was generated in this study to
determine whether PG0382 can modulate the host immune response by subverting TLR
signalling. The phenotype of P. gingivalis ΔPG0382 were first examined. The mean
generation time of P. gingivalis ΔPG0382 (approximately 3 h) was comparable to wildtype
P. gingivalis, suggesting that the PG0382 gene is not important for P. gingivalis growth. Like
wildtype P. gingivalis, the P. gingivalis ΔPG0382 mutant grew as black-pigmented colonies
when cultured on horse-blood agar. P. gingivalis expresses extracellular proteases
(e.g. Kgp and RgpA/B gingipain proteases) that degrade haemoglobin. The subsequent
accumulation of µ-oxo bishaem-containing pigments accounts for the black pigmentation
of P. gingivalis colonies (Zambon, Reynolds and Slots, 1981; Smalley et al., 1998). In
addition to normal pigmentation, cryo-EM revealed that P. gingivalis ΔPG0382 also
exhibits a normal EDSL. Although the exact protein components of the EDSL are still
unclear, the reduced EDSL of the P. gingivalis gingipain protease-deficient mutant,
P. gingivalis W50ABK, suggests that Kgp and RgpA/B are required for the formation of the
EDSL (Chen et al., 2011; Gorasia et al., 2015). Accordingly, the absence of black
pigmentation and EDSL can indicate impaired gingipain protease activity, or disrupted
transportation and/or attachment of gingipain proteases to the outer cell membrane of
P. gingivalis. The importance of PG0382 for gingipain protease activity was also directly
tested, and P. gingivalis ΔPG0382 was found to retain wildtype levels of Kgp and RgpA/B
activity. The gingipain proteases play a central role in the dysregulation of the host
immune response by P. gingivalis. Consequently, a different host immune response to
P. gingivalis ΔPG0382 would not be attributable to altered gingipain protease activity.
160
Studies have shown that P. gingivalis can regulate TLR-mediated cytokine responses in
different cell types, including oral epithelial cells (Hajishengallis et al., 2008; Lu et al., 2009;
Maekawa et al., 2014). Therefore, the stimulation of inflammatory gene expression in oral
epithelial cells (e.g. OKF6 cells) by P. gingivalis ΔPG0382 was investigated. The findings
presented above suggest that PG0382 does not function to repress the inflammatory
response (e.g. TNF and IL-36G expression) of oral epithelial cells towards P. gingivalis.
Mammalian TIR domains are intracellular signalling modules present in TLRs and adaptor
proteins (e.g. MYD88 and MAL). As such, bacterial Tcps need to be translocated into the
host cell cytoplasm to exert inhibitory activity on TLR signalling. In the case of B. melitensis,
TcpB was shown to be delivered into the cytoplasm of RAW 264.7 cells, possibly via a type
IV secretion system (Salcedo et al., 2013). TcpC has been shown to be secreted by E. coli
CFT037 and then taken up by macrophages (e.g. mouse bone marrow-derived
macrophages) (Cirl et al., 2008). While S. aureus TirS was detected in cell culture medium,
its mechanism of secretion is not known (Askarian et al., 2014). The mechanisms of
secretion and host cell entry of other bacterial Tcps, including TlpA from S. enterica and
YpTdp from Y. pestis, are yet to be elucidated. P. gingivalis has a type IX secretion system,
which is involved in the extracellular secretion of proteins with a C-terminal domain (CTD)
(Nakayama, 2015; Seers et al., 2006; Slakeski et al., 2011). This system is unlikely to
mediate the secretion of PG0382 because it does not have a CTD. Although the precise
mechanism for the delivery of the P. gingivalis SerB phosphatase into host cells is not
known, SerB has been shown to be secreted as well as produced intracellularly following
the invasion of gingival keratinocytes by P. gingivalis (Takeuchi et al., 2013). The efficient
invasion of gingival epithelial cells by P. gingivalis is dependent on its fimbriae (Yilmaz et
al., 2002, 2003). P. gingivalis W50 is an afimbriated strain and appears to exhibit reduced
invasion efficiency, relative to other P. gingivalis strains (Duncan et al., 1993; Watanabe et
al., 1992). Consequently, the ability of PG0382 to inhibit TLR signalling in oral epithelial
cells might largely depend on the secretion and subsequent uptake of PG0382. Additional
studies, including single-cell analysis, will therefore be required to determine whether
PG0382 is secreted by P. gingivalis, and if so, whether it is delivered into the cytoplasm of
oral epithelial cells where it can interact with TLR adaptor proteins.
P. gingivalis has also been shown to modulate TLR-mediated cytokine responses in
macrophages (Hajishengallis et al., 2007; Wang et al., 2007, 2010). Thus, the effects of
PG0382 gene deletion on the cytokine responses of RAW 264.7 cells to P. gingivalis were
determined. Although the responses elicited by wildtype P. gingivalis and P. gingivalis
ΔPG0382 were not statistically different, P. gingivalis ΔPG0382 appeared to consistently
stimulate weaker IL-6, CCL2 and IL-10 responses. These findings go against the
hypothesised role of PG0382 functioning as an inhibitor of TLR-mediated inflammatory
161
responses. Indeed, they contrast with findings from experiments where RAW 264.7 cells
challenged with a TcpC-deficient E. coli CFT037 mutant mounted stronger inflammatory
cytokine responses (e.g. TNF and IL-6) when compared to cells challenged with wildtype
E. coli CFT037 (Cirl et al., 2008). Moreover, the inhibitory effect of TcpC was shown to be
MYD88-dependent, because TcpC suppressed TNF secretion in wildtype mouse bone
marrow-derived macrophages but not in MYD88-deficient macrophages (Cirl et al., 2008).
Mouse bone marrow-derived dendritic cells have also been found to exhibit increased TNF
secretion when challenged with a TcpB-deficient B. melitensis mutant (Salcedo et al., 2013).
Interestingly, TcpB has also been shown in vitro to cause restructuring of the endoplasmic
reticulum in macrophages to trigger an unfolded protein response (Smith et al., 2013). The
stimulation of the unfolded protein response is proposed to support B. melitensis
replication by mobilising amino acid transport to support lipid biogenesis. In addition,
mice infected with a TcpB-deficient B. melitensis mutant had reduced bacterial burden and
colonisation of the liver and spleen, compared to mice infected with wildtype B. melitensis
(Radhakrishnan et al., 2009). While PG0382 may have a direct immunomodulatory role,
the possibility that PG0382 may affect the inflammatory response indirectly, for instance
through the modulation of cell-surface expression of proteins and/or lipids that stimulate
the host inflammatory response to P. gingivalis, cannot be excluded. Accordingly,
additional functional experiments will be required to understand how PG0382 might
modulate the inflammatory response of macrophages to P. gingivalis, or whether PG0382
may possess other functions that extend beyond modulating cytokine responses.
The potential immunomodulatory function of PG0382 was further investigated in a mouse
model of peritoneal infection. P. gingivalis infection stimulated an increase in the numbers
of inflammatory monocytes (albeit not statistically significant due to variation between
mice) and neutrophils. Interestingly, the numbers of activated macrophages did not
appear to increase following P. gingivalis infection. Intraperitoneal injection of heat-killed
P. gingivalis has previously been reported to cause a decline in the numbers of activated
macrophages in the first 24 h, followed by an increase in macrophage numbers that
peaked 5 days-post infection (Lam et al., 2014). The reduction in macrophage numbers
has been coined the “macrophage disappearance reaction”, which is attributable to the
adhesion of macrophages to the peritoneal lining following their activation by
inflammatory stimuli (Barth et al., 1995). Notably, peritoneal infection with wildtype
P. gingivalis and P. gingivalis ΔPG0382 induced comparable increase in the numbers of
inflammatory monocytes and neutrophils. The effects of other bacterial Tcps on the
recruitment of immune cells in vivo have yet to be directly investigated. However, analysis
of skin lesions resulting from cutaneous infection of mice with a S. aureus TirS-deficient
mutant revealed increased levels of myeloperoxidase activity and inflammatory gene
162
expression (e.g. IL-1, IL-6 and CXCL1), relative to mice infected with wildtype S. aureus
(Patot et al., 2017). This suggests that TirS may play a role in suppressing immune cell
(e.g. neutrophil) infiltration and/or activity. In addition, results from an in vitro study with
a B. melitensis TcpB-deficient mutant suggest that TcpB can interfere with dendritic cell
maturation by inhibiting TLR2 signalling (Salcedo et al., 2008). Although P. gingivalis
ΔPG0382 stimulated comparable recruitment of inflammatory monocytes and neutrophils
as wildtype P. gingivalis, there is a possibility that P. gingivalis ΔPG0382 may compromise
their activity (e.g. phagocytosis and nitric oxide production). Therefore, it would be
worthwhile investigating whether there are any differences in the kinetics of immune cell
recruitment and activity between the two groups of cells isolated from mice infected with
wildtype P. gingivalis or P. gingivalis ΔPG0382 in a comprehensive model with an extended
time-course.
In conclusion, the results presented in this Chapter indicate that deletion of the PG0382
gene does not affect the growth rate, EDSL or gingipain protease activity of P. gingivalis.
Furthermore, the in vitro assays performed suggest that PG0382 does not modulate the
inflammatory response of oral epithelial cells towards P. gingivalis. Intriguingly, PG0382
may in fact enhance cytokine responses in macrophages; however, further studies will be
required to determine whether this is a direct or indirect effect. It is also unclear at this
stage whether PG0382 is delivered into host cells, and how the delivery process may
impact on the ability of PG0382 to modulate host inflammatory responses. Peritoneal
infection of mice with P. gingivalis stimulated the rapid recruitment of neutrophils and
inflammatory monocytes. The same response was seen when mice were infected with the
P. gingivalis PG0382-deficient mutant. Thus, further in vivo studies, including the mouse
model of P. gingivalis-induced periodontitis, might provide insight into whether PG0382
plays a role in modulating the host immune response to P. gingivalis, and hence
contributes to the progression of chronic periodontitis.
163
General Discussion
164
7.1 Summary
This thesis explored two facets of the interaction between the host and P. gingivalis,
including their effects on the host immune response. Specifically, the regulation of the
orphan chemokine, CXCL14, in oral epithelial cells by P. gingivalis illustrates how the
interplay between host- and pathogen-derived factors can potentially modulate the host
immune response. CXCL14 was shown to exhibit bactericidal activity against health-
associated Streptococcus species, whilst P. gingivalis was largely resistant to killing by
CXCL14. This thesis also identified and investigated a potential P. gingivalis TIR
domain-containing protein (Tcp), namely PG0382. P. gingivalis PG0382 shares similar
sequence and structural characteristics with TLR adaptor proteins and bacterial Tcps.
Moreover, PG0382 was demonstrated to exert a negative impact on the TLR adaptor
proteins MAL and MYD88. A PG0382-deficient P. gingivalis mutant (P. gingivalis ΔPG0382)
was created to investigate how PG0382 might influence the host immune response to
P. gingivalis. Interestingly, P. gingivalis ΔPG0382 stimulated a weaker cytokine response in
macrophages. Studies in mice revealed that peritoneal infection with P. gingivalis
ΔPG0382 stimulated a similar innate immune response as wildtype P. gingivalis. Taken
together, this thesis has identified and defined novel interactions between host-derived
and pathogen-derived factors in modulating the host immune response. In particular, it
has provided a molecular basis for exploring potential roles for CXCL14 and P. gingivalis
PG0382 in the development and progression of chronic periodontitis.
7.2 Implications of a dysregulated CXCL14 response for chronic
inflammation and microbial dysbiosis
The expression of chemokines by oral epithelial cells is important for the recruitment of
immune cells to prevent or clear infection. In this study, oral epithelial cells (e.g. OKF6
cells) were shown to upregulate the expression of CXCL14 in response to P. gingivalis. The
stimulation of CXCL14 expression was primarily mediated by the gingipain proteases
produced by P. gingivalis, and was dependent on the host protease-activated receptor,
PAR-3. Significantly, the activation of MEK-ERK1/2 signalling by epidermal growth factor
(EGF) was shown to suppress CXCL14 gene transcription. However, the gingipain
proteases can antagonise the EGF-mediated suppression of CXCL14 by proteolytically
degrading EGF. P. gingivalis can therefore not only directly stimulate CXCL14 expression
via PAR-3 but also promotes its expression by antagonising EGF signalling. Accordingly,
this is likely to result in a dysregulated CXCL14 response in the context of P. gingivalis
infection/colonisation. Furthermore, the ability of the gingipain proteases to degrade
CXCL14 would likely result in a suboptimal CXCL14 response, whereby the impaired
recruitment of CXCL14 target cells may compromise the immune response to P. gingivalis.
Importantly, the gingipain proteases associated with outer-membrane vesicles (OMVs)
165
released by P. gingivalis may also provide a mechanism to dysregulate CXCL14 expression
and/or activity at sites distant from the tooth-accreted biofilm. Although CXCL14 is
expressed by oral epithelial cells, it may possess context-dependent functions based on its
localised expression at specific sites in the oral mucosal epithelium (e.g. junctional
epithelium vs. sulcular epithelium), and thus this aspect should be taken into
consideration when further establishing a role for CXCL14 in chronic periodontitis.
This is the first study to demonstrate PAR3-dependent stimulation of CXCL14 expression.
Other bacterial species, such as the respiratory pathogen Pseudomonas aeruginosa, have
been demonstrated to exert agonistic and antagonistic effects on the host immune
response by differentially modulating PAR signalling. For example, the P. aeruginosa
extracellular protease, LepA, was shown to activate NF-B in bronchial epithelial cells by
stimulating PAR-1, PAR-2 and PAR-4 signalling (Kida et al., 2008), whereas the
extracellular LasB protease produced by P. aeruginosa can degrade the extracellular
domain of PAR-2 to prevent receptor signalling (Dulon et al., 2005). As such, it would be
interesting to determine whether proteases produced by other oral bacterial species
(e.g. T. denticola) can also regulate CXCL14 via PAR-3. Accordingly, localised expression of
CXCL14 within specific sites in the oral mucosa (e.g. junctional epithelium) might be
dependent on the protease milieu.
The findings presented in this thesis indicate that CXCL14 is bactericidal against certain
oral bacteria (e.g. S. gordonii). However, P. gingivalis is resistant to killing by CXCL14
killing, most likely because the gingipain proteases can degrade CXCL14. CXCL14 is
constitutively expressed in various mucosal tissues, including in the oral epithelium, and
hence has been proposed to also play a role in homeostatic defence. For instance, CXCL14
may function as a mediator of cutaneous antimicrobial defence to prevent infections that
might otherwise arise following minor skin abrasions (Frick et al., 2011). CCL28 is another
homeostatic chemokine, and is constitutively expressed by the epithelial cells of the
salivary glands (Hieshima et al., 2003). Like CXCL14, CCL28 has been shown to have
bactericidal activity against periodontal pathogens, including P. gingivalis and
A. actinomycetemcomitans (Watkins et al., 2007). CXCL14, and other homeostatic
chemokines, are therefore likely to have important roles in maintaining periodontal
health. Importantly, oral commensal species can participate in maintaining periodontal
health by acting as antigenic stimulants to facilitate the activity of an effective
non-destructive inflammatory barrier against potential pathogens. The stimulation of
hBD-2 expression in human oral epithelial cells by F. nucleatum has been proposed to be
important for maintaining periodontal tissue homeostasis by inhibiting the overgrowth of
the tooth-accreted biofilm (plaque) (Ghosh et al., 2018). In contrast, P. gingivalis appears
166
to stimulate a weaker hBD-2 response and thereby potentially compromise host defence
against the biofilm (Ghosh et al., 2018; Lu et al., 2009). The role of oral commensal species
in regulating the expression of homeostatic chemokines, including CXCL14, has yet to be
investigated. Therefore, the ability of oral commensal species (e.g. S. mitis) and pathogens
(e.g. A. actinomycetemcomitans) to regulate CXCL14 may provide further important insight
into the role of CXCL14 in maintaining tissue homeostasis.
The potential “double-edged” nature of host immunomodulatory factors (e.g. cytokines
and chemokines) can make it difficult to clearly define their role as being host-protective
and host-destructive when dysregulated. Although typically protective, the persistence of
innate immune cells (e.g. neutrophils) due to unresolved infection can be highly
detrimental to the host. CXCL14 exerts pleiotropic chemotactic activity towards various
innate immune cells (e.g. neutrophils, macrophages and dendritic cells) (Shellenberger et
al. 2004; Shurin et al. 2005; Kurth et al. 2001; Cao et al. 2000). Thus, the dysregulation of
CXCL14 by P. gingivalis might contribute to the development of chronic inflammation by
promoting dysregulated immune cell recruitment, and thereby promote oral dysbiosis.
CXCL14 bactericidal activity may also contribute to microbial dysbiosis. For example,
appropriately regulated CXCL14 expression might be important for limiting the growth of
susceptible bacterial species in the tooth-accreted biofilm. However, the ability of the
P. gingivalis gingipain proteases to proteolytically degrade CXCL14 may enable
P. gingivalis to not only protect itself, but also protect closely associated bacterial species
(e.g. accessory pathogens) in the biofilm, which might otherwise be susceptible to killing
by CXCL14. The Gram-negative oral pathogen, P. intermedia expresses the protease,
interpain A, which is homologous to the Streptococcus pyogenes protease, speB.
Significantly, speB has been shown to protect S. pyogenes from CXCL14-mediated killing by
degrading CXCL14 (Frick et al., 2011). Therefore, proteases produced by other oral
bacteria may also be able to compromise CXCL14 activity. Taken together, the
dysregulation of CXCL14 may potentially contribute to chronic periodontitis by promoting
chronic inflammation and oral dysbiosis.
The findings from this thesis have provided important molecular insights into the
regulation and role of CXCL14. However, the regulation and role of CXCL14 also needs to
be considered in a broader context in vivo. Experimental mouse model studies could
provide pre-clinical evidence with respect to the relevance of CXCL14 in chronic
periodontitis. Periodontal inflammation and alveolar bone loss are key indicators of
disease progression in chronic periodontitis. Therefore, the potential contribution and
role of CXCL14 in the development of chronic periodontitis could be investigated with
CXCL14-deficient mice in the P. gingivalis-induced mouse model of periodontitis. In
167
addition to assessing the impact on alveolar bone resorption, analysis of the oral
microbiome, for example, by next generation sequencing, might also provide further
insight into the role of CXCL14 in promoting host-microbe homeostasis, and specific
consequences of its dysregulation by P. gingivalis.
7.3 CXCL14 in tissue regeneration
Current treatment for chronic periodontitis involves scaling and root planing to remove
plaque (Pihlstrom et al., 2005). This results in at least the temporary resolution of
inflammation and attenuation of disease progression (Cobb, 2002). Antibiotics are
sometimes employed as an adjuvant to inhibit biofilm growth when scaling and root
planing are not sufficient to resolve inflammation and prevent disease progression (Jepsen
and Jepsen, 2016; Slots and Rams, 1990). Stem cell transplantation is being explored as a
therapeutic avenue to promote the repair of the periodontium in chronic periodontitis
(Chen et al., 2012). Notably, CXCL14 was recently suggested to be a trophic factor
produced by dental pulp-derived mesenchymal stem cells (Hayashi et al., 2015). In
particular, CXCL14 was proposed to facilitate tissue regeneration, in an ectopic mouse
model of tooth root transplantation, by promoting endogenous cell migration (Hayashi et
al., 2015). However, the target migratory cells in which CXCL14 might act to promote
tissue regeneration were not identified. The results from this thesis suggest that CXCL14
does not regulate the migration of oral epithelial cells, or at least OKF6 cells in an in vitro
setting. Interestingly, a study by Shellenberger et al. suggests that CXCL14 can inhibit
angiogenesis by blocking IL-8-mediated endothelial cell migration (Shellenberger et al.,
2004). This may have implications in regeneration therapy, as angiogenesis is crucial for
tissue regeneration. Therefore, further studies will be required to definitively establish
whether CXCL14 is a tropic factor, and hence how it could potentially be therapeutically
exploited to promote tissue regeneration in chronic periodontitis.
7.4 A potential role for PG0382 in immune subversion
The family of Toll-like receptors (TLRs) share a common modular intracellular TIR
domain that initiates downstream signalling to induce changes in gene expression by
forming heterotypic TIR-TIR interactions with TLR adaptor proteins (e.g. MAL, MYD88,
TRAM and TRIF). Bacterial Tcps are proposed to facilitate bacterial immune subversion by
blocking TLR signalling. The bioinformatic analyses performed in this thesis suggest that
several P. gingivalis strains express putative Tcps (e.g. PG0382). Through bioinformatic
analysis, PG0382 from P. gingivalis W83/W50 was found to share similar properties to
TLR adaptor proteins and bacterial Tcps. Significantly, PG0382 caused MAL and MYD88
levels to be markedly reduced when co-expressed in mammalian cells (e.g. HEK293T
cells). The mechanism underlying the ability of PG0382 to reduce MAL and MYD88 levels
168
has yet to be determined. However, studies of other bacterial Tcps might provide further
insight. TcpB from B. melitensis was shown to promote the ubiquitination MAL, and
thereby target MAL for degradation by the proteasome (Sengupta et al., 2010). P. gingivalis
has been shown to suppress neutrophil antimicrobial responses by inducing signalling
crosstalk between the C5aR and TLR2 to promote the ubiquitination and proteasomal
degradation of MYD88 (Maekawa et al., 2014). In addition to being degraded via the
proteasomal pathway, cellular proteins can be degraded via the lysosomal pathway
(Ciechanover, 2005). Thus, these two pathways could potentially be exploited by PG0382
to reduce MAL and MYD88 levels. This could be investigated with pharmacological agents
that can selectively inhibit the proteasomal and lysosomal degradation pathways (e.g.
MG132 and chloroquine, respectively).
Recent studies have also reported roles for bacterial Tcps in modulating inflammasome
activation (Jakka et al., 2017; Waldhuber et al., 2016). As described in Chapter 1,
inflammasome activation is critical for IL-1 maturation. Canonical inflammasome
activation is mediated by NLRP1, NLRP3 or NLRC4 (Latz et al., 2013), whereas
non-canonical inflammasome activation is mediated by caspase-4 (Kayagaki et al., 2011).
Upon binding intracellular LPS, caspase-4 oligomerises and induces caspase-1-mediated
IL-1 maturation. TcpB has been shown to interact with caspase-4, resulting in the
ubiquitination and degradation by the proteasome. Moreover, TcpB was shown to
attenuate IL-1 secretion by macrophages (e.g. THP-1 cells and RAW 264.7 cells) (Jakka et
al., 2017). In contrast, TcpC can inhibit canonical caspase-1 processing of IL-1 by
inhibiting NLRP3-mediated inflammasome activation (Waldhuber et al., 2016).
Significantly, in a mouse model of urinary tract infection, IL-1 levels in the urine of mice
infected with wildtype E. coli CFT073 were lower than those infected with a TcpC-deficient
mutant. These findings suggest that bacterial Tcps may also execute immunomodulatory
activity through TLR-independent signalling pathways. Therefore, it would be interesting
to determine whether PG0382 can interact and modulate the functions of other immune
signalling molecules, including specific components of inflammasome pathways.
Intriguingly, instead of heightened inflammatory cytokine responses, RAW 264.7 cells
appeared to mount weaker responses when challenged with an isogenic PG0382-deficient
P. gingivalis mutant, suggesting that PG0382 may promote a pro-inflammatory response.
This contrasts with other bacterial Tcps, which have shown to exert anti-inflammatory
effects on the host immune response (Cirl et al., 2008). P. gingivalis ΔPG0382 has normal
Kgp and Rgp activity, and therefore the apparent reduced cytokine activity in RAW 264.7
cells challenged with P. gingivalis ΔPG0382 is unlikely to be attributable to impaired
gingipain protease activity. In addition to the gingipain proteases, P. gingivalis produces an
169
array of surface-associated virulence factors (e.g. haemagglutinins) (Holt et al., 1999; How
et al., 2016), and therefore changes to the expression of surface-associated proteins could
potentially affect the host inflammatory response. The ability of PG0382 to modulate,
either directly or indirectly, the expression and/or function of P. gingivalis
surface-associated proteins has yet to be elucidated. It will therefore be important to
analyse the proteomic profiles of different cellular compartments of P. gingivalis ΔPG0382
(e.g. outer membrane and periplasm), for example by applying mass spectrometry-based
approaches (Gorasia et al., 2015).
A mouse model of peritoneal infection was used to assess the potential role of PG0382 in
modulating the innate immune response to P. gingivalis. However, the absence of PG0382
expression by P. gingivalis did not appear to affect the responses elicited. The
manipulation of TLR signalling by P. gingivalis can result in reduced macrophage and
neutrophil activity (Maekawa et al., 2014; Wang et al., 2010). To that end, it would be
interesting to determine whether PG0382 can affect the antimicrobial functions of the
immune cells recruited, for instance, macrophage phagocytic activity and reactive oxygen
species production by neutrophils. Although the peritonitis model is useful for
investigating immune cell recruitment, it does not represent the natural course of
P. gingivalis colonisation/infection and proliferation that occurs in chronic periodontitis.
Thus, the P. gingivalis-induced mouse model of periodontitis might provide more relevant
insight into the potential role of PG0382 in virulence.
7.5 The TIR domain as a primordial microbial signalling module
Many proteins involved in regulating innate immunity (e.g. TLRs and NLRs) have a
modular structure comprising different domains. In addition to the TIR domain, other
examples include LRR, ATPase/NTPase and NACHT domains (Dunin-Horkawicz et al.,
2014; Koonin and Aravind, 2002). These protein domains often have homologs of
unknown function in bacteria (Koonin and Aravind, 2002). The bioinformatic searches
performed in Chapter 5 revealed that there are eleven putative P. gingivalis Tcps, and
some P. gingivalis strains were found to have two or more Tcps. The propagation of
P. gingivalis Tcps between strains may have occurred through DNA exchange by natural
competence and conjugation (Tribble et al., 2007). Other bacterial Tcps are largely found
in genomic regions within phage origins, and thus their exchange between bacteria was
likely to have occurred through horizontal gene transfer events (Zhang et al., 2011).
Interestingly, cluster analysis suggests that protein domains associated with mammalian
innate immune signalling components, including TIR and NACHT domains, are of bacterial
origin, and were acquired through mitochondrial endosymbiosis and additional horizontal
gene transfer events (Koonin and Aravind, 2002). Therefore, the likely bacterial origin of
170
the TIR domain suggests that not all bacterial Tcps have evolved to mediate the
subversion of the host immune system.
The TIR domain has a broad phylogenetic distribution and widespread within the
genomes of microorganisms, including many that are not pathogenic. For instance, there is
an over-representation of TIR domains within cyanobacteria (e.g. Anabaena variabilis and
Nodularia spumigena), which rarely engage in immune subversion (Spear et al., 2009).
Although experimental evidence points to an immune subversive role for at least some
bacterial Tcps, it is important to acknowledge that in vitro systems may not always
accurately reflect the role of bacterial Tcps in immune subversion. For instance, YpTdp
from Y. pestis was shown to bind MYD88 and inhibit LPS-stimulated NF-B activation in an
in vitro expression system (e.g. HEK293T cells). However, YpTdp was not required for
Y. pestis virulence in mice (Spear et al., 2012). Therefore, in vivo experiments are crucial to
determine the importance of bacterial Tcps in causing disease.
Most studies exploring bacterial Tcps have largely focused on their potential function as
virulence factors. However, bacterial Tcps often contain other functional domains, and
therefore it is possible that they have evolved to mediate specific physiological purposes.
Specifically, given that the TIR domain is a modular interactive domain, it is possible that
bacterial Tcps engage in protein-protein interactions to mediate intrinsic physiological
processes. The identification of multiple Tcps in the same strain of P. gingivalis, for
instance, PG0382 and PG1864 in P. gingivalis W83/50, and A343_0215 and A343_1154 in
P. gingivalis JCVISC001, therefore raises the possibility that the Tcps might form both
homotypic and/or heterotypic interactions through their TIR domains. The generation of a
complemented P. gingivalis ΔPG0382 strain in which the re-expressed PG0382 protein is
tagged with an appropriate epitope (e.g. V5 epitope) would be useful for not only studying
the localisation of PG0382 in P. gingivalis but also identifying potential binding partners.
Such information would provide greater insight into the likely function(s) of PG0382.
7.6 Bacterial Tcps as therapeutic agents for inflammation
In addition to their host-protective roles, TLRs can also contribute to the pathogenesis of
infectious and/or inflammatory diseases. The subversion of TLR signalling by
Mycobacterium tuberculosis contributes to pathology in tuberculosis (Harding and Boom,
2010), while the ability of P. gingivalis to subvert TLR2 is central to the pathogenesis of
chronic periodontitis (Hajishengallis and Lamont, 2014). Manipulating the unique
interactions between TLRs and adaptor proteins could therefore potentially be exploited
to provide new therapeutic opportunities for treating some diseases (O’Neill et al., 2009).
For example, a dominant-negative form of MYD88 was shown in rats to protect the
171
myocardium from tissue damage following ischemia/reperfusion injury by inhibiting
NF-B activation (Hua et al., 2005). In addition, chemical compounds (e.g. compound 4a)
that mimic the BB loop in the TIR domain of MYD88 have been shown in mice to attenuate
IL-1-induced fever (Bartfai et al., 2003). The ability of bacterial Tcps to inhibit TLR
signalling might therefore be utilised for therapeutic intervention in TLR-mediated
inflammatory diseases. Bacterial Tcps studied thus far for their ability to modulate TLR
signalling have been shown to contain coiled-coil motifs (Alaidarous et al., 2014; Cirl et al.,
2008; Rana et al., 2011). The addition of an artificial coiled-coil domain to the TIR domain
of MYD88 resulted in potent inhibition of TLR signalling (Fekonja, Bencina and Jerala,
2012). Significantly, the TcpC TIR domain was shown to ameliorate inflammation in the
mouse model of collagen-induced arthritis (Pasi et al., 2016). The therapeutic effects were
attributed to the ability of the TcpC TIR domain to block MYD88 signalling, and thus
inhibit pathogenic Th17 cell responses. Therefore, elucidating the mechanism by which
bacterial Tcps interact with TLRs and/or TLR adaptor proteins to suppress TLR signalling
could potentially be useful for the design of novel therapeutics to inhibit TLR-mediated
pathogenic inflammatory responses.
7.7 Conclusion
The ecological properties of the oral cavity are complex, dynamic and highly variable
between individuals. The interactions between host cells and oral microbiota is important
for shaping various microbial communities in the oral cavity. P. gingivalis causes an
imbalance in microbial distribution and shifts microbiota homeostasis to dysbiosis,
whereby tissue-destructive chronic inflammation ensues. The novel host cell-P. gingivalis
interactions identified and explored in this thesis provide further insight into the role of
P. gingivalis as a major pathogen in chronic periodontitis. Finally, the findings from this
thesis should also be examined in a broader context to provide greater perspective into
the potential involvement of CXCL14 and PG0382 in the development of chronic
periodontitis.
172
Bibliography
Aas, J. a, Paster, B.J., Stokes, L.N., Olsen, I. and Dewhirst, F.E. (2005), “Defining the Normal Bacterial Flora of the Oral Cavity Defining the Normal Bacterial Flora of the Oral Cavity”, Journal of Clinical Microbiology, Vol. 43 No. 11, pp. 5721–5732.
Abbott, D.W., Wilkins, A., Asara, J.M. and Cantley, L.C. (2004), “The Crohn’s disease protein, NOD2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO”, Current Biology, Vol. 14 No. 24, pp. 2217–2227.
Abe, T., Hosur, K.B., Hajishengallis, E., Reis, E.S., Ricklin, D., Lambris, J.D. and Hajishengallis, G. (2012), “Local complement-targeted intervention in periodontitis: proof-of-concept using a C5a receptor (CD88) antagonist.”, Journal of Immunology, Vol. 189 No. 11, pp. 5442–5548.
Abusleme, L., Dupuy, A. K., Dutzan, N., Silva, N., Burleson, J. A., Strausbaugh, L. D., Gamonal, J. and Diaz, P. I. (2013) ‘The subgingival microbiome in health and periodontitis and its relationship with community biomass and inflammation’, ISME Journal, 7(5), pp. 1016–1025
Aguilera, O., Andrés, M.T., Heath, J., Fierro, J.F. and Douglas, C.W.I. (1998), “Evaluation of the antimicrobial effect of lactoferrin on Porphyromonas gingivalis, prevotella intermedia and prevotella nigrescens”, FEMS Immunology and Medical Microbiology, Vol. 21 No. 1, pp. 29–36.
Al-Ahmad, A., Wunder, A., Auschill, T.M., Follo, M., Braun, G., Hellwig, E. and Arweiler, N.B. (2007), “The in vivo dynamics of Streptococcus spp., Actinomyces naeslundii, Fusobacterium nucleatum and Veillonella spp. in dental plaque biofilm as analysed by five-colour multiplex fluorescence in situ hybridization”, Journal of Medical Microbiology, Vol. 56 No. 5, pp. 681–687.
Al-Qutub, M.N., Braham, P.H., Karimi-Naser, L.M., Liu, X., Genco, C.A. and Darveau, R.P. (2006), “Hemin-dependent modulation of the lipid A structure of Porphyromonas gingivalis lipopolysaccharide”, Infection and Immunity, Vol. 74 No. 8, pp. 4474–4485.
Alaidarous, M., Ve, T., Casey, L.W., Valkov, E., Ericsson, D.J., Ullah, M.O., Schembri, M. a., et al. (2014), “Mechanism of bacterial interference with TLR4 signaling by brucella toll/interleukin-1 receptor domain-containing protein TcpB”, Journal of Biological Chemistry, Vol. 289 No. 2, pp. 654–668.
Alexopoulou, L., Czopik Holt, A., Medzhitov, R. and Flavell, R.A. (2001), “Recognition of double-stranded RNA and activation of NF-kappa B by Toll-like receptor 3”, Nature, Vol. 413 No. 6857, pp. 732–738.
Allen, S., Crown, T. and Handel. (2007), “Chemokine:Receptor Structure, Interactions, and Antagonism”, Annual Review of Immunology, Vol. 25 No. 1, pp. 787–820.
Amano, A., Nakagawa, I., Okahashi, N. and Hamada, N. (2004), “Variations of Porphyromonas gingivalis fimbriae in relation to microbial pathogenesis”, Journal of Periodontal Research, Vol. 39 No. 2, pp. 136–142.
Andersen-Nissen, E., Smith, K.D., Strobe, K.L., Barrett, S.L., Cookson, B.T., Logan, S.M. and Aderem, A. (2005), “Evasion of Toll-like receptor 5 by flagellated bacteria”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 102 No. 26, pp. 9247–9252.
Andrian, E., Mostefaoui, Y., Rouabhia, M. and Grenier, D. (2007), “Regulation of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases by Porphyromonas gingivalis in an engineered human oral mucosa model”, Journal of Cellular Physiology, Vol. 207 No. 211, pp. 26–62.
Ara, T., Tokoyoda, K., Sugiyama, T., Egawa, T., Kawabata, K. and Nagasawa, T. (2003), “Long-term hematopoietic stem cells require stromal cell-derived factor-1 for
173
colonizing bone marrow during ontogeny”, Immunity, Vol. 19 No. 2, pp. 257–267.
Arbibe, L., Kim, D.W., Batsche, E., Pedron, T., Mateescu, B., Muchardt, C., Parsot, C., et al. (2007), “An injected bacterial effector targets chromatin access for transcription factor NF-κB to alter transcription of host genes involved in immune responses”, Nature Immunology, Vol. 8 No. 1, pp. 47–56.
Arizon, M., Nudel, I., Segev, H., Mizraji, G., Elnekave, M., Furmanov, K., Eli-Berchoer, L., et al. (2012), “Langerhans cells down-regulate inflammation-driven alveolar bone loss”, Proceedings of the National Academy of Sciences, Vol. 109 No. 18, pp. 7043–7048.
Arnold, R.R., Brewer, M. and Gauthier, J.J. (1980), “Bactericidal activity of human lactoferrin: Sensitivity of a variety of microorganisms”, Infection and Immunity, Vol. 28 No. 3, pp. 893–898.
Arslan, S.Y., Leung, K.P. and Wu, C.D. (2009), “The effect of lactoferrin on oral bacterial attachment”, Oral Microbiology and Immunology, Vol. 24 No. 5, pp. 411–416.
Aruni, W., Vanterpool, E., Osbourne, D., Roy, F., Muthiah, A., Dou, Y. and Fletcher, H.M. (2011), “Sialidase and sialoglycoproteases can modulate virulence in Porphyromonas gingivalis”, Infection and Immunity, Vol. 79 No. 7, pp. 2779–2791.
Asai, Y., Ohyama, Y., Gen, K. and Ogawa, T. (2001), “Bacterial fimbriae and their peptides activate human gingival epithelial cells through Toll-like receptor 2”, Infection and Immunity, Vol. 69 No. 12, pp. 7387–7395.
Askarian, F., Sorge, N.M. van, Sangvik, M., Beasley, F.C., Jørn R. Henriksen b Johanna U.E. Sollid, C, J.A.G. van S., Nizet, V., et al. (2014), “A Staphylococcus aureus TIR Domain Protein Virulence Factor Blocks TLR2-Mediated NF-κB Signaling”, Karger Open Access, Vol. 29 No. 6, pp. 997–1003.
Assuma, R., Oates, T., Cochran, D., Amar, S. and Graves, D.T. (1998), “IL-1 and TNF antagonists inhibit the inflammatory response and bone loss in experimental periodontitis”, Journal of Immunology, Vol. 160 No. 1, pp. 403–409.
Attströum, R., Laurel, A. ‐B, Lahsson, U. and Sjöuholm, A. (1975), “Complement factors in gingival crevice material from healthy and inflamed gingiva in humans”, Journal of Periodontal Research, Vol. 10 No. 1, pp. 19–27.
Augsten, M., Hägglöf, C., Olsson, E., Stolz, C., Tsagozis, P., Levchenko, T., Frederick, M., et al. (2009), “CXCL14 is an autocrine growth factor for fibroblasts and acts as a multi-modal stimulator of prostate tumor growth”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 106 No. 9, pp. 3414–3419.
Austyn, J.M. and Gordon, S. (1981), “F4/80, a monoclonal antibody directed specifically against the mouse macrophage”, European Journal of Immunology, Vol. 11 No. 10, pp. 805–815.
Avraham, R. and Yarden, Y. (2011), “Feedback regulation of EGFR signalling: Decision making by early and delayed loops”, Nature Reviews Molecular Cell Biology, Vol. 12 No. 2, pp. 104–117.
Bachrach, G., Altman, H., Kolenbrander, P.E., Chalmers, N.I., Gabai-Gutner, M., Mor, A., Friedman, M., et al. (2008), “Resistance of Porphyromonas gingivalis ATCC 33277 to direct killing by antimicrobial peptides is protease independent”, Antimicrobial Agents and Chemotherapy, Vol. 52 No. 2, pp. 638–642.
Banbula, A., Banbula, M., Bugno, A., Kuster, P., Heinrich, J., Travis, J. and Potempa. (1999), “Rapid and Efficient Inactivation of IL-6 Gingipains, Lysine- and Arginine-Specific Proteinases from Porphyromonas Gingivalis”, Biochemical and Biophysical Research Communications, Vol. 261 No. 3, pp. 598–602.
Banchereau, J. and Steinman, R.M. (1998), “Dendritic cells and the control of immunity.”, Nature, Vol. 392 No. March, pp. 245–252.
174
Barksby, H.E., Nile, C.J., Jaedicke, K.M., Taylor, J.J. and Preshaw, P.M. (2009), “Differential expression of immunoregulatory genes in monocytes in response to Porphyromonas gingivalis and Escherichia coli lipopolysaccharide”, Clinical and Experimental Immunology, Vol. 156 No. 3, pp. 479–487.
Barron, L. and Wynn, T. a. (2011), “Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages.”, American Journal of Physiology. Gastrointestinal and Liver Physiology, Vol. 300 No. 5, pp. 723–728.
Bartfai, T., Behrens, M.M., Gaidarova, S., Pemberton, J., Shivanyuk, A. and Rebek, J. (2003), “A low molecular weight mimic of the Toll/IL-1 receptor/resistance domain inhibits IL-1 receptor-mediated responses.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 100, pp. 7971–7976.
Barth, M.W., Hendrzak, J.A., Melnicoff, M.J. and Morahan, P.S. (1995), “Review of the macrophage disappearance reaction.”, Journal of Leukocyte Biology, Vol. 57 No. 3, pp. 361–7.
Becker, M.R., Paster, B.J., Leys, E.J., Moeschberger, M.L., Kenyon, S.G., Galvin, J.L., Boches, S.K., et al. (2002), “Molecular analysis of bacterial species associated with childhood caries”, J Clin Microbiol, Vol. 40 No. 3, pp. 1001–1009.
Beklen, A., Hukkanen, M., Richardson, R. and Konttinen, Y.T. (2008), “Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis”, Oral Microbiology and Immunology, Vol. 23 No. 5, pp. 425–431.
Bella, J., Hindle, K.L., McEwan, P.A. and Lovell, S.C. (2008), “The leucine-rich repeat structure”, Cellular and Molecular Life Sciences, Vol. 65 No. 15, pp. 2307–2333.
Berglundh, T., Liljenberg, B. and Lindhe, J. (2002), “Some cytokine profiles of T-helper cells in lesions of advanced periodontitis.”, Journal of Clinical Periodontology, Vol. 29 No. 8, pp. 705–709.
Berglundh, T., Liljenberg, B., Tarkowski, A. and Lindhe, J. (2002), “The presence of local and circulating autoreactive B cells in patients with advanced periodontitis.”, Journal of Clinical Periodontology, Vol. 29 No. 4, pp. 281–286.
Bishop, R.E., Gibbons, H.S., Guina, T., Trent, M.S., Miller, S.I. and Raetz, C.R.H. (2000), “Transfer of palmitate from phospholipids to lipid A in outer membranes of Gram-negative bacteria”, The EMBO Journal, Vol. 19 No. 19, pp. 5071–5080.
Boone, D.L., Turer, E.E., Lee, E.G., Ahmad, R.C., Wheeler, M.T., Tsui, C., Hurley, P., et al. (2004), “The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses”, Nature Immunology, Vol. 5 No. 10, pp. 1052–1060.
Bowie, A., Kiss-Toth, E., Symons, J.A., Smith, G.L., Dower, S.K. and O’Neill, L.A.J. (2000), “A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling”, Proceedings of the National Academy of Sciences, Vol. 97 No. 18, pp. 10162–10167.
Bradshaw, D.J., Marsh, P.D., Watson, G.K. and Allison, C. (1998), “Role of Fusobacterium nucleatum and coaggregation in anaerobe survival in planktonic and biofilm oral microbial communities during aeration”, Infection and Immunity, Vol. 66 No. 10, pp. 4729–4732.
Brissette, C. a. and Lukehart, S. a. (2007), “Mechanisms of decreased susceptibility to beta-defensins by Treponema denticola”, Infection and Immunity, Vol. 75 No. 5, pp. 2307–2315.
Burkhard, P., Stetefeld, J. and Strelkov, S. V. (2001), “Coiled coils: A highly versatile protein folding motif”, Trends in Cell Biology, Vol. 11 No. 2, pp. 82–88.
Burnier, L. and Mosnier, L.O. (2013), “Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated
175
receptor 3”, Blood, Vol. 122 No. 5, pp. 807–816.
Burns, E., Eliyahu, T., Uematsu, S., Akira, S. and Nussbaum, G. (2010), “TLR2-dependent inflammatory response to Porphyromonas gingivalis is MyD88 independent, whereas MyD88 is required to clear infection.”, Journal of Immunology, Vol. 184 No. 3, pp. 1455–1462.
Burns, K., Janssens, S., Brissoni, B., Olivos, N., Beyaert, R. and Tschopp, J. (2003), “Inhibition of Interleukin 1 Receptor/Toll-like Receptor Signaling through the Alternatively Spliced, Short Form of MyD88 Is Due to Its Failure to Recruit IRAK-4”, The Journal of Experimental Medicine, Vol. 197 No. 2, pp. 263–268.
Byrne, S.J., Dashper, I.B., Darby, G.G., Adams, B., Hoffmann, E.C. and Reynolds. (2009), “Progression of chronic periodontitis can be predicted by the levels ofPorphyromonas gingivalisandTreponema denticolain subgingival plaque”, Oral Microbiology and Immunology, Vol. 24 No. 6, pp. 469–477.
Calkins, C.C., Platt, K., Potempa, J. and Travis, J. (1998), “Inactivation of tumor necrosis factor-α by proteinases (gingipains) from the periodontal pathogen, Porphyromonas gingivalis. Implications of immune evasion”, Journal of Biological Chemistry, Vol. 273 No. 12, pp. 6611–6614.
Cao, X., Zhang, W., Wan, T., He, L., Chen, T., Yuan, Z., Ma, S., et al. (2000), “Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells.”, Journal of Immunology, Vol. 165 No. 5, pp. 2588–2595.
Carlsson, E., Ding, J.L. and Byrne, B. (2016), “SARM modulates MyD88-mediated TLR activation through BB-loop dependent TIR-TIR interactions”, Biochimica et Biophysica Acta - Molecular Cell Research, Vol. 1863 No. 2, pp. 244–253.
Carlsson, J. (1997), “Bacterial metabolism in dental biofilms”, Advances in Dental Research, Vol. 11 No. 1, pp. 75–80.
Carty, M., Goodbody, R., Schröder, M., Stack, J., Moynagh, P.N. and Bowie, A.G. (2006), “The human adaptor SARM negatively regulates adaptor protein TRIF-dependent Toll-like receptor signaling.”, Nature Immunology, Vol. 7 No. 10, pp. 1074–81.
Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y., et al. (2003), “An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid”, Nature Immunology, Vol. 4 No. 7, pp. 702–707.
Chan, S.L., Low, L.Y., Hsu, S., Li, S., Liu, T., Santelli, E., Le Negrate, G., et al. (2009), “Molecular mimicry in innate immunity. Crystal structure of a bacterial TIR domain”, Journal of Biological Chemistry, Vol. 284 No. 32, pp. 21386–21392.
Chang, K.M., Lehrhaupt, N., Lin, L.M., Feng, J., Wu-Wang, C.Y. and Wang, S.L. (1996), “Epidermal growth factor in gingival crevicular fluid and its binding capacity in inflamed and non-inflamed human gingiva”, Archives of Oral Biology, Vol. 41 No. 7, pp. 719–724.
Chen, F.M., Sun, H.H., Lu, H. and Yu, Q. (2012), “Stem cell-delivery therapeutics for periodontal tissue regeneration”, Biomaterials, Vol. 33 No. 27, pp. 6320–6344.
Chen, L., Guo, L., Tian, J., He, H., Marinova, E., Zhang, P., Zheng, B., et al. (2012), “Overexpression of CXC Chemokine Ligand 14 Exacerbates Collagen-Induced Arthiritis”, Journal of Immunology, Vol. 29 No. 6, pp. 997–1003.
Chen, T., Hosogi, Y., Nishikawa, K., Fleischmann, R.D., Walling, J., Duncan, M.J. and Abbey, K. (2004), “Comparative Whole-Genome Analysis of Virulent and Avirulent Strains of Porphyromonas gingivalis Comparative Whole-Genome Analysis of Virulent and Avirulent Strains of Porphyromonas gingivalis”, Vol. 186 No. 16, pp. 5473–5479.
Chen, Y.Y., Peng, B., Yang, Q., Glew, M.D., Veith, P.D., Cross, K.J., Goldie, K.N., et al. (2011),
176
“The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis”, Molecular Microbiology, Vol. 79 No. 5, pp. 1380–1401.
Chi, B., Qi, M. and Kuramitsu, H.K. (2003), “Role of dentilisin in Treponema denticola epithelial cell layer penetration”, Research in Microbiology, Vol. 154 No. 9, pp. 637–643.
Chuang, T.H. and Ulevitch, R.J. (2004), “Triad3A, an E3 ubiquitin-protein ligase regulating Toll-like receptors”, Nature Immunology, Vol. 5 No. 5, pp. 495–502.
Chung, W.O. and Dale, B. a. (2008), “Differential utilization of nuclear factor-kappaB signaling pathways for gingival epithelial cell responses to oral commensal and pathogenic bacteria.”, Oral Microbiology and Immunology, Vol. 23 No. 2, pp. 119–126.
Chung, W.O., Hansen, S.R., Rao, D. and Dale, B.A. (2004), “Protease-Activated Receptor Signaling Increases Epithelial Antimicrobial Peptide Expression”, Journal of Immunology, Vol. 173 No. 8, pp. 5165–5170.
Ciechanover, A. (2005), “Proteolysis: From the lysosome to ubiquitin and the proteasome”, Nature Reviews Molecular Cell Biology, Vol. 6 No. 1, pp. 79–86.
Cionca, N., Giannopoulou, C., Ugolotti, G., & Mombelli, A. (2009). Amoxicillin and Metronidazole as an Adjunct to Full-Mouth Scaling and Root Planing of Chronic Periodontitis. Journal of Periodontology.
Cirl, C., Cirl, A., Wieser, M., Yadav, S., Duerr, S., Schubert, H., Fischer, D., et al. (2008), “Subversion of Toll-like receptor signaling by a unique family of bacterial Toll/interleukin-1 receptor domain–containing proteins”, Nature Medicine, Vol. 14 No. 4, pp. 399–406.
Cirl, C. and Miethke, T. (2010), “Microbial Toll/interleukin 1 receptor proteins: A new class of virulence factors”, International Journal of Medical Microbiology, Elsevier GmbH., Vol. 300 No. 6, pp. 396–401.
Coats, S.R., Jones, J.W., Do, C.T., Braham, P.H., Bainbridge, B.W., To, T.T., Goodlett, D.R., et al. (2009), “Human Toll-like receptor 4 responses to P. gingivalis are regulated by lipid A 1-and 4’-phosphatase activities”, Cellular Microbiology, Vol. 11 No. 11, pp. 1587–1599.
Cobb, C.M. (2002), “Clinical significance of non-surgical periodontal therapy: an evidence-based perspective of scaling and root planing.”, Journal of Clinical Periodontology, Vol. 29 Suppl 2, pp. 6–16.
Collins, P.J., McCully, M.L., Martínez-Muñoz, L., Santiago, C., Wheeldon, J., Caucheteux, S., Thelen, S., et al. (2017), “Epithelial chemokine CXCL14 synergizes with CXCL12 via allosteric modulation of CXCR4”, The FASEB Journal, Vol. 13 No. 7, pp. 3084–3097.
Cooper, P.R., Palmer, L.J. and Chapple, I.L.C. (2013), “Neutrophil extracellular traps as a new paradigm in innate immunity: friend or foe?”, Periodontology 2000, Vol. 63 No. 1, pp. 165–197.
Corbin, B.D., Seeley, E.H., Raab, A., Feldmann, J., Miller, M.R., Torres, V.J., Anderson, K.L., et al. (2008), “Metal chelation and inhibition of bacterial growth in tissue abscesses”, Science, Vol. 319 No. 5865, pp. 962–965.
Coughlin, S.R. and Camerer, E. (2003), “PARticipation in inflammation”, Journal of Clinical Investigation, Vol. 111 No. 1, pp. 25–27.
Couper, K.N., Blount, D.G. and Riley, E.M. (2008), “IL-10: The Master Regulator of Immunity to Infection”, Journal of Immunology, Vol. 180 No. 9, pp. 5771–5777.
Croft, M., Duncan, D.D. and Swain, S.L. (1992), “Response of naive antigen-specific CD4+ T cells in vitro: characteristics and antigen-presenting cell requirements.”, The Journal
177
of Experimental Medicine, Vol. 176 No. 5, pp. 1431–1437.
Cuenda, A., Rouse, J., Doza, Y.N., Meier, R., Cohen, P., Gallagher, T.F., Young, P.R., et al. (1995), “SB 203580 is a specific inhibitor of a MAP kinase homologue which is stimulated by cellular stresses and interleukin-1”, FEBS Letters, Vol. 364 No. 2, pp. 229–233.
Cutler, C.W., Kalmar, J.R. and Arnold, R.R. (1991), “Phagocytosis of virulent Porphyromonas gingivalis by human polymorphonuclear leukocytes requires specific immunoglobulin G”, Infection and Immunity, Vol. 59 No. 6, pp. 2097–2104.
D’Aiuto, F., Casas, J.P., Shah, T., Humphries, S.E., Hingorani, A.D. and Tonetti, M.S. (2005), “C-reactive protein (+1444C > T) polymorphism influences CRP response following a moderate inflammatory stimulus”, Atherosclerosis, Vol. 179 No. 2, pp. 413–417.
Dai, C., Basilico, P., Cremona, T.P., Collins, P., Moser, B., Benarafa, C. and Wolf, M. (2015), “CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection”, Journal of Immunology, Vol. 194 No. 12, pp. 5980–5989.
Dale, B.A., Presland, R.B., Lewis, S.P., Underwood, R.A. and Fleckman, P. (1997), “Transient expression of epidermal filaggrin in cultured cells causes collapse of intermediate filament networks with alteration of cell shape and nuclear integrity”, Journal of Investigative Dermatology, Vol. 108 No. 2, pp. 179–187.
Dale, B. a. and Fredericks, L.P. (2005), “Antimicrobial peptides in the oral environment: expression and function in health and disease”, Curr Issues Mol Biol., Vol. 7 No. 2, pp. 119–133.
Dale, B. a, Kimball, J.R., Krisanaprakornkit, S., Roberts, F., Robinovitch, M., O’Neal, R., Valore, E. V, et al. (2001), “Localized antimicrobial peptide expression in human gingiva.”, Journal of Periodontal Research, Vol. 36 No. 5, pp. 285–94.
Dalod, M., Chelbi, R., Malissen, B. and Lawrence, T. (2014), “Dendritic cell maturation: Functional specialization through signaling specificity and transcriptional programming”, EMBO Journal, Vol. 33 No. 10, pp. 1104–1116.
Darveau, R., Pham, T.-T., Lemley, K., Reife, R., Bainbridge, B., Coats, S., Howald, W., et al. (2004), “Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4”, Infection and Immunity, Vol. 72 No. 9, pp. 5041–5051.
Darveau, R.P. (2010), “Periodontitis: a polymicrobial disruption of host homeostasis.”, Nature Reviews. Microbiology, Nature Publishing Group, Vol. 8 No. 7, pp. 481–490.
Darveau, R.P., Belton, C.M., Reife, R.A. and Lamont, R.J. (1998), “Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis”, Infection and Immunity, Vol. 66 No. 4, pp. 1660–1665.
Dashper, S.G., Ang, C.S., Veith, P.D., Mitchell, H.L., Lo, A.W.H., Seers, C. a., Walsh, K. a., et al. (2009), “Response of Porphyromonas gingivalis to heme limitation in continuous culture”, Journal of Bacteriology, Vol. 191 No. 3, pp. 1044–1055.
De-Gennaro, L.A., Lopes, J.D. and Mariano, M. (2006), “Autoantibodies directed to extracellular matrix components in patients with different clinical forms of periodontitis”, J Periodontol, Vol. 77 No. 12, pp. 2025–2030.
Deasy, M.J., Vogel, R.I., Macedo-Sobrinho, B., Gertzman, G. and Simon, B. (1980), “Familial benign chronic neutropenia associated with periodontal disease. A case report”, J Periodontol, Vol. 51 No. 4, pp. 206–210.
DeFea, K.A., Zalevsky, J., Thoma, M.S., Dery, O., Mullins, R.D. and Bunnett, N.W. (2000), “Beta-Arrestin-dependent endocytosis of proteinase-activated receptor 2 is required for intracellular targeting of activated ERK1/2”, Journal of Cell Biology, Vol. 148 No. 6,
178
pp. 1267–1281.
Devine, D.A., Marsh, P.D., Percival, R.S., Rangarajan, M. and Curtis, M.A. (1999), “Modulation of antibacterial peptide activity by products of Porphyromonas gingivalis and Prevotella spp.”, Microbiology, Vol. 145 No. 4, pp. 965–971.
Diaz, P.I., Zilm, P.S. and Rogers, A.H. (2002), “Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments”, Microbiology, Vol. 148 No. 2, pp. 467–472.
Dickson, M. a, Hahn, W.C., Ino, Y., Ronfard, V., Wu, J.Y., Weinberg, R. a, Louis, D.N., et al. (2000), “Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics.”, Molecular and Cellular Biology, Vol. 20 No. 4, pp. 1436–1447.
Dieu-Nosjean, M.C., Massacrier, C., Vanbervliet, B., Fridman, W.H. and Caux, C. (2001), “IL-10 induces CCR6 expression during Langerhans cell development while IL-4 and IFN-gamma suppress it.”, Journal of Immunology, Vol. 167 No. 10, pp. 5594–5602.
Dige, I., Raarup, M.K., Nyengaard, J.R., Kilian, M. and Nyvad, B. (2009), “Actinomyces naeslundii in initial dental biofilm formation”, Microbiology, Vol. 155 No. 7, pp. 2116–2126.
Dixon, D.R. and Darveau, R.P. (2005), “Lipopolysaccharide Heterogeneity: Innate Host Responses to Bacterial Modification of Lipid A Structure”, Journal of Dental Research, Vol. 84 No. 7, pp. 584–595.
Dommisch, H., Chung, W., Rohani, M., Williams, D., Rangarajan, M., Curtis, M. and Dale, B. (2007), “Protease-activated receptor 2 mediates human beta-defensin 2 and CC chemokine ligand 20 mRNA expression in response to proteases secreted by Porphyromonas gingivalis”, Infection and Immunity, Vol. 75 No. 9, pp. 4326–4333.
Domon, H., Honda, T., Oda, T., Yoshie, H. and Yamazaki, K. (2008), “Early and preferential induction of IL-1 receptor-associated kinase-M in THP-1 cells by LPS derived from Porphyromonas gingivalis.”, Journal of Leukocyte Biology, Vol. 83 No. 3, pp. 672–679.
Dulon, S., Leduc, D., Cottrell, G.S., D’Alayer, J., Hansen, K.K., Bunnett, N.W., Hollenberg, M.D., et al. (2005), “Pseudomonas aeruginosa elastase disables proteinase-activated receptor 2 in respiratory epithelial cells”, American Journal of Respiratory Cell and Molecular Biology, Vol. 32 No. 5, pp. 411–419.
Duncan, M.J., Nakao, S., Skobe, Z. and Xie, H. (1993), “Interactions of Porphyromonas gingivalis with epithelial cells”, Infection and Immunity, Vol. 61 No. 5, pp. 2260–2265.
Duncia, J. V., Santella, J.B., Higley, C.A., Pitts, W.J., Wityak, J., Frietze, W.E., Rankin, F.W., et al. (1998), “MEK inhibitors: The chemistry and biological activity of U0126, its analogs, and cyclization products”, Bioorganic & Medicinal Chemistry Letters, Pergamon, Vol. 8 No. 20, pp. 2839–2844.
Dunin-Horkawicz, S., Kopec, K.O. and Lupas, A.N. (2014), “Prokaryotic ancestry of eukaryotic protein networks mediating innate immunity and apoptosis”, Journal of Molecular Biology, Vol. 426 No. 7, pp. 1568–1582.
Dunne, A., Ejdebäck, M., Ludidi, P.L., O’Neill, L.A.J. and Gay, N.J. (2003), “Structural Complementarity of Toll/Interleukin-1 Receptor Domains in Toll-like Receptors and the Adaptors Mal and MyD88”, Journal of Biological Chemistry, Vol. 278 No. 42, pp. 41443–41451.
Dutzan, N., Vernal, R., Hernandez, M., Dezerega, A., Rivera, O., Silva, N., Aguillon, J.C., et al. (2009), “Levels of Interferon-Gamma and Transcription Factor T-Bet in Progressive Periodontal Lesions in Patients With Chronic Periodontitis”, Journal of Periodontology, Vol. 80 No. 2, pp. 290–296.
179
Ebersole, J.L., Cappelli, D. and Holt, S.C. (2001), “Periodontal diseases: to protect or not to protect is the question?”, Acta Odontologica Scandinavica, Vol. 59 No. 3, pp. 161–166.
Elkaim, R., Bugueno-Valdebenito, I.M., Benkirane-Jessel, N. and Tenenbaum, H. (2017), “Porphyromonas gingivalis and its lipopolysaccharide differently modulate epidermal growth factor-dependent signaling in human gingival epithelial cells.”, Journal of Oral Microbiology, Vol. 9 No. 1, p. 1334503.
Eskan, M.A., Jotwani, R., Abe, T., Chmelar, J., Lim, J.H., Liang, S., Ciero, P.A., et al. (2012), “The leukocyte integrin antagonist Del-1 inhibits IL-17-mediated inflammatory bone loss”, Nat Immunol, Vol. 13 No. 5, pp. 465–473.
Eskan, M. A., Hajishengallis, G. and Kinane, D. F. (2007) ‘Differential activation of human gingival epithelial cells and monocytes by Porphyromonas gingivalis fimbriae’, Infection and Immunity, 75(2), pp. 892–898.
Faurschou, M. and Borregaard, N. (2003), “Neutrophil granules and secretory vesicles in inflammation”, Microbes and Infection, Vol. 5 No. 14, pp. 1317–1327.
Fekonja, O., Benc•ina, M. and Jerala, R. (2012), “Toll/interleukin-1 receptor domain dimers as the platform for activation and enhanced inhibition of Toll-like receptor signaling”, Journal of Biological Chemistry, Vol. 287 No. 37, pp. 30993–31002.
Fitzgerald, K. (2010), “NLR-containing inflammasomes: Central mediators of host defense and inflammation”, European Journal of Immunology, Vol. 40 No. 3, pp. 595–598.
Fitzgerald, K., Fitzgerald, E., Palsson McDermott, A., Bowie, C., Jefferies, A., Mansell, G., Brady, E., et al. (2001), “Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction”, Nature, Vol. 413 No. 6851, pp. 78–83.
Fletcher, H.M., Schenkein, H. a., Morgan, R.M., Bailey, K. a., Berry, C.R. and Macrina, F.L. (1995), “Virulence of a Porphyromonas gingivalis W83 mutant defective in the prtH gene”, Infection and Immunity, Vol. 63 No. 4, pp. 1521–1528.
Ford, P.J., Gemmell, E., Hamlet, S.M., Hasan, A., Walker, P.J., West, M.J., Cullinan, M.P., et al. (2005), “Cross-reactivity of GroEL antibodies with human heat shock protein 60 and quantification of pathogens in atherosclerosis”, Oral Microbiology and Immunology, Vol. 20 No. 5, pp. 296–302.
Franchi, L., Franchi, N., Warner, K., Viani, G. and Nuñez. (2009), “Function of Nod-like receptors in microbial recognition and host defense”, Immunological Reviews, Vol. 227 No. 1, pp. 106–128.
Frick, I.-M., Nordin, S.L., Baumgarten, M., Mörgelin, M., Sørensen, O.E., Olin, A.I. and Egesten, A. (2011), “Constitutive and inflammation-dependent antimicrobial peptides produced by epithelium are differentially processed and inactivated by the commensal Finegoldia magna and the pathogen Streptococcus pyogenes.”, Journal of Immunology, Vol. 187 No. 8, pp. 4300–4309.
Fuchs, E. and Green, H. (1980), “Changes in keratin gene expression during terminal differentiation of the keratinocyte”, Cell, Vol. 19 No. 4, pp. 1033–1042.
Gangloff, M. (2012), “Different dimerisation mode for TLR4 upon endosomal acidification?”, Trends in Biochemical Sciences, Vol. 37 No. 3, pp. 92–98.
Garlet, G.P., Martins, W., Ferreira, B.R., Milanezi, C.M. and Silva, J.S. (2003), “Patterns of chemokines and chemokine receptors expression in different forms of human periodontal disease.”, Journal of Periodontal Research, Vol. 38 No. 2, pp. 210–217.
Gaspersic, R., Stiblar-Martincic, D., Osredkar, J. and Skaleric, U. (2003), “Influence of subcutaneous administration of recombinant TNF-alpha on ligature-induced periodontitis in rats.”, Journal of Periodontal Research, Vol. 38 No. 2, pp. 198–203.
Gay, N.J., Symmons, M.F., Gangloff, M. and Bryant, C.E. (2014), “Assembly and localization
180
of Toll-like receptor signalling complexes”, Nature Reviews Immunology, Vol. 14 No. 8, pp. 546–558.
Geissmann, F., Auffray, C., Palframan, R., Wirrig, C., Ciocca, A., Campisi, L., Narni-Mancinelli, E., et al. (2008), “Blood monocytes: distinct subsets, how they relate to dendritic cells, and their possible roles in the regulation of T-cell responses”, Immunol Cell Biol, Vol. 86 No. 5, pp. 398–408.
Geissmann, F., Jung, S. and Littman, D.R. (2003), “Blood monocytes consist of two principal subsets with distinct migratory properties”, Immunity, Vol. 19 No. 1, pp. 71–82.
Gemmell, E. and Seymour, G.J. (1998), “Cytokine Profiles of Cells Extracted from Humans with Periodontal Diseases”, Journal of Dental Research, Vol. 77 No. 1, pp. 16–26.
Genco, R.J. and Van Dyke, T.E. (2010), “Prevention: Reducing the risk of CVD in patients with periodontitis.”, Nature Reviews. Cardiology, Vol. 7 No. 9, pp. 479–480.
Ghosh, S.K., Feng, Z., Fujioka, H., Lux, R., McCormick, T.S. and Weinberg, A. (2018), “Conceptual Perspectives: Bacterial Antimicrobial Peptide Induction as a Novel Strategy for Symbiosis with the Human Host”, Frontiers in Microbiology, Vol. 9 No. February, pp. 1–8.
Giacaman, R. a., Asrani, A.C., Ross, K.F. and Herzberg, M.C. (2009), “Cleavage of protease-activated receptors on an immortalized oral epithelial cell line by Porphyromonas gingivalis gingipains”, Microbiology, Vol. 155 No. 10, pp. 3238–3246.
Gibbs, R.S. (2001), “The relationship between infections and adverse pregnancy outcomes: an overview.”, Annals of Periodontology, Vol. 6 No. 1, pp. 153–163.
Gorasia, D.G., Veith, P.D., Chen, D., Seers, C.A., Mitchell, H.A., Chen, Y.Y., Glew, M.D., et al. (2015), “Porphyromonas gingivalis Type IX Secretion Substrates Are Cleaved and Modified by a Sortase-Like Mechanism”, PLoS Pathogens, Vol. 11 No. 9, pp. 1–31.
Gordon, S. (2003), “Alternative activation of macrophages.”, Nature Reviews. Immunology, Vol. 3 No. 1, pp. 23–35.
Graham, F.L., Smiley, J., Russell, W.C. and Nairn, R. (1977), “Characteristics of a human cell line transformed by DNA from human adenovirus type 5.”, The Journal of General Virology, Vol. 36 No. 1, pp. 59–74.
Grassi, F., Cristino, S., Toneguzzi, S., Piacentini, A., Facchini, A. and Lisignoli, G. (2004), “CXCL12 Chemokine Up-Regulates Bone Resorption and MMP-9 Release by Human Osteoclasts: CXCL12 Levels Are Increased in Synovial and Bone Tissue of Rheumatoid Arthritis Patients”, Journal of Cellular Physiology, Vol. 199 No. 2, pp. 244–251.
Graves, D. (2008), “Cytokines That Promote Periodontal Tissue Destruction”, Journal of Periodontology, Vol. 79 No. 8s, pp. 1585–1591.
Graves, D.T. and Cochran. (2003), “The Contribution of Interleukin-1 and Tumor Necrosis Factor to Periodontal Tissue Destruction”, Journal of Periodontology, Vol. 74 No. 3, pp. 391–401.
Grayson, R., Douglas, C.W.I., Heath, J., Rawlinson, a and Evans, G.S. (2003), “Activation of human matrix metalloproteinase 2 by gingival crevicular fluid and Porphyromonas gingivalis.”, Journal of Clinical Periodontology, Vol. 30 No. 6, pp. 542–50.
Greer, A., Zenobia, C. and Darveau, R.P. (2013), “Defensins And LL-37: A Review Of Function In The Gingival Epithelium”, Periodontology 2000, Vol. 63 No. 1, pp. 67–79.
Griffen, A.L., Becker, M.R., Lyons, S.R., Moeschberger, M.L. and Leys, E.J. (1998), “Prevalence of Porphyromonas gingivalis and periodontal health status.”, Journal of Clinical Microbiology, Vol. 36 No. 11, pp. 3239–42.
Griffen, A. L., Beall, C. J., Campbell, J. H., Firestone, N. D., Kumar, P. S., Yang, Z. K., Leys, E. J. (2012). Distinct and complex bacterial profiles in human periodontitis and health
181
revealed by 16S pyrosequencing. ISME Journal.
Groom, J.R. and Luster, A.D. (2011), “CXCR3 ligands: redundant, collaborative and antagonistic functions”, Immunology and Cell Biology, Vol. 89 No. 2, pp. 207–215.
Gui, M.J., Dashper, S.G., Slakeski, N., Chen, Y.Y. and Reynolds, E.C. (2015), “Spheres of influence: Porphyromonas gingivalis outer membrane vesicles”, Molecular Oral Microbiology, pp. 1–14.
Haffajee, a D., Cugini, M. a, Dibart, S., Smith, C., Kent, R.L. and Socransky, S.S. (1997), “Clinical and microbiological features of subjects with adult periodontitis who responded poorly to scaling and root planing.”, Journal of Clinical Periodontology, Vol. 24 No. 10, pp. 767–76.
Hajishengallis, G. (2009), “Porphyromonas gingivalis-host interactions: open war or intelligent guerilla tactics?”, Microbes and Infection, Vol. 11 No. 6–7, pp. 637–645.
Hajishengallis, G. (2010), “Complement and periodontitis”, Biochemical Pharmacology.
Hajishengallis, G. (2014), “Immunomicrobial pathogenesis of periodontitis: Keystones, pathobionts, and host response”, Trends in Immunology, Vol. 35 No. 1, pp. 3–11.
Hajishengallis, G. (2015), “Periodontitis : from microbial immune subversion to systemic inflammation”, Nature Publishing Group, Vol. 15 No. 1, pp. 30–44.
Hajishengallis, G., Hajishengallis, R., Darveau, M. and Curtis. (2012), “The keystone-pathogen hypothesis”, Nature Reviews Microbiology, Vol. 10 No. 10, pp. 717–725.
Hajishengallis, G. and Lambris, J.D. (2010), “Crosstalk pathways between Toll-like receptors and the complement system”, Trends in Immunology, Vol. 31 No. 4, pp. 154–163.
Hajishengallis, G. and Lamont, R.J. (2012), “Beyond the red complex and into more complexity: The polymicrobial synergy and dysbiosis (PSD) model of periodontal disease etiology”, Molecular Oral Microbiology, Vol. 27 No. 6, pp. 409–419.
Hajishengallis, G. and Lamont, R.J. (2014), “Breaking bad: Manipulation of the host response by Porphyromonas gingivalis”, European Journal of Immunology, Vol. 44 No. 2, pp. 328–338.
Hajishengallis, G., Liang, S., Payne, M.A., Hashim, A., Jotwani, R., Eskan, M.A., McIntosh, M.L., et al. (2011), “Low-abundance biofilm species orchestrates inflammatory periodontal disease through the commensal microbiota and complement”, Cell Host and Microbe, Vol. 10 No. 5, pp. 497–506.
Hajishengallis, G., Shakhatreh, M.-A.K., Wang, M. and Liang, S. (2007), “Complement Receptor 3 Blockade Promotes Complement Receptor 3 Blockade Promotes IL-12-Mediated Clearance of Porphyromonas gingivalis and Negates Its Virulence In Vivo 1”, Journal of Immunology, Vol. 179 No. 4, pp. 2359–2367.
Hajishengallis, G., Wang, M. and Liang, S. (2009), “Induction of distinct TLR2-mediated proinflammatory and proadhesive signaling pathways in response to Porphyromonas gingivalis fimbriae.”, Journal of Immunology, Vol. 182 No. 11, pp. 6690–6696.
Hajishengallis, G., Wang, M., Liang, S., Triantafilou, M. and Triantafilou, K. (2008), “Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 105 No. 36, pp. 13532–13537.
Hamilton, J.A. (2008), “Colony-stimulating factors in inflammation and autoimmunity”, Nature Reviews Immunology, Vol. 8 No. 7, pp. 533–544.
Hansen, K.K., Saifeddine, M. and Hollenberg, M.D. (2004), “Tethered ligand-derived peptides of proteinase-activated receptor 3 (PAR3) activate PAR1 and PAR2 in Jurkat T cells”, Immunology, Vol. 112 No. 2, pp. 183–190.
182
Hara, T. and Tanegashima, K. (2014), “CXCL14 antagonizes the CXCL12-CXCR4 signaling axis”, Biomolecular Concepts, Vol. 5 No. 2, pp. 167–173.
Haraszthy, V.I., Zambon, J.J., Trevisan, M., Zeid, M. and Genco, R.J. (2000), “Identification of Periodontal Pathogens in Atheromatosus Plaque”, J Periodontol, Vol. 71 No. 10, pp. 1554–1560.
Harding, C. V. and Boom, W.H. (2010), “Regulation of antigen presentation by Mycobacterium tuberculosis: A role for Toll-like receptors”, Nature Reviews Microbiology, Vol. 8 No. 4, pp. 296–307.
Harokopakis, E. and Hajishengallis, G. (2005), “Integrin activation by bacterial fimbriae through a pathway involving CD14, tool-like receptor 2, and phosphatidylinositol-3-kinase”, European Journal of Immunology, Vol. 35 No. 4, pp. 1201–1210.
Harrington, L.E., Hatton, R.D., Mangan, P.R., Turner, H., Murphy, T.L., Murphy, K.M. and Weaver, C.T. (2005), “Interleukin 17–producing CD4+ effector T cells develop via a lineage distinct from the T helper type 1 and 2 lineages”, Nature Immunology, Vol. 6 No. 11, pp. 1123–1132.
Hasegawa, Y., Tribble, G.D., Baker, H. V., Mans, J.J., Handfield, M. and Lamont, R.J. (2008), “Role of Porphyromonas gingivalis SerB in gingival epithelial cell cytoskeletal remodeling and cytokine production”, Infection and Immunity, Vol. 76 No. 6, pp. 2420–2427.
Hashimoto, M., Ogawa, S., Asai, Y., Takai, Y. and Ogawa, T. (2003), “Binding of Porphyromonas gingivalis fimbriae to Treponema denticola dentilisin”, FEMS Microbiology Letters, Vol. 226 No. 2, pp. 267–271.
Hastürk, H., Tezcan, I., Yel, L., Ersoy, F., Sanal, O., Yamalik, N. and Berker, E. (1998), “A case of chronic severe neutropenia: oral findings and consequences of short-term granulocyte colony-stimulating factor treatment.”, Australian Dental Journal, Vol. 43 No. 1, pp. 9–13.
Hatakeyama, S., Yaegashi, T., Oikawa, Y., Fujiwara, H., Mikami, T., Takeda, Y., & Satoh, M. (2006). Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epithelia. Journal of Periodontal Research.
Havens, A.M., Chiu, E., Taba, M., Wang, J., Shiozawa, Y., Jung, Y., Taichman, L.S., et al. (2008), “Stromal-derived factor-1alpha (CXCL12) levels increase in periodontal disease.”, Journal of Periodontology, Vol. 79 No. 5, pp. 845–853.
Hayashi, F., Smith, K.D., Ozinsky, a, Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng, J.K., et al. (2001), “The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5.”, Nature, Vol. 410 No. 6832, pp. 1099–1103.
Hayashi, Y., Murakami, M., Kawamura, R., Ishizaka, R., Fukuta, O. and Nakashima, M. (2015), “CXCL14 and MCP1 are potent trophic factors associated with cell migration and angiogenesis leading to higher regenerative potential of dental pulp side population cells.”, Stem Cell Research & Therapy, Vol. 6, p. 111.
Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., et al. (2000), “A Toll-like receptor recognizes bacterial DNA.”, Nature, Vol. 408 No. 6813, pp. 740–745.
Henikoff, S. and Henikoff, J.G. (1992), “Amino acid substitution matrices from protein blocks.”, Proceedings of the National Academy of Sciences, Vol. 89 No. 22, pp. 10915–10919.
Henkart, P.A. (1994), “Lymphocyte-mediated cytotoxicity: Two pathways and multiple effector molecules”, Immunity, Vol. 1 No. 5, pp. 343–346.
Hieshima, K., Ohtani, H., Shibano, M., Izawa, D., Nakayama, T., Kawasaki, Y., Shiba, F., et al. (2003), “CCL28 has dual roles in mucosal immunity as a chemokine with broad-
183
spectrum antimicrobial activity.”, Journal of Immunology, Vol. 170 No. 3, pp. 1452–61.
Highfield, J. (2009), “Diagnosis and classification of periodontal disease.”, Australian Dental Journal, Vol. 54 Suppl 1, pp. S11-26.
Hinode, D., Nagata, A., Ichimiya, S., Hayashi, H., Morioka, M. and Nakamura, R. (1992), “Generation of plasma kinin by three types of protease isolated from Porphyromonas gingivalis 381”, Archives of Oral Biology, Vol. 37 No. 10, pp. 859–861.
Hofbauer, L.C., Lacey, D.L., Dunstan, C.R., Spelsberg, T.C., Riggs, B.L. and Khosla, S. (1999), “Interleukin-1B and tumor necrosis factor-a, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells”, Bone, Vol. 25 No. 3, pp. 255–259.
Holden, J.A., Attard, T.J., Laughton, K.M., Mansell, A., O’Brien-Simpson, N.M. and Reynolds, E.C. (2014), “Porphyromonas gingivalis lipopolysaccharide weakly activates M1 and M2 polarized mouse macrophages but induces inflammatory cytokines”, Infection and Immunity, Vol. 82 No. 10, pp. 4190–4203.
Holt, S.C., Kesavalu, L., Walker, S. and Genco, C. a. (1999), “Virulence factors of Porphyromonas gingivalis.”, Periodontology 2000, Vol. 20, pp. 168–238.
Holzhausen, M., Spolidorio, L.C., Ellen, R.P., Jobin, M.-C., Steinhoff, M., Andrade-Gordon, P. and Vergnolle, N. (2006), “Protease-Activated Receptor-2 Activation: A major role in the pathogenesis of Porphyromonas gingivalis Infection”, The American Journal of Pathology, Vol. 168 No. 4, pp. 1189–1199.
Hong, B.Y., Araujo, M.V.F., Strausbaugh, L.D., Terzi, E., Ioannidou, E. and Diaz, P.I. (2015), “Microbiome profiles in periodontitis in relation to host and disease characteristics”, PLoS ONE, Vol. 10 No. 5, p. e0127077.
Hoover, D.M., Boulegue, C., Yang, D., Oppenheim, J.J., Tucker, K., Lu, W. and Lubkowski, J. (2002), “The structure of human macrophage inflammatory protein-3alpha /CCL20. Linking antimicrobial and CC chemokine receptor-6-binding activities with human beta-defensins.”, The Journal of Biological Chemistry, Vol. 277 No. 40, pp. 37647–54.
Horng, T., Barton, G.M., Flavell, R.A. and Medzhitov, R. (2002), “The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors”, Nature, Vol. 420 No. 6913, pp. 329–333.
Hoshino, K., Takeuchi, O., Kawai, T., Sanjo, H., Ogawa, T., Takeda, Y., Takeda, K., et al. (1999), “Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product.”, Journal of Immunology, Vol. 162 No. 7, pp. 3749–3752.
Hou, L., Sasakj, H. and Stashenko, P. (2000), “B-cell deficiency predisposes mice to disseminating anaerobic infections: Protection by passive antibody transfer”, Infection and Immunity, Vol. 68 No. 10, pp. 5645–5651.
How, K.Y., Song, K.P. and Chan, K.G. (2016), “Porphyromonas gingivalis: An Overview of Periodontopathic Pathogen below the Gum Line”, Frontiers in Microbiology, Vol. 7 No. February, pp. 1–14.
Hromas, R., Hromas, H., Broxmeyer, C., Kim, H., Nakshatri, K., Christopherson, M., Azam, Y.-H., et al. (1999), “Cloning of BRAK, a Novel Divergent CXC Chemokine Preferentially Expressed in Normal versus Malignant Cells”, Biochemical and Biophysical Research Communications, Vol. 255 No. 3, pp. 703–706.
Hua, F., Ha, T., Ma, J., Gao, X., Kelley, J., Williams, D.L., Browder, I.W., et al. (2005), “Blocking the MyD88-dependent pathway protects the myocardium from ischemia/reperfusion injury in rat hearts”, Biochemical and Biophysical Research Communications, Vol. 338 No. 2, pp. 1118–1125.
Hunter, S., Apweiler, R., Attwood, T.K., Bairoch, A., Bateman, A., Binns, D., Bork, P., et al.
184
(2009), “InterPro: The integrative protein signature database”, Nucleic Acids Research, Vol. 37, available at:https://doi.org/10.1093/nar/gkn785.
Huq, N.L., Seers, C.A., Toh, E.C.Y., Dashper, S.G., Slakeski, N., Zhang, L., Ward, B.R., et al. (2013), “Propeptide-Mediated Inhibition of Cognate Gingipain Proteinases”, PLoS ONE, Vol. 8 No. 6, available at:https://doi.org/10.1371/journal.pone.0065447.
Husebye, H., Halaas, Ø., Stenmark, H., Tunheim, G., Sandanger, Ø., Bogen, B., Brech, A., et al. (2006), “Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity”, The EMBO Journal, Vol. 25 No. 4, pp. 683–692.
Huynh, J., Scholz, G.M., Aw, J., Kwa, M.Q., Achuthan, A., Hamilton, J.A. and Reynolds, E.C. (2016), “IRF6 Regulates the Expression of IL-36 by Human Oral Epithelial Cells in Response to Porphyromonas gingivalis”, Journal of Immunology, Vol. 196 No. 5, pp. 2230–2238.
Huynh, K.K. and Grinstein, S. (2007), “Regulation of Vacuolar pH and Its Modulation by Some Microbial Species”, Microbiology and Molecular Biology Reviews, Vol. 71 No. 3, pp. 452–462.
Imamura, T. (2003), “The role of gingipains in the pathogenesis of periodontal disease.”, Journal of Periodontology, Vol. 74 No. 1, pp. 111–118.
Imamura, T., Pike, R.N., Potempa, J. and Travis, J. (1994), “Pathogenesis of periodontitis: A major arginine-specific cysteine proteinase from Porphyromonas gingivalis induces vascular permeability enhancement through activation of the kallikrein/kinin pathway”, Journal of Clinical Investigation, Vol. 94 No. 1, pp. 361–367.
Imamura, T., Travis, J. and Potempa, J. (2003), “The Biphasic Virulence Activities of Gingipains: Activation and Inactivation of Host Proteins”, Current Protein & Peptide Science, Vol. 4 No. 6, pp. 443–450.
Inohara, N., Ogura, Y., Fontalba, A., Gutierrez, O., Pons, F., Crespo, J., Fukase, K., et al. (2003), “Host recognition of bacterial muramyl dipeptide mediated through NOD2: Implications for Crohn’s disease”, Journal of Biological Chemistry, Vol. 278 No. 8, pp. 5509–5512.
Into, T., Inomata, M., Niida, S., Murakami, Y. and Shibata, K.I. (2010), “Regulation of MyD88 aggregation and the MyD88-dependent signaling pathway by sequestosome 1 and histone deacetylase 6”, Journal of Biological Chemistry, Vol. 285 No. 46, pp. 35759–35769.
Ivanyi, L. and Lehner, T. (1970), “Stimulation of lymphocyte transformation by bacterial antigens in patients with periodontal disease”, Archives of Oral Biology, Vol. 15 No. 11, pp. 1089–1096.
Jakka, P., Namani, S., Murugan, S., Rai, N. and Radhakrishnan, G. (2017), “The Brucella effector protein TcpB induces degradation of inflammatory caspases and thereby subverts noncanonical inflammasome activation in macrophages”, Journal of Biological Chemistry, Vol. 292 No. 50, pp. 20613–20627.
Janssens, S., Burns, K., Tschopp, J. and Beyaert, R. (2002), “Regulation of interleukin-1- and lipopolysaccharide-induced NF-??B activation by alternative splicing of MyD88”, Current Biology, Vol. 12 No. 6, pp. 467–471.
Jeffcoat, M.K., Geurs, N.C., Reddy, M.S., Cliver, S.P., Goldenberg, R.L., Hauth, J.C., McCormick, M., et al. (2001), “Periodontal infection and preterm birth: results of a prospective study.”, Journal of the American Dental Association (1939), Vol. 132 No. 7, pp. 875–80.
Jenkins, K., Jenkins, A. and Mansell. (2010), “TIR-containing adaptors in Toll-like receptor signalling”, Cytokine, Vol. 49 No. 3, pp. 237–244.
Jepsen, K. and Jepsen, S. (2016), “Antibiotics/antimicrobials: Systemic and local administration in the therapy of mild to moderately advanced periodontitis”,
185
Periodontology 2000, Vol. 71 No. 1, pp. 82–112.
Jiang, Z., Mak, T.W., Sen, G. and Li, X. (2004), “Toll-like receptor 3-mediated activation of NF-kappaB and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-beta.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 101 No. 10, pp. 3533–3538.
Joly, S., Maze, C., McCray, P.B. and Guthmiller, J.M. (2004), “Human β-Defensins 2 and 3 Demonstrate Strain-Selective Activity against Oral Microorganisms”, Journal of Clinical Microbiology, Vol. 42 No. 3, pp. 1024–1029.
Jotwani, R., Eswaran, S.V.K., Moonga, S. and Cutler, C.W. (2010), “MMP-9/TIMP-1 imbalance induced in human dendritic cells by Porphyromonas gingivalis”, FEMS Immunology and Medical Microbiology, Vol. 58 No. 3, pp. 314–321.
Jotwani, R., Pulendran, B., Agrawal, S. and Cutler, C.W. (2003), “Human dendritic cells respond to Porphyromonas gingivalis LPS by promoting a Th2 effector response in vitro”, European Journal of Immunology, Vol. 33 No. 11, pp. 2980–2986.
Kadowaki, T., Nakayama, K., Yoshimura, F., Okamoto, K., Abe, N. and Yamamoto, K. (1998), “Arg-gingipain Acts as a Major Processing Enzyme for Various Cell Surface Proteins in Porphyromonas gingivalis”, Journal of Biological Chemistry, Vol. 273 No. 44, pp. 29072–29076.
Kagan, J., Kagan, R. and Medzhitov. (2006), “Phosphoinositide-Mediated Adaptor Recruitment Controls Toll-like Receptor Signaling”, Cell, Vol. 125 No. 5, pp. 943–955.
Kagan, J.C., Su, T., Horng, T., Chow, A., Akira, S. and Medzhitov, R. (2008), “TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-β”, Nature Immunology, Vol. 9 No. 4, pp. 361–368.
Kägi, D., Vignaux, F., Ledermann, B., Bürki, K., Depraetere, V., Nagata, S., Hengartner, H., et al. (1994), “Fas and perforin pathways as major mechanisms of T cell-mediated cytotoxicity.”, Science, Vol. 265 No. 5171, pp. 528–530.
Kanaya, S., Nemoto, E., Ogawa, T. and Shimauchi, H. (2009), “Porphyromonas gingivalis fimbriae induce unique dendritic cell subsets via Toll-like receptor 2”, Journal of Periodontal Research, Vol. 44 No. 4, pp. 543–549.
Kanaya, S., Nemoto, E., Tomohiko, O. and Shimauchi, H. (2004), “Porphyromonas gingivalis lipopolysaccharides induce maturation of dendritic cells with CD14+CD16+ phenotype”, European Journal of Immunology, Vol. 34 No. 5, pp. 1451–1460.
Kaplan, C.W., Lux, R., Haake, S.K. and Shi, W. (2009), “The Fusobacterium nucleatum outer membrane protein RadD is an arginine-inhibitable adhesin required for inter-species adherence and the structured architecture of multispecies biofilm”, Molecular Microbiology, Vol. 71 No. 1, pp. 35–47.
Kathariou, S., Kanenaka, R., Allen, R.D., Fok, A.K. and Mizumoto, C. (1995), “Repression of motility and flagellin production at 37 degrees C is stronger in Listeria monocytogenes than in the nonpathogenic species Listeria innocua.”, Canadian Journal of Microbiology, Vol. 41 No. 7, pp. 572–577.
Katz, J., Yang, Q.B., Zhang, P., Potempa, J., Travis, J., Michalek, S.M. and Balkovetz, D.F. (2002), “Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains”, Infection and Immunity, Vol. 70 No. 5, pp. 2512–2518.
Kawada, M., Yoshida, A., Suzuki, N., Nakano, Y., Saito, T., Oho, T. and Koga, T. (2004), “Prevalence of Porphyromonas gingivalis in relation to periodontal status assessed by real-time PCR”, Oral Microbiology and Immunology, Vol. 19 No. 5, pp. 289–292.
Kawai, T., Adachi, O., Ogawa, T., Takeda, K. and Akira, S. (1999), “Unresponsiveness of MyD88-deficient mice to endotoxin”, Immunity, Vol. 11 No. 1, pp. 115–122.
186
Kawasaki, K., Ernst, R.K. and Miller, S.I. (2004), “3-O-Deacylation of Lipid A by PagL, a PhoP/PhoQ-regulated Deacylase of Salmonella typhimurium, Modulates Signaling through Toll-like Receptor 4”, Journal of Biological Chemistry, Vol. 279 No. 19, pp. 20044–20048.
Kayagaki, N., Warming, S., Lamkanfi, M., Walle, L. Vande, Louie, S., Dong, J., Newton, K., et al. (2011), “Non-canonical inflammasome activation targets caspase-11”, Nature, Vol. 479 No. 7371, pp. 117–121.
Kellermann, S.A., Hudak, S., Oldham, E.R., Liu, Y.J. and McEvoy, L.M. (1999), “The CC chemokine receptor-7 ligands 6Ckine and macrophage inflammatory protein-3 beta are potent chemoattractants for in vitro- and in vivo-derived dendritic cells.”, Journal of Immunology, Vol. 162 No. 7, pp. 3859–3864.
Khlgatian, M., Nassar, H., Chou, H.-H., Gibson, F.C. and Genco, C.A. (2002), “Fimbria-dependent activation of cell adhesion molecule expression in Porphyromonas gingivalis-infected endothelial cells.”, Infection and Immunity, Vol. 70 No. 1, pp. 257–67.
Kida, Y., Higashimoto, Y., Inoue, H., Shimizu, T. and Kuwano, K. (2008), “A novel secreted protease from Pseudomonas aeruginosa activates NF-KB through protease-activated receptors”, Cellular Microbiology, Vol. 10 No. 7, pp. 1491–1504.
Kidd, P. (2003), “Th1/Th2 balance: The hypothesis, its limitations, and implications for health and disease”, Alternative Medicine Review, Vol. 8 No. 3, pp. 223–246.
Kirschner, N., Rosenthal, R., Furuse, M., Moll, I., Fromm, M. and Brandner, J.M. (2013), “Contribution of Tight Junction Proteins to Ion, Macromolecule, and Water Barrier in Keratinocytes”, Journal of Investigative Dermatology, Vol. 133 No. 10, pp. 1161–1169.
Kishore, S.P., Bungum, M.K., Platt, J.L. and Brunn, G.J. (2005), “Selective suppression of Toll-like receptor 4 activation by chemokine receptor 4”, FEBS Letters, Vol. 579 No. 3, pp. 699–704.
Klausen, B., Hougen, H.P. and Fiehn, N.E. (1989), “Increased periodontal bone loss in temporarily B lymphocyte-deficient rats.”, Journal of Periodontal Research, Vol. 24 No. 6, pp. 384–390.
Kobayashi, Y. (2006), “Neutrophil infiltration and chemokines.”, Critical Reviews in Immunology, Vol. 26 No. 4, pp. 307–16.
Kobe, B. and Kajava, A. V. (2001), “The leucine-rich repeat as a protein recognition motif”, Current Opinion in Structural Biology, Vol. 11 No. 6, pp. 725–732.
Kolaczkowska, E. and Kubes, P. (2013), “Neutrophil recruitment and function in health and inflammation”, Nature Reviews. Immunology, Vol. 13 No. 3, pp. 159–75.
Kolenbrander, P.E., Palmer, R.J., Periasamy, S. and Jakubovics, N.S. (2010), “Oral multispecies biofilm development and the key role of cell–cell distance”, Nature Reviews Microbiology, Nature Publishing Group, Vol. 8 No. 7, pp. 471–480.
Koonin, E. V. and Aravind, L. (2002), “Origin and evolution of eukaryotic apoptosis: The bacterial connection”, Cell Death and Differentiation, Vol. 9 No. 4, pp. 394–404.
Koziel, J., Mydel, P. and Potempa, J. (2014), “The link between periodontal disease and rheumatoid arthritis: an updated review.”, Current Rheumatology Reports, Vol. 16 No. 3, p. 408.
Krautgartner, W.D. and Vitkov, L. (2008), “Visualization of neutrophil extracellular traps in TEM”, Micron, Vol. 39 No. 4, pp. 367–372.
Krieg, A., Correa, R.G., Garrison, J.B., Le Negrate, G., Welsh, K., Huang, Z., Knoefel, W.T., et al. (2009), “XIAP mediates NOD signaling via interaction with RIP2”, Proceedings of the National Academy of Sciences, Vol. 106 No. 34, pp. 14524–14529.
187
Krisanaprakornkit, S., Kimball, J.R., Weinberg, a, Darveau, R.P., Bainbridge, B.W. and Dale, B. a. (2000), “Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier”, Infection and Immunity, Vol. 68 No. 5, pp. 2907–2915.
Kurth, I., Willimann, K., Schaerli, P., Hunziker, T., Clark Lewis, I. and Moser, B. (2001), “Monocyte selectivity and tissue localization suggests a role for breast and kidney-expressed chemokine (BRAK) in macrophage development”, The Journal of Experimental Medicine, Vol. 194 No. 6, pp. 855–861.
Kwa, M.Q., Nguyen, T., Huynh, J., Ramnath, D., De Nardo, D., Lam, P.Y., Reynolds, E.C., et al. (2014), “Interferon regulatory factor 6 differentially regulates toll-like receptor 2-dependent chemokine gene expression in epithelial cells”, Journal of Biological Chemistry, Vol. 289 No. 28, pp. 19758–19768.
Lam, R.S., O’Brien-Simpson, N.M., Lenzo, J.C., Holden, J.A., Brammar, G.C., Walsh, K.A., McNaughtan, J.E., et al. (2014), “Macrophage Depletion Abates Porphyromonas gingivalis –Induced Alveolar Bone Resorption in Mice”, Journal of Immunology, Vol. 193 No. 5, pp. 2349–2362.
Lamont, R.J. and Hajishengallis, G. (2015), “Polymicrobial synergy and dysbiosis in inflammatory disease”, Trends in Molecular Medicine, Vol. 21 No. 3, pp. 172–183.
Lamont, R.J., Lamont, C.A., Bevan, S., Gil, R.E., Persson, B. and Rosan. (1993), “Involvement of Porphyromonas gingivalis fimbriae in adherence to Streptococcus gordonii”, Oral Microbiology and Immunology, Vol. 8 No. 5, pp. 272–276.
Lamster, I.B., Celenti, R. and Ebersole, J.L. (1990), “The relationship of serum IgG antibody titers to periodontal pathogens to indicators of the host response in crevicular fluid”, J Clin Periodontol, Vol. 17 No. 7 Pt 1, pp. 419–425.
Lappin, D., Kjeldsen, M., Sander, L. and Df, K. (2000), “Inducible nitric oxide synthase expression in periodontitis”, Journal of Periodontal Research, Vol. 35 No. 6, pp. 369–373.
Lappin, D.F., Macleod, C.P., Kerr, A., Mitchell, T. and Kinane, D.F. (2001), “Anti-inflammatory cytokine IL-10 and T cell cytokine profile in periodontitis granulation tissue”, Clinical and Experimental Immunology, Vol. 123 No. 2, pp. 294–300.
Latz, E., Xiao, T.S. and Stutz, A. (2013), “Activation and regulation of the inflammasomes.”, Nature Reviews. Immunology, Vol. 13 No. 6, pp. 397–411.
Lee, P.Y., Wang, J.-X., Parisini, E., Dascher, C.C. and Nigrovic, P.A. (2013), “Ly6 family proteins in neutrophil biology”, Journal of Leukocyte Biology, Vol. 94 No. 4, pp. 585–594.
Lee, Y.S., Park, J.S., Kim, J.H., Jung, S.M., Lee, J.Y., Kim, S.-J. and Park, S.H. (2011), “Smad6-specific recruitment of Smurf E3 ligases mediates TGF-β1-induced degradation of MyD88 in TLR4 signalling”, Nature Communications, available at:https://doi.org/10.1038/ncomms1469.
Li, J., Helmerhorst, E.J., Leone, C.W., Troxler, R.F., Yaskell, T., Haffajee, A.D., Socransky, S.S., et al. (2004), “Identification of early microbial colonizers in human dental biofilm”, Journal of Applied Microbiology, Vol. 97 No. 6, pp. 1311–1318.
Li, M., Firth, J.D. and Putnins, E.E. (2005), “Keratinocyte growth factor-1 expression in healthy and diseased human periodontal tissues”, Journal of Periodontal Research, Vol. 40 No. 2, pp. 118–128.
Liang, C.-C., Park, A.Y. and Guan, J.-L. (2007), “In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro”, Nature Protocols, Vol. 2 No. 2, pp. 329–333.
188
Liang, S., Krauss, J.L., Domon, H., McIntosh, M.L., Hosur, K.B., Qu, H., Li, F., et al. (2011), “The C5a Receptor Impairs IL-12-Dependent Clearance of Porphyromonas gingivalis and Is Required for Induction of Periodontal Bone Loss”, Journal of Immunology, Vol. 186 No. 2, pp. 869–877.
Liang, S.C., Tan, X.-Y., Luxenberg, D.P., Karim, R., Dunussi-Joannopoulos, K., Collins, M. and Fouser, L.A. (2006), “Interleukin (IL)-22 and IL-17 are coexpressed by Th17 cells and cooperatively enhance expression of antimicrobial peptides”, The Journal of Experimental Medicine, Vol. 203 No. 10, pp. 2271–2279.
Lichtenberger, B.M., Gerber, P. a, Holcmann, M., Buhren, B. a, Amberg, N., Smolle, V., Schrumpf, H., et al. (2013), “Epidermal EGFR controls cutaneous host defense and prevents inflammation.”, Science Translational Medicine, Vol. 5 No. 199, p. 199ra111.
Lin, C.L., Suri, R.M., Rahdon, R. a, Austyn, J.M. and Roake, J. a. (1998), “Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation.”, European Journal of Immunology, Vol. 28 No. 12, pp. 4114–22.
Lindberg, J., af Klint, E., Catrina, A.I., Nilsson, P., Klareskog, L., Ulfgren, A.-K. and Lundeberg, J. (2006), “Effect of infliximab on mRNA expression profiles in synovial tissue of rheumatoid arthritis patients.”, Arthritis Research & Therapy, Vol. 8 No. 6, p. R179.
Liu, C., Hashizume, T., Kurita-Ochiai, T., Fujihashi, K. and Yamamoto, M. (2010), “Oral immunization with Porphyromonas gingivalis outer membrane protein and CpGoligodeoxynucleotides elicits T helper 1 and 2 cytokines for enhanced protective immunity.”, Molecular Oral Microbiology, Vol. 25 No. 3, pp. 178–89.
Loesche, W.J. (1979), “Clinical and microbiological aspects of chemotherapeutic agents used according to the specific plaque hypothesis.”, Journal of Dental Research, Vol. 58 No. 12, pp. 2404–2412.
Loesche, W.J., Bradbury, D.R. and Woolfolk, M.P. (1977), “Reduction of Dental Decay in Rampant Caries Individuals Following Short-Term Kanamycin Treatment”, Journal of Dental Research, Vol. 56 No. 3, pp. 254–265.
Loos, B.G., Craandijk, J., Hoek, F.J., Wertheim-van Dillen, P.M. and van der Velden, U. (2000), “Elevation of systemic markers related to cardiovascular diseases in the peripheral blood of periodontitis patients.”, Journal of Periodontology, Vol. 71 No. 10, pp. 1528–1534.
Lopatin, D.E. and Blackburn, E. (1992), “Avidity and titer of immunoglobulin G subclasses to Porphyromonas gingivalis in adult periodontitis patients.”, Oral Microbiology and Immunology, Vol. 7 No. 6, pp. 332–7.
López, N.J., Da Silva, I., Ipinza, J. and Gutiérrez, J. (2005), “Periodontal therapy reduces the rate of preterm low birth weight in women with pregnancy-associated gingivitis”, The Journal of Periodontology, Vol. 76 No. 11 Suppl, pp. 2144–2153.
López, N.J., Smith, P.C. and Gutierrez, J. (2002), “Higher risk of preterm birth and low birth weight in women with periodontal disease.”, Journal of Dental Research, Vol. 81 No. 1, pp. 58–63.
Lourbakos, A., Potempa, J., Travis, J., D’Andrea, M.R., Andrade Gordon, P., Santulli, R., Mackie, E.J., et al. (2001), “Arginine-specific protease from Porphyromonas gingivalis activates protease-activated receptors on human oral epithelial cells and induces interleukin-6 secretion”, Infection and Immunity, Vol. 69 No. 8, pp. 5121–5130.
Low, L.Y., Mukasa, T., Reed, J.C. and Pascual, J. (2007), “Characterization of a TIR-like protein from Paracoccus denitrificans”, Biochemical and Biophysical Research Communications, Vol. 356 No. 2, pp. 481–486.
Lu, J., Lu, G., Song, Q., Tang, C., Zou, F., Han, Z., Zhao, B., et al. (2015), “IRX1
189
hypomethylation promotes osteosarcoma metastasis via induction of CXCL14/NF-κB signaling”, The Journal of Clinical Investigation, Vol. 125 No. 5, pp. 1839–1856.
Lu, Q., Darveau, R.P., Samaranayake, L.P., Wang, C.Y. and Jin, L. (2009), “Differential modulation of human β-defensins expression in human gingival epithelia by Porphyromonas gingivalis lipopolysaccharide with tetra- And penta-acylated lipid A structures”, Innate Immunity, Vol. 15 No. 6, pp. 325–335.
Lupas, A., Van Dyke, M. and Stock, J. (1991), “Predicting coiled coils from protein sequences”, Science, Vol. 252 No. 5009, pp. 1162–1164.
Madrigal, A., Madrigal, K., Barth, G., Papadopoulos, C., Genco, R. and Isberg. (2012), “Pathogen-Mediated Proteolysis of the Cell Death Regulator RIPK1 and the Host Defense Modulator RIPK2 in Human Aortic Endothelial Cells”, PLOS Pathogens, Vol. 8 No. 6, p. e1002723.
Maekawa, T., Maekawa, J., Krauss, T., Abe, R., Jotwani, M., Triantafilou, K., Triantafilou, A., et al. (2014), “Porphyromonas gingivalis Manipulates Complement and TLR Signaling to Uncouple Bacterial Clearance from Inflammation and Promote Dysbiosis”, Cell Host & Microbe, Vol. 15 No. 6, pp. 768–778.
Maerki, C., Meuter, S., Liebi, M., Mühlemann, K., Frederick, M.J., Yawalkar, N., Moser, B., et al. (2009), “Potent and broad-spectrum antimicrobial activity of CXCL14 suggests an immediate role in skin infections.”, Journal of Immunology, Vol. 182 No. 1, pp. 507–514.
Maisetta, G., Batoni, G., Esin, S., Luperini, F., Pardini, M., Bottai, D., Florio, W., et al. (2003), “Activity of human beta-defensin 3 alone or combined with other antimicrobial agents against oral bacteria.”, Antimicrobial Agents and Chemotherapy, Vol. 47 No. 10, pp. 3349–3351.
Maloney, G., Schröder, M. and Bowie, A.G. (2005), “Vaccinia virus protein A52R activates p38 mitogen-activated protein kinase and potentiates lipopolysaccharide-induced interleukin-10”, Journal of Biological Chemistry, Vol. 280 No. 35, pp. 30838–30844.
Mansell, A., Brint, E., Gould, J. a., O’Neill, L. a. and Hertzog, P.J. (2004), “Mal interacts with tumor necrosis factor receptor-associated factor (TRAF)-6 to mediate NF-kappaB activation by toll-like receptor (TLR)-2 and TLR4”, Journal of Biological Chemistry, Vol. 279 No. 36, pp. 37227–37230.
Mansell, A., Smith, R., Doyle, S.L., Gray, P., Fenner, J.E., Crack, P.J., Nicholson, S.E., et al. (2006), “Suppressor of cytokine signaling 1 negatively regulates Toll-like receptor signaling by mediating Mal degradation”, Nature Immunology, Vol. 7 No. 2, pp. 148–155.
Marques, R.E., Guabiraba, R., Russo, R.C. and Teixeira, M.M. (2013), “Targeting CCL5 in inflammation”, Expert Opinion on Therapeutic Targets, Vol. 17 No. 12, pp. 1439–1460.
Marsh, P.D. (1994), “Microbial ecology of dental plaque and its significance in health and disease.”, Advances in Dental Research, Vol. 8 No. 2, pp. 263–271.
Marsh, P.D. (2003), “Are dental diseases examples of ecological catastrophes?”, Microbiology, Vol. 149 No. 2, pp. 279–294.
Marsh, P.D. and Devine, D.A. (2011), “How is the development of dental biofilms influenced by the host?”, Journal of Clinical Periodontology, Vol. 38 No. 11, pp. 28–35.
Mascia, F., Mascia, V., Mariani, G., Girolomoni, S. and Pastore. (2003), “Blockade of the EGF Receptor Induces a Deranged Chemokine Expression in Keratinocytes Leading to Enhanced Skin Inflammation”, The American Journal of Pathology, Vol. 163 No. 1, pp. 303–312.
Mathews, M., Jia, H.P., Guthmiller, J.M., Losh, G., Graham, S., Johnson, G.K., Tack, B.F., et al. (1999), “Production of beta-defensin antimicrobial peptides by the oral mucosa and
190
salivary glands.”, Infection and Immunity, Vol. 67 No. 6, pp. 2740–5.
Matsuda, N., Lin, W.L., Kumar, N.M., Cho, M.I. and Genco, R.J. (1992), “Mitogenic, chemotactic, and synthetic responses of rat periodontal ligament fibroblastic cells to polypeptide growth factors in vitro.”, Journal of Periodontology, Vol. 63 No. 6, pp. 515–25.
McCrudden, M.T.C., Orr, D.F., Yu, Y., Coulter, W.A., Manning, G., Irwin, C.R. and Lundy, F.T. (2013), “LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid”, Journal of Clinical Periodontology, Vol. 40 No. 10, pp. 933–941.
McGettrick, A.F., Brint, E.K., Palsson-McDermott, E.M., Rowe, D.C., Golenbock, D.T., Gay, N.J., Fitzgerald, K. a, et al. (2006), “Trif-related adapter molecule is phosphorylated by PKC{epsilon} during Toll-like receptor 4 signaling.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 103 No. 24, pp. 9196–201.
Mclaughlin, J.N., Patterson, M.M. and Malik, A.B. (2007), “Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 104 No. 13, pp. 5662–5667.
Meghji, S., Henderson, B. and Wilson, M. (1993), “Higher-titer antisera from patients with periodontal disease inhibit bacterial capsule-induced bone breakdown”, Journal of Periodontal Research, Vol. 28 No. 2, pp. 115–121.
Metcalf, D. (2008), “Hematopoietic cytokines”, Blood, Vol. 111 No. 2, pp. 485–491.
Meuter, S. and Moser, B. (2008), “Constitutive expression of CXCL14 in healthy human and murine epithelial tissues”, Cytokine, Vol. 44 No. 2, pp. 248–255.
Meuter, S., Schaerli, P., Roos, R.S., Brandau, O., Bösl, M.R., von Andrian, U.H. and Moser, B. (2007), “Murine CXCL14 is dispensable for dendritic cell function and localization within peripheral tissues.”, Molecular and Cellular Biology, Vol. 27 No. 3, pp. 983–992.
Mitchell-Lewis, D., Engebretson, S.P., Chen, J., Lamster, I.B. and Papapanou, P.N. (2001), “Periodontal infections and pre-term birth: early findings from a cohort of young minority women in New York”, Eur J Oral Sci, Vol. 109 No. 1, pp. 34–39.
Moen, K., Brun, J.G., Valen, M., Skartveit, L., Ribs Eribe, E.K., Olsen, I. and Jonsson, R. (2006), “Synovial inflammation in active rheumatoid arthritis and psoriatic arthritis facilitates trapping of a variety of oral bacterial DNAs”, Clinical and Experimental Rheumatology, Vol. 24 No. 6, pp. 656–663.
Moffatt, C.E., Inaba, H., Hirano, T. and Lamont, R.J. (2012), “Porphyromonas gingivalis SerB-mediated dephosphorylation of host cell cofilin modulates invasion efficiency”, Cellular Microbiology, Vol. 14 No. 4, pp. 577–588.
Mogi, M., Otogoto, J., Ota, N., Inagaki, H., Minami, M. and Kojima, K. (1999), “Interleukin 1β, interleukin 6, β2-microglobulin, and transforming growth factor-α in gingival crevicular fluid from human periodontal disease”, Archives of Oral Biology, Vol. 44 No. 6, pp. 535–539.
Moser, R., Schleiffenbaum, B., Groscurth, P. and Fehr, J. (1989), “Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage”, Journal of Clinical Investigation, Vol. 83 No. 2, pp. 444–455.
Mosmann, T.R. and Coffman, R.L. (1989), “Heterogeneity of cytokine secretion patterns and functions of helper T cells”, Advances in Immunology, Vol. 46, pp. 111–147.
Mosser, D.M. and Edwards, J.P. (2008), “Exploring the full spectrum of macrophage activation”, Nature Reviews Immunology, Vol. 8 No. 12, pp. 958–969.
191
Muraguchi, A., Hirano, T., Tang, B., Matsuda, T., Horii, Y., Nakajima, K. and Kishimoto, T. (1988), “The essential role of B cell stimulatory factor 2 (BSF-2/IL-6) for the terminal differentiation of B cells.”, The Journal of Experimental Medicine, Vol. 167 No. 2, pp. 332–344.
Murray, P.J. and Wynn, T.A. (2012), “Protective and pathogenic functions of macrophage subsets”, Nature Reviews Immunology, Vol. 11 No. 11, pp. 723–737.
Nakagawa, I., Amano, A., Kuboniwa, M., Nakamura, T., Kawabata, S. and Hamada, S. (2002), “Functional Differences among FimA Variants of Porphyromonas gingivalis and Their Effects on Adhesion to and Invasion of Human Epithelial Cells”, Infection and Immunity, Vol. 70 No. 1, pp. 277–285.
Nakamura, T., Kido, J., Kido, R., Ohishi, K., Yamauchi, N., Kataoka, M. and Nagata, T. (2000), “The association of calprotectin level in gingival crevicular fluid with gingival index and the activities of collagenase and aspartate aminotransferase in adult periodontitis patients.”, Journal of Periodontology, Vol. 71 No. 3, pp. 361–367.
Nakayama, K. (2015), “Porphyromonas gingivalis and related bacteria: From colonial pigmentation to the type IX secretion system and gliding motility”, Journal of Periodontal Research, Vol. 50 No. 1, pp. 1–8.
Nakayama, K., Yoshimura, F., Kadowaki, T. and Yamamoto, K. (1996), “Involvement of arginine-specific cysteine proteinase (Arg-gingipain) in fimbriation of Porphyromonas gingivalis.”, Journal of Bacteriology, Vol. 178 No. 10, pp. 2818–24.
Nemoto, E., Kanaya, S., Minamibuchi, M. and Shimauchi, H. (2005), “Cleavage of PDGF receptor on periodontal ligament cells by elastase”, Journal of Dental Research, Vol. 84 No. 7, pp. 629–633.
Newman, R., Salunkhe, P., Godzik, A. and Reed, J. (2006), “Identification and characterization of a novel bacterial virulence factor that shares homology with mammalian Toll/interleukin-1 receptor family proteins”, Infection and Immunity, Vol. 74 No. 1, pp. 594–601.
Nicu, E.A., Van Der Velden, U., Everts, V., Van Winkelhoff, A.J., Roos, D. and Loos, B.G. (2007), “Hyper-reactive PMNs in FcγRIIa 131 H/H genotype periodontitis patients”, Journal of Clinical Periodontology, Vol. 34 No. 11, pp. 938–945.
Niebuhr, M., Baumert, K. and Werfel, T. (2010), “TLR-2-mediated cytokine and chemokine secretion in human keratinocytes”, Experimental Dermatology, Vol. 19 No. 10, pp. 873–877.
Niessen, C.M. (2007), “Tight junctions/adherens junctions: basic structure and function”, Journal of Investigative Dermatology, Vol. 127 No. 11, pp. 2525–2532.
Nisapakultorn, K., Ross, K. and Herzberg, M. (2001), “Calprotectin expression in vitro by oral epithelial cells confers resistance to infection by Porphyromonas gingivalis”, Infection and Immunity, Vol. 69 No. 7, pp. 4242–7.
Nishiya, T., Kajita, E., Horinouchi, T., Nishimoto, A. and Miwa, S. (2007), “Distinct roles of TIR and non-TIR regions in the subcellular localization and signaling properties of MyD88”, FEBS Letters, Vol. 581 No. 17, pp. 3223–3229.
Njoroge, T., Genco, R.J., Sojar, H.T., Hamada, N. and Genco, C.A. (1997), “A role for fimbriae in Porphyromonas gingivalis invasion of oral epithelial cells”, Infection and Immunity, Vol. 65 No. 5, pp. 1980–1984.
Noack, B., Genco, R.J., Trevisan, M., Grossi, S., Zambon, J.J. and De Nardin, E. (2001), “Periodontal Infections Contribute to Elevated Systemic C-Reactive Protein Level”, Journal of Periodontology, Vol. 72 No. 9, pp. 1221–1227.
North, B.J. and Verdin, E. (2004), “Sirtuins: Sir2-related NAD-dependent protein deacetylases”, Genome Biology, Vol. 5 No. 5, p. 224.
192
O’ Brien-Simpson, N., Veith, P.D., Dashper, S.G. and Reynolds, E.C. (2003), “Porphyromonas gingivalis gingipains: the molecular teeth of a microbial vampire”, Curr Protein Pept Sci, Vol. 4 No. 6, pp. 409–426.
O’Neill, L.A. (2006), “DisSARMing Toll-like receptor signaling”, Nature Immunology, Vol. 7 No. 10, pp. 1023–1025.
O’Neill, L.A.J., Bryant, C.E. and Doyle, S.L. (2009), “Therapeutic targeting of Toll-like receptors for infectious and inflammatory diseases and cancer.”, Pharmacological Reviews, Vol. 61 No. 2, pp. 177–97.
O’Toole, G., Kaplan, H.B. and Kolter, R. (2000), “Biofilm Formation as Microbial Development”, Annual Review of Immunology, Vol. 54, pp. 49–79.
Ogrendik, M. (2009), “Rheumatoid arthritis is linked to oral bacteria: Etiological association”, Modern Rheumatology, Vol. 19 No. 5, pp. 453–456.
Ostrowska, E. and Reiser, G. (2008), “The protease-activated receptor-3 (PAR-3) can signal autonomously to induce interleukin-8 release”, Cellular and Molecular Life Sciences, Vol. 65 No. 6, pp. 970–981.
Otogoto, J.I. and Kuramitsu, H.K. (1993), “Isolation and characterization of the Porphyromonas gingivalis prtT gene, coding for protease activity”, Infection and Immunity, Vol. 61 No. 1, pp. 117–123.
Otte, M., Kliewer, A., Schütz, D., Reimann, C., Schulz, S. and Stumm, R. (2014), “CXCL14 is no direct modulator of CXCR4”, FEBS Letters, Vol. 588 No. 24, pp. 4769–4775.
Ouhara, K., Komatsuzawa, H., Yamada, S., Shiba, H., Fujiwara, T., Ohara, M., Sayama, K., et al. (2005), “Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells.”, The Journal of Antimicrobial Chemotherapy, Vol. 55 No. 6, pp. 888–96.
Ozawa, S., Ozawa, Y., Kato, R., Komori, Y., Maehata, E., Kubota, R.-I. and Hata. (2006), “BRAK/CXCL14 expression suppresses tumor growth in vivo in human oral carcinoma cells”, Biochemical and Biophysical Research Communications, Vol. 348 No. 2, pp. 406–412.
Ozinsky, A., Underhill, D.M., Fontenot, J.D., Hajjar, A.M., Smith, K.D., Wilson, C.B., Schroeder, L., et al. (2000), “The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 97 No. 25, pp. 13766–71.
de Pablo, P., Chapple, I.L., Buckley, C.D. and Dietrich, T. (2009), “Periodontitis in systemic rheumatic diseases”, Nat Rev Rheumatol, Vol. 5 No. 4, pp. 218–224.
Paing, M.M., Stutts, A.B., Kohout, T.A., Lefkowitz, R.J. and Trejo, J. (2002), “β-arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation”, Journal of Biological Chemistry, Vol. 277 No. 2, pp. 1292–1300.
Palmer, R.J., Gordon, S.M., Cisar, J.O. and Kolenbrander, P.E. (2003), “Coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque”, Journal of Bacteriology, Vol. 185 No. 11, pp. 3400–3409.
Papadopoulos, G., Weinberg, E.O., Massari, P., Gibson, F.C., Wetzler, L.M., Morgan, E.F. and Genco, C.A. (2013), “Macrophage-Specific TLR2 Signaling Mediates Pathogen-Induced TNF-Dependent Inflammatory Oral Bone Loss”, Journal of Immunology, Vol. 190 No. 3, pp. 1148–1157.
Park, Y. and McBride, B.C. (1993), “Characterization of the tpr gene product and isolation of a specific protease-deficient mutant of Porphyromonas gingivalis W83”, Infection and Immunity, Vol. 61 No. 10, pp. 4139–4146.
193
Park, Y., Simionato, M.R., Sekiya, K., Murakami, Y., James, D., Chen, W., Hackett, M., et al. (2005), “Short fimbriae of Porphyromonas gingivalis and their role in coadhesion with Streptococcus gordonii”, Infection and Immunity, Vol. 73 No. 7, pp. 3983–3989.
Parkar, M.H., Kuru, L., Giouzeli, M. and Olsen, I. (2001), “Expression of growth-factor receptors in normal and regenerating human periodontal cells”, Archives of Oral Biology, Vol. 46 No. 3, pp. 275–284.
Pasi, S., Kant, R. and Surolia, A. (2016), “TIR-TcpC ameliorates experimental autoimmune arthritis by down-modulating Th17 cell response”, Journal of Biological Chemistry, Vol. 291 No. 23, pp. 12358–12369.
Paster, B.J., Boches, S.K., Galvin, J.L., Ericson, R.E., Lau, C.N., Levanos, V.A., Sahasrabudhe, A., et al. (2001), “Bacterial diversity in human subgingival plaque”, Journal of Bacteriology, Vol. 183 No. 12, pp. 3770–3783.
Pastore, S., Mascia, F., Mariani, V. and Girolomoni, G. (2008), “The epidermal growth factor receptor system in skin repair and inflammation”, Journal of Investigative Dermatology, Vol. 128 No. 6, pp. 1365–1374.
Pastore, S., Mascia, F., Mariotti, F., Dattilo, C., Mariani, V. and Girolomoni, G. (2005), “ERK1/2 Regulates Epidermal Chemokine Expression and Skin Inflammation”, Journal of Immunology, Vol. 174 No. 8, pp. 5047–5056.
Patot, S., Imbert, P., Baude, J., Martins Simões, P., Campergue, J.B., Louche, A., Nijland, R., et al. (2017), “The TIR Homologue Lies near Resistance Genes in Staphylococcus aureus, Coupling Modulation of Virulence and Antimicrobial Susceptibility”, PLoS Pathogens, Vol. 13 No. 1, pp. 1–23.
Patters, M.R., Niekrash, C.E. and Lang, N.P. (1989), “Assessment of complement cleavage in gingival fluid during experimental gingivitis in man”, Journal of Clinical Periodontology, Blackwell Publishing Ltd, Vol. 16 No. 1, pp. 33–37.
Patterson, N.J. and Werling, D. (2013), “To con protection: TIR-domain containing proteins (Tcp) and innate immune evasion.”, Veterinary Immunology and Immunopathology, Elsevier B.V., Vol. 155 No. 3, pp. 147–54.
Pelicano, H., Lu, W., Zhou, Y., Zhang, W., Chen, Z., Hu, Y. and Huang, P. (2009), “Mitochondrial dysfunction and reactive oxygen species imbalance promote breast cancer cell motility through a CXCL14-mediated mechanism”, Cancer Research, Vol. 69 No. 6, pp. 2375–2383.
Pelletier, M., Maggi, L., Micheletti, A., Lazzeri, E., Tamassia, N., Costantini, C., Cosmi, L., et al. (2010), “Evidence for a cross-talk between human neutrophils and Th17 cells”, Blood, Vol. 115 No. 2, pp. 335–343.
Peng, J., Yuan, Q., Lin, B., Panneerselvam, P., Wang, X., Luan, X.L., Lim, S.K., et al. (2010), “SARM inhibits both TRIF- and MyD88-mediated AP-1 activation”, European Journal of Immunology, Vol. 40 No. 6, pp. 1738–1747.
Perfetto, S.P., Chattopadhyay, P.K., Lamoreaux, L., Nguyen, R., Ambrozak, D., Koup, R.A. and Roederer, M. (2006), “Amine reactive dyes: An effective tool to discriminate live and dead cells in polychromatic flow cytometry”, Journal of Immunological Methods, Vol. 313 No. 1–2, pp. 199–208.
Pfaffl, M.W. (2001), “A new mathematical model for relative quantification in real-time RT-PCR”, Nucleic Acids Research, Vol. 29 No. 9, p. e45.
Phillipson, M., Heit, B., Colarusso, P., Liu, L., Ballantyne, C.M. and Kubes, P. (2006), “Intraluminal crawling of neutrophils to emigration sites: a molecularly distinct process from adhesion in the recruitment cascade.”, The Journal of Experimental Medicine, Vol. 203 No. 12, pp. 2569–75.
Pihlstrom, B.L., Michalowicz, B.S. and Johnson, N.W. (2005), “Periodontal diseases”, Lancet,
194
Vol. 366, pp. 1809–1820.
Pober, J.S., Gimbrone Jr, M.A., Lapierre, L.A., Mendrick, D.L., Fiers, W., Rothlein, R. and Springer, T.A. (1986), “Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon”, Journal of Immunology, Vol. 137 No. 6, pp. 1893–1896.
Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., et al. (1998), “Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene”, Science, Vol. 282 No. 5396, pp. 2085–2088.
Popadiak, K., Potempa, J., Riesbeck, K. and Blom, A.M. (2007), “Biphasic Effect of Gingipains from Porphyromonas gingivalis on the Human Complement System”, Journal of Immunology, Vol. 178 No. 11, pp. 7242–7250.
Potempa, J., Pike, R. and Travis, J. (1995), “The multiple forms of trypsin-like activity present in various strains of Porphyromonas gingivalis are due to the presence of either Arg-gingipain or Lys-gingipain”, Infection and Immunity, Vol. 63 No. 4, pp. 1176–1182.
Potempa, J., Sroka, A., Imamura, T. and Travis, J. (2003), “Gingipains, the major cysteine proteinases and virulence factors of Porphyromonas gingivalis: structure, function and assembly of multidomain protein complexes.”, Current Protein & Peptide Science, Vol. 4 No. 6, pp. 397–407.
Potempa, M., Potempa, J., Kantyka, T., Nguyen, K.A., Wawrzonek, K., Manandhar, S.P., Popadiak, K., et al. (2009), “Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3”, PLoS Pathogens, Vol. 5 No. 2, p. e1000316.
Potten, C.S. and Morris, R.J. (1988), “Epithelial stem cells in vivo.”, J Cell Sci Suppl, Vol. 10, pp. 45–62.
Pulendran, B., Kumar, P., Cutler, C.W., Mohamadzadeh, M., Van Dyke, T. and Banchereau, J. (2001), “Lipopolysaccharides from Distinct Pathogens Induce Different Classes of Immune Responses In Vivo”, Journal of Immunology, Vol. 167 No. 9, pp. 5067–5076.
Pyrc, K., Milewska, A., Kantyka, T., Sroka, A., Maresz, K., Kozieł, J., Nguyen, K.-A., et al. (2013), “Inactivation of epidermal growth factor by Porphyromonas gingivalis as a potential mechanism for periodontal tissue damage.”, Infection and Immunity, Vol. 81 No. 1, pp. 55–64.
Qi, M., Miyakawa, H. and Kuramitsu, H.K. (2003), “Porphyromonas gingivalis induces murine macrophage foam cell formation”, Microbial Pathogenesis, Vol. 35 No. 6, pp. 259–267.
Quinchia-Rios, B.H., Guerrero, M., Abozeid, S., Bainbridge, B., Darveau, R., Compton, T. and Bertics, P.J. (2008), “Down-regulation of epidermal growth factor receptor-dependent signaling by Porphyromonas gingivalis lipopolysaccharide in life-expanded human gingival fibroblasts”, Journal of Periodontal Research, Vol. 43 No. 3, pp. 290–304.
Radhakrishnan, G., Yu, Q., Harms, J. and Splitter, G. (2009), “Brucella TIR Domain-containing Protein Mimics Properties of the Toll-like Receptor Adaptor Protein TIRAP”, Journal of Biological Chemistry, Vol. 284 No. 15, pp. 9892–9898.
Rahman, A., True, A.L., Anwar, K.N., Ye, R.D., Voyno-Yasenetskaya, T.A. and Malik, A.B. (2002), “Gαq and Gβγ regulate PAR-1 signaling of thrombin-induced NF-κB activation and ICAM-1 transcription in endothelial cells”, Circulation Research, Vol. 91 No. 5, pp. 398–405.
Rana, R., Rana, M., Zhang, A., Spear, H., Atkins, B. and Byrne. (2013), “Bacterial TIR-containing proteins and host innate immune system evasion”, Medical Microbiology
195
and Immunology, Vol. 202 No. 1, pp. 1–10.
Rana, R.R., Simpson, P., Zhang, M., Jennions, M., Ukegbu, C., Spear, A.M., Alguel, Y., et al. (2011), “Yersinia pestis TIR-domain protein forms dimers that interact with the human adaptor protein MyD88”, Microbial Pathogenesis, Elsevier Ltd, Vol. 51 No. 3, pp. 89–95.
Raschke, W.C., Baird, S., Ralph, P. and Nakoinz, I. (1978), “Functional macrophage cell lines transformed by abelson leukemia virus”, Cell, Vol. 15 No. 1, pp. 261–267.
Reife, R.A., Coats, S.R., Al-Qutub, M., Dixon, D.M., Braham, P.A., Billharz, R.J., Howald, W.N., et al. (2006), “Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity: Differential activities of tetra- and penta-acylated lipid A structures on E-selectin expression and TLR4 recognition”, Cellular Microbiology, Vol. 8 No. 5, pp. 857–868.
Reife, R.A., Shapiro, R.A., Bamber, B.A., Berry, K.K., Mick, G.E. and Darveau, R.P. (1995), “Porphyromonas gingivalis lipopolysaccharide is poorly recognized by molecular components of innate host defense in a mouse model of early inflammation”, Infection and Immunity, Vol. 63 No. 12, pp. 4686–4694.
Rice, W., Ganz, T., Kinkade, J., Selsted, M., Lehrer, R. and Parmley, R. (1987), “Defensin-rich dense granules of human neutrophils”, Blood, Vol. 70 No. 3, pp. 757–765.
Rogers, J.D., Palmer, R.J., Kolenbrander, P.E. and Scannapieco, F.A. (2001), “Role of Streptococcus gordonii amylase-binding protein A in adhesion to hydroxyapatite, starch metabolism, and biofilm formation”, Infection and Immunity, Vol. 69 No. 11, pp. 7046–7056.
Roop, D.R., Huitfeldt, H., Kilkenny, A. and Yuspa, S.H. (1987), “Regulated expression of differentiation-associated keratins in cultured epidermal cells detected by monospecific antibodies to unique peptides of mouse epidermal keratins”, Differentiation, Vol. 35 No. 2, pp. 143–150.
Rowe, D.C., McGettrick, A.F., Latz, E., Monks, B.G., Gay, N.J., Yamamoto, M., Akira, S., et al. (2006), “The myristoylation of TRIF-related adaptor molecule is essential for Toll-like receptor 4 signal transduction.”, Proceedings of the National Academy of Sciences of the United States of America, Vol. 103 No. 16, pp. 6299–6304.
Ruggiero, S., Cosgarea, R., Potempa, J., Potempa, B., Eick, S. and Chiquet, M. (2013), “Cleavage of extracellular matrix in periodontitis: Gingipains differentially affect cell adhesion activities of fibronectin and tenascin-C”, Biochimica et Biophysica Acta - Molecular Basis of Disease, Elsevier B.V., Vol. 1832 No. 4, pp. 517–526.
de Saint-Vis, B., Fugier-Vivier, I., Massacrier, C., Gaillard, C., Vanbervliet, B., Aït-Yahia, S., Banchereau, J., et al. (1998), “The cytokine profile expressed by human dendritic cells is dependent on cell subtype and mode of activation.”, Journal of Immunology, Vol. 160 No. 4, pp. 1666–1676.
Salcedo, S.P., Marchesini, M.I., Degos, C., Terwagne, M., Von Bargen, K., Lepidi, H., Herrmann, C.K., et al. (2013), “BtpB, a novel Brucella TIR-containing effector protein with immune modulatory functions.”, Frontiers in Cellular and Infection Microbiology, Vol. 3 No. 28, available at:https://doi.org/10.3389/fcimb.2013.00028.
Salcedo, S.P., Marchesini, M.I., Lelouard, H., Fugier, E., Jolly, G., Balor, S., Muller, A., et al. (2008), “Brucella control of dendritic cell maturation is dependent on the TIR-containing protein Btp1”, PLoS Pathogens, Vol. 4, p. 21.
Sallusto, F., Palermo, B., Lenig, D., Miettinen, M., Matikainen, S., Julkunen, I., Forster, R., et al. (1999), “Distinct patterns and kinetics of chemokine production regulate dendritic cell function”, European Journal of Immunology, Vol. 29 No. 5, pp. 1617–1625.
Sánchez, R. and Šali, A. (1997), “Advances in comparative protein-structure modelling”, Current Opinion in Structural Biology, Vol. 7 No. 2, pp. 206–214.
196
Sasai, M., Oshiumi, H., Matsumoto, M., Inoue, N., Fujita, F., Nakanishi, M. and Seya, T. (2005), “Cutting Edge: NF-kappaB-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation.”, Journal of Immunology, Vol. 174 No. 1, pp. 27–30.
Sasaki, H., Okamatsu, Y., Kawai, T., Kent, R., Taubman, M. and Stashenko, P. (2004), “The interleukin-10 knockout mouse is highly susceptible to Porphyromonas gingivalis-induced alveolar bone loss”, Journal of Periodontal Research, Vol. 39 No. 6, pp. 432–441.
Sato, S., Sugiyama, M., Yamamoto, M. and Watanabe, Y. (2003), “TRIF associates with TRAF6 and TBK1, and activates two distinct transcription factors, NF-kappa B and IRF-3, in the Toll-like receptor signaling.”, Journal of Immunology, Vol. 71 No. 8, pp. 4304–4310.
Schroder, K. and Tschopp, J. (2010), “The Inflammasomes”, Cell, Vol. 140 No. 6, pp. 821–832.
Schultz, C., Wolf, V., Lange, R. and Mertens, E. (1998), “Evidence for a new type of outer membrane lipid in oral spirochete Treponema denticola”, Journal of Biological Chemistry, Vol. 273 No. 25, pp. 15661–15666.
Schwede, T., Kopp, J., Guex, N. and Peitsch, M.C. (2003), “SWISS-MODEL: An automated protein homology-modeling server”, Nucleic Acids Research, Vol. 31 No. 13, pp. 3381–3385.
Scott, D.A. and Krauss, J.L. (2013), “Neutrophils in periodontal inflammation”, Frontiers of Oral Biology, available at:https://doi.org/10.1159/000329672.
Seers, C. a., Slakeski, N., Veith, P.D., Nikolof, T., Chen, Y.Y., Dashper, S.G. and Reynolds, E.C. (2006), “The RgpB C-terminal domain has a role in attachment of RgpB to the outer membrane and belongs to a novel C-terminal-domain family found in Porphyromonas gingivalis”, Journal of Bacteriology, Vol. 188 No. 17, pp. 6376–6386.
Seinost, G., Wimmer, G., Skerget, M., Thaller, E., Brodmann, M., Gasser, R., Bratschko, R.O., et al. (2005), “Periodontal treatment improves endothelial dysfunction in patients with severe periodontitis”, American Heart Journal, Vol. 149 No. 6, pp. 1050–1054.
Sengupta, D., Koblansky, A., Gaines, J., Brown, T., West, a P., Zhang, D., Nishikawa, T., et al. (2010), “Subversion of innate immune responses by Brucella through the targeted degradation of the TLR signaling adapter, MAL.”, Journal of Immunology, Vol. 184 No. 2, pp. 956–964.
Serrano, C., Torres, N., Valdivieso, C., Castaño, C., Barrera, M. and Cabrales, A. (2009), “Antibiotic resistance of periodontal pathogens obtained from frequent antibiotic users.”, Acta Odontológica Latinoamericana : AOL, Vol. 22 No. 2, pp. 99–104.
Seymour, G.J., Ford, P.J., Cullinan, M.P., Leishman, S. and Yamazaki, K. (2007), “Relationship between periodontal infections and systemic disease”, Clinical Microbiology and Infection.
Shellenberger, T., Wang, M., Gujrati, M., Jayakumar, A., Strieter, R., Burdick, M., Ioannides, C., et al. (2004), “BRAK/CXCL14 is a potent inhibitor of angiogenesis and a chemotactic factor for immature dendritic cells”, Cancer Research, Vol. 64 No. 22, pp. 8262–8270.
Shi, J., Blundell, T.L. and Mizuguchi, K. (2001), “FUGUE: Sequence-structure homology recognition using environment-specific substitution tables and structure-dependent gap penalties”, Journal of Molecular Biology, Vol. 310 No. 1, pp. 243–257.
Shi, Y., Ratnayake, D.B., Okamoto, K., Abe, N., Yamamoto, K. and Nakayama, K. (1999), “Genetic analyses of proteolysis, hemoglobin binding, and hemagglutination of Porphyromonas gingivalis: Construction of mutants with a combination of rgpA,
197
rgpB, kgp, and hagA”, Journal of Biological Chemistry, Vol. 274 No. 25, pp. 17955–17960.
Shimazu, R., Akashi, S., Ogata, H., Nagai, Y., Fukudome, K., Miyake, K. and Kimoto, M. (1999), “MD-2, a Molecule that Confers Lipopolysaccharide Responsiveness on Toll-like Receptor 4”, The Journal of Experimental Medicine, Vol. 189 No. 11, pp. 1777–1782.
Shin, J., Ji, S. and Choi, Y. (2008), “Ability of oral bacteria to induce tissue-destructive molecules from human neutrophils”, Oral Diseases, Vol. 14 No. 4, pp. 327–334.
Shurin, G. V, Ferris, R.L., Tourkova, I.L., Perez, L., Lokshin, A., Balkir, L., Collins, B., et al. (2005), “Loss of new chemokine CXCL14 in tumor tissue is associated with low infiltration by dendritic cells (DC), while restoration of human CXCL14 expression in tumor cells causes attraction of DC both in vitro and in vivo.”, Journal of Immunology, Vol. 174 No. 9, pp. 5490–5498.
Sims, J.E. and Smith, D.E. (2010), “The IL-1 family: regulators of immunity”, Nature Reviews Immunology 2010 10:2, Nature Publishing Group, Vol. 10 No. 2, p. 89.
Slack, J.L., Schooley, K., Bonnert, T.P., Mitcham, J.L., Qwarnstrom, E.E., Sims, J.E. and Dower, S.K. (2000), “Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways”, Journal of Biological Chemistry, Vol. 275 No. 7, pp. 4670–4678.
Slakeski, N., Seers, C. a., Ng, K., Moore, C., Cleal, S.M., Veith, P.D., Lo, A.W., et al. (2011), “C-terminal domain residues important for secretion and attachment of RgpB in Porphyromonas gingivalis”, Journal of Bacteriology, Vol. 193 No. 1, pp. 132–142.
Sleeman, M. a, Fraser, J.K., Murison, J.G., Kelly, S.L., Prestidge, R.L., Palmer, D.J., Watson, J.D., et al. (2000), “B cell- and monocyte-activating chemokine (BMAC), a novel non-ELR alpha-chemokine.”, International Immunology, Vol. 12 No. 5, pp. 677–689.
Slocum, C., Coats, S.R., Hua, N., Kramer, C., Papadopoulos, G., Weinberg, E.O., Gudino, C. V., et al. (2014), “Distinct Lipid A Moieties Contribute to Pathogen-Induced Site-Specific Vascular Inflammation”, PLoS Pathogens, Vol. 10 No. 7, p. e1004215.
Slots, J. and Rams, T.E. (1990), “Antibiotics in periodontal therapy: advantages and disadvantages”, Journal of Clinical Periodontology, Vol. 17, pp. 479–493.
Slots, J. and Ting, M. (2002) ‘Systemic antibiotics in the treatment of periodontal disease’, Periodontology 2000, 28(1), pp. 106–176.
Smalley, J.W., Silver, J., Marsh, P.J. and Birss, A.J. (1998), “The periodontopathogen Porphyromonas gingivalis binds iron protoporphyrin IX in the mu-oxo dimeric form: an oxidative buffer and possible pathogenic mechanism.”, The Biochemical Journal, Vol. 331 (Pt 3), pp. 681–685.
Smith, J. a., Khan, M., Magnani, D.D., Harms, J.S., Durward, M., Radhakrishnan, G.K., Liu, Y.P., et al. (2013), “Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages”, PLoS Pathogens, Vol. 9 No. 12, pp. 1–12.
Snyder, G., Deredge, D., Waldhuber, A., Fresquez, T., Wilkins, D., Smith, P., Durr, S., et al. (2014), “Crystal structures of the Toll/Interleukin-1 receptor (TIR) domains from the Brucella protein TcpB and host adaptor TIRAP reveal mechanisms of molecular mimicry”, Journal of Biological Chemistry, Vol. 289 No. 2, pp. 669–679.
Socransky, S.S., Haffajee, a D., Cugini, M. a, Smith, C. and Kent, R.L. (1998), “Microbial complexes in subgingival plaque.”, Journal of Clinical Periodontology, Vol. 25 No. 2, pp. 134–144.
Soh, U.J., Dores, M.R., Chen, B. and Trejo, J. (2010), “Signal transduction by protease-activated receptors”, British Journal of Pharmacology, Vol. 160 No. 2, pp. 191–203.
198
Sørensen, O.E., Follin, P., Johnsen, A.H., Calafat, J., Sandra Tjabringa, G., Hiemstra, P.S. and Borregaard, N. (2001), “Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3”, Blood, Vol. 97 No. 12, pp. 3951–3959.
Souto, G.R., Queiroz, C.M., Costa, F.O. and Mesquita, R.A. (2014), “Relationship Between Chemokines and Dendritic Cells in Human Chronic Periodontitis”, Journal of Periodontology, Vol. 85 No. 10, pp. 1416–1423.
Spear, A.M., Loman, N.J., Atkins, H.S. and Pallen, M.J. (2009), “Microbial TIR domains: not necessarily agents of subversion?”, Trends in Microbiology, Vol. 17 No. 9, pp. 393–398.
Spear, A.M., Rana, R.R., Jenner, D.C., Flick-Smith, H.C., Oyston, P.C.F., Simpson, P., Matthews, S.J., et al. (2012), “A Toll/interleukin (IL)-1 receptor domain protein from Yersinia pestis interacts with mammalian IL-1/Toll-like receptor pathways but does not play a central role in the virulence of Y. pestis in a mouse model of bubonic plague”, Microbiology (United Kingdom), Vol. 158 No. 6, pp. 1593–1606.
Squier, C. and Kremer, M. (2001), “Biology of oral mucosa and esophagus.”, Journal of the National Cancer Institute. Monographs, Vol. 29, pp. 7–15.
Starnes, T., Starnes, K., Rasila, M., Robertson, Z., Brahmi, R., Dahl, K., Christopherson, R., et al. (2006), “The chemokine CXCL14 (BRAK) stimulates activated NK cell migration: Implications for the downregulation of CXCL14 in malignancy”, Experimental Hematology, Vol. 34 No. 8, pp. 1101–1105.
Stathopoulou, P. G., Benakanakere, M. R., Galicia, J. C., & Kinane, D. F. (2009). The host cytokine response to Porphyromonas gingivalis is modified by gingipains. Oral Microbiology and Immunology.
Stein, M., Keshav, S., Harris, N. and Gordon, S. (1992), “Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.”, The Journal of Experimental Medicine, Vol. 176 No. 1, pp. 287–92.
Steinman, R.M., Hawiger, D., Liu, K., Bonifaz, L., Bonnyay, D., Mahnke, K., Iyoda, T., et al. (2003), “Dendritic cell function in vivo during the steady state: a role in peripheral tolerance.”, Annals of the New York Academy of Sciences, Vol. 987, pp. 15–25.
Stöver, A.G., Da Silva Correia, J., Evans, J.T., Cluff, C.W., Elliott, M.W., Jeffery, E.W., Johnson, D.A., et al. (2004), “Structure-Activity Relationship of Synthetic Toll-like Receptor 4 Agonists”, Journal of Biological Chemistry, Vol. 279 No. 6, pp. 4440–4449.
Strieter, R.M., Burdick, M.D., Gomperts, B.N., Belperio, J.A. and Keane, M.P. (2005), “CXC chemokines in angiogenesis”, Cytokine and Growth Factor Reviews, Vol. 16 No. 6, pp. 593–609.
Strieter, R.M., Polverini, P.J., Kunkel, S.L., Arenberg, D.A., Burdick, M.D., Kasper, J., Dzuiba, J., et al. (1995), “The functional role of the ELR motif in CXC chemokine-mediated angiogenesis”, Journal of Biological Chemistry, Vol. 270 No. 45, pp. 27348–27357.
Su, X., Li, S., Meng, M., Qian, W., Xie, W., Chen, D., Zhai, Z., et al. (2006), “TNF receptor-associated factor-1 (TRAF1) negatively regulates Toll/IL-1 receptor domain-containing adaptor inducing IFN-?? (TRIF)-mediated signaling”, European Journal of Immunology, Vol. 36 No. 1, pp. 199–206.
Sugawara, S., Nemoto, E., Tada, H., Miyake, K., Imamura, T. and Takada, H. (2000), “Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness”, Journal of Immunology, Vol. 165 No. 1, pp. 411–418.
Tabeta, K., Yamazaki, K., Hotokezaka, H., Yoshie, H. and Hara, K. (2000), “Elevated humoral immune response to heat shock protein 60 (hsp60) family in periodontitis patients.”,
199
Clinical and Experimental Immunology, Vol. 120, pp. 285–293.
Tada, H., Sugawara, S., Nemoto, E., Takahashi, N., Imamura, T., Potempa, J., Travis, J., et al. (2002), “Proteolysis of CD14 on human gingival fibroblasts by arginine-specific cysteine proteinases from Porphyromonas gingivalis leading to down-regulation of lipopolysaccharide-induced interleukin-8 production”, Infection and Immunity, Vol. 70 No. 6, pp. 3304–3307.
Takatsu, K. (1997), “Cytokines Involved in B-Cell Differentiation and Their Sites of Action”, Experimental Biology and Medicine, Vol. 215 No. 2, pp. 121–133.
Takeda, K., Kaisho, T. and Akira, S. (2003), “Toll-like receptors.”, Annual Review of Immunology, Vol. 21 No. 1, pp. 335–76.
Takeichi, O., Haber, J., Kawai, T., Smith, D.J., Moro, I. and Taubman, M.A. (2000), “Cytokine profiles of T-lymphocytes from gingival tissues with pathological pocketing.”, Journal of Dental Research, Vol. 79 No. 8, pp. 1548–1555.
Takeuchi, H., Takeuchi, T., Hirano, S., Whitmore, I., Morisaki, A., Amano, R., Lamont, R., et al. (2013), “The Serine Phosphatase SerB of Porphyromonas gingivalis Suppresses IL-8 Production by Dephosphorylation of NF-κB RelA/p65”, PLOS Pathogens, Vol. 9 No. 4, p. e1003326.
Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R.L., et al. (2002), “Cutting edge: role of Toll-like receptor 1 in mediating immune response to microbial lipoproteins.”, Journal of Immunology, Vol. 169 No. 1, pp. 10–14.
Tanegashima, K., Suzuki, K., Nakayama, Y., Tsuji, K., Shigenaga, A., Otaka, A. and Hara, T. (2013), “CXCL14 is a natural inhibitor of the CXCL12-CXCR4 signaling axis”, FEBS Letters, Vol. 587 No. 12, pp. 1731–1735.
Tao, X., Tao, Y., Xu, Y., Zheng, A., Beg, L. and Tong. (2002), “An extensively associated dimer in the structure of the C713S mutant of the TIR domain of human TLR2”, Biochemical and Biophysical Research Communications, Vol. 299 No. 2, pp. 216–221.
Teng, Y.-T. a. (2003), “The role of acquired immunity and periodontal disease progression.”, Critical Reviews in Oral Biology and Medicine : An Official Publication of the American Association of Oral Biologists, Vol. 14 No. 4, pp. 237–252.
Tessema, M., Klinge, D.M., Yingling, C.M., Do, K., Van Neste, L. and Belinsky, S.A. (2010), “Re-expression of CXCL14, a common target for epigenetic silencing in lung cancer, induces tumor necrosis.”, Oncogene, Vol. 29 No. 37, pp. 5159–70.
Thacher, S.M. and Rice, R.H. (1985), “Keratinocyte-specific transglutaminase of cultured human epidermal cells: Relation to cross-linked envelope formation and terminal differentiation”, Cell, Vol. 40 No. 3, pp. 685–695.
Theilade, E. (1986), “The non-specific theory in microbial etiology of inflammatory periodontal diseases.”, Journal of Clinical Periodontology, Vol. 13 No. 10, pp. 905–911.
Tonetti, M.S., Imboden, M.A. and Lang, N.P. (1998), “Neutrophil migration into the gingival sulcus is associated with transepithelial gradients of interleukin-8 and ICAM-1.”, Journal of Periodontology, Vol. 69 No. 10, pp. 1139–47.
Trent, M.S., Pabich, W., Raetz, C.R.H. and Miller, S.I. (2001), “A PhoP/PhoQ-induced Lipase (PagL) that Catalyzes 3-O-Deacylation of Lipid A Precursors in Membranes of Salmonella typhimurium”, Journal of Biological Chemistry, Vol. 276 No. 12, pp. 9083–9092.
Tribble, G.D., Lamont, G.J., Progulske-Fox, A. and Lamont, R.J. (2007), “Conjugal transfer of chromosomal DNA contributes to genetic variation in the oral pathogen Porphyromonas gingivalis”, Journal of Bacteriology, Vol. 189 No. 17, pp. 6382–6388.
Tribble, G.D., Mao, S., James, C.E. and Lamont, R.J. (2006), “A Porphyromonas gingivalis
200
haloacid dehalogenase family phosphatase interacts with human phosphoproteins and is important for invasion”, Proc Natl Acad Sci U S A, Vol. 103 No. 29, pp. 11027–11032.
Trinchieri, G. (2003), “Interleukin-12 and the regulation of innate resistance and adaptive immunity”, Nature Reviews Immunology, Vol. 3 No. 2, pp. 133–146.
Tsukamoto, Y., Usui, M., Yamamoto, G., Takagi, Y., Tachikawa, T., Yamamoto, M. and Nakamura, M. (2012), “Role of the junctional epithelium in periodontal innate defense and homeostasis”, Journal of Periodontal Research, Vol. 47 No. 6, pp. 750–757.
Uehara, A., Imamura, T., Potempa, J., Travis, J. and Takada, H. (2008), “Gingipains from Porphyromonas gingivalis synergistically induce the production of proinflammatory cytokines through protease-activated receptors with Toll-like receptor and NOD1/2 ligands in human monocytic cells”, Cellular Microbiology, Vol. 10 No. 5, pp. 1181–1189.
Uehara, A., Muramoto, K., Imamura, T., Nakayama, K., Potempa, J., Travis, J., Sugawara, S., et al. (2005), “Arginine-Specific Gingipains from Porphyromonas gingivalis Stimulate Production of Hepatocyte Growth Factor (Scatter Factor) through Protease-Activated Receptors in Human Gingival Fibroblasts in Culture”, Journal of Immunology, Vol. 175 No. 9, pp. 6076–6084.
Underhill, D.M., Ozinsky, A., Hajjar, A.M., Stevens, A., Wilson, C.B., Bassetti, M. and Aderem, A. (1999), “The Toll-like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens”, Nature, Vol. 401 No. 6755, pp. 811–815.
Veith, P.D., Chen, Y.Y., Gorasia, D.G., Chen, D., Glew, M.D., O’Brien-Simpson, N.M., Cecil, J.D., et al. (2014), “Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors”, Journal of Proteome Research, Vol. 13 No. 5, pp. 2420–2432.
Vernal, R., Dutzan, N., Chaparro, A., Puente, J., Valenzuela, M.A. and Gamonal, J. (2005), “Levels of interleukin-17 in gingival crevicular fluid and in supernatants of cellular cultures of gingival tissue from patients with chronic periodontitis”, Journal of Clinical Periodontology, Vol. 32 No. 4, pp. 383–389.
Vetter, I.R. and Wittinghofer, A. (1999), “Nucleoside triphosphate-binding proteins: Different scaffolds to achieve phosphoryl transfer”, Quarterly Reviews of Biophysics, Vol. 32 No. 1, pp. 1–56.
Vincents, B., Guentsch, A., Kostolowska, D., von Pawel-Rammingen, U., Eick, S., Potempa, J. and Abrahamson, M. (2011), “Cleavage of IgG1 and IgG3 by gingipain K from Porphyromonas gingivalis may compromise host defense in progressive periodontitis”, The FASEB Journal , Vol. 25 No. 10, pp. 3741–3750.
Visintin, a, Mazzoni, A., Spitzer, J.H., Wyllie, D.H., Dower, S.K. and Segal, D.M. (2001), “Regulation of Toll-like receptors in human monocytes and dendritic cells.”, Journal of Immunology, Vol. 166 No. 1, pp. 249–255.
Vitkov, L., Klappacher, M., Hannig, M. and Krautgartner, W.D. (2009), “Extracellular neutrophil traps in periodontitis”, Journal of Periodontal Research, Vol. 44 No. 5, pp. 664–672.
Wakabayashi, H., Yamauchi, K., Kobayashi, T., Yaeshima, T., Iwatsuki, K. and Yoshie, H. (2009), “Inhibitory effects of lactoferrin on growth and biofilm formation of Porphyromonas gingivalis and Prevotella intermedia”, Antimicrobial Agents and Chemotherapy, Vol. 53 No. 8, pp. 3308–3316.
Waldhuber, A., Puthia, M., Wieser, A., Cirl, C., Dürr, S., Neumann-pfeifer, S., Albrecht, S., et al. (2016), “Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation”, The Journal of Clinical Investigation, Vol. 126 No. 12, pp. 1–12.
201
Wang, M., Krauss, J.L., Domon, H., Hosur, K.B., Liang, S., Magotti, P., Triantafilou, M., et al. (2010), “Microbial hijacking of complement-toll-like receptor crosstalk”, Science Signaling, Vol. 3 No. 109, p. ra11.
Wang, M., Shakhatreh, M.A., James, D., Liang, S., Nishiyama, S., Yoshimura, F., Demuth, D.R., et al. (2007), “Fimbrial proteins of porphyromonas gingivalis mediate in vivo virulence and exploit TLR2 and complement receptor 3 to persist in macrophages”, Journal of Immunology, Vol. 179 No. 4, pp. 2349–2358.
Watanabe, K., Yamaji, Y. and Umemoto, T. (1992), “Correlation between cell‐adherent activity and surface structure in Porphyromonas gingivalis”, Oral Microbiology and Immunology, Vol. 7 No. 6, pp. 357–363.
Watanabe, T., Kitani, A., Murray, P.J. and Strober, W. (2004), “NOD2 is a negative regulator of Toll-like receptor 2-mediated T helper type 1 responses”, Nat Immunol, Vol. 5 No. 8, pp. 800–808.
Watkins, H.R., Lapp, C. a, Hanes, P.J., Dickinson, D.P., Volkmann, K.R., Newman, C.L. and Konzelman, J.L. (2007), “CCL28 effects on periodontal pathogens.”, Journal of Periodontology, Vol. 78 No. 12, pp. 2356–2363.
Watters, T., Watters, E., Kenny, L.A.J. and O’Neill. (2007), “Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins”, Immunology and Cell Biology, Vol. 85 No. 6, pp. 411–419.
Wegner, N., Wait, R., Sroka, A., Eick, S., Nguyen, K.-A., Lundberg, K., Kinloch, A., et al. (2010), “Peptidylarginine deiminase from Porphyromonas gingivalis citrullinates human fibrinogen and α-enolase: implications for autoimmunity in rheumatoid arthritis.”, Arthritis and Rheumatism, Vol. 62 No. 9, pp. 2662–72.
Weinberg, A., Belton, C.M., Park, Y. and Lamont, R.J. (1997), “Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells”, Infection and Immunity, Vol. 65 No. 1, pp. 313–316.
Wente, M., Wente, C., Mayer, M., Gaida, C., Michalski, T., Giese, F., Bergmann, N., et al. (2008), “CXCL14 expression and potential function in pancreatic cancer”, Cancer Letters, Vol. 259 No. 2, pp. 209–217.
Werner, H. and Katz, J. (2004), “The Emerging Role of the Insulin-like Growth Factors in Oral Biology”, Journal of Dental Research, Vol. 83 No. 11, pp. 832–836.
Wesche, H., Henzel, W.J., Shillinglaw, W., Li, S. and Cao, Z. (1997), “MyD88: An adapter that recruits IRAK to the IL-1 receptor complex”, Immunity, Vol. 7 No. 6, pp. 837–847.
Xu, Y., Tao, X., Shen, B., Horng, T., Medzhitov, R., Manley, J.L. and Tong, L. (2000), “Structural basis for signal transduction by the Toll / interleukin-1 receptor domains”, Nature, Vol. 408, pp. 111–115.
Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., et al. (2003a), “Role of Adaptor TRIF in the MyD88-Independent Toll-Like Receptor Signaling Pathway”, Science, Vol. 301 No. 5633, pp. 640–643.
Yamamoto, M., Sato, S., Hemmi, H., Hoshino, K., Kaisho, T., Sanjo, H., Takeuchi, O., et al. (2003b), “TRAM is specifically involved in the Toll-like receptor 4–mediated MyD88-independent signaling pathway”, Nature Immunology, Vol. 4 No. 11, pp. 1144–1150.
Yamamoto, M., Sato, S., Hemmi, H., Sanjo, H., Uematsu, S., Kaisho, T., Hoshino, K., et al. (2002), “Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4”, Nature, Vol. 420 No. 6913, pp. 324–329.
Yamasaki, K. (2006), “Kallikrein-mediated proteolysis regulates the antimicrobial effects of cathelicidins in skin”, The FASEB Journal, Vol. 20 No. 12, pp. 2068–2080.
Yamazaki, K., Nakajima, T. and Hara, K. (1995), “Immunohistological analysis of T cell
202
functional subsets in chronic inflammatory periodontal disease.”, Clinical and Experimental Immunology, Vol. 99 No. 3, pp. 384–391.
Yang, D., Chen, Q., Hoover, D.M., Staley, P., Tucker, K.D., Lubkowski, J. and Oppenheim, J.J. (2003), “Many chemokines including CCL20/MIP-3alpha display antimicrobial activity.”, Journal of Leukocyte Biology, Vol. 74 No. 3, pp. 448–455.
Yang, D., Chen, Q., Schmidt, A.P., Anderson, G.M., Wang, J.M., Wooters, J., Oppenheim, J.J., et al. (2000), “LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells.”, The Journal of Experimental Medicine, Vol. 192 No. 7, pp. 1069–1074.
Yao, Y., Berg, E.A., Costello, C.E., Troxler, R.F. and Oppenheim, F.G. (2003), “Identification of protein components in human acquired enamel pellicle and whole saliva using novel proteomics approaches”, Journal of Biological Chemistry, Vol. 278 No. 7, pp. 5300–5308.
Yilmaz, Ö., Watanabe, K. and Lamont, R.J. (2002), “Involvement of integrins in fimbriae-mediated binding and invasion by Porphyromonas gingivalis”, Cellular Microbiology, Vol. 4 No. 5, pp. 305–314.
Yilmaz, Ö., Young, P.A., Lamont, R.J. and Kenny, G.E. (2003), “Gingival epithelial cell signalling and cytoskeletal responses to Porphyromonas gingivalis invasion”, Microbiology, Vol. 149 No. 9, pp. 2417–2426.
Yu, J.J., Ruddy, M.J. and Gaffen, S.L. (2007), “An essential role for IL-17 in preventing pathogen initiated bone destruction : recruitment of neutrophils to inflamed bone requires IL-17 receptor – dependent signals”, Blood, Vol. 109 No. 9, pp. 1–16.
Yuki, T., Haratake, A., Koishikawa, H., Morita, K., Miyachi, Y. and Inoue, S. (2007), “Tight junction proteins in keratinocytes: Localization and contribution to barrier function”, Experimental Dermatology, Vol. 16 No. 4, pp. 324–330.
Zambon, J.J., Reynolds, H.S. and Slots, J. (1981), “Black-pigmented Bacteroides Spp. in the human oral cavity”, Infection and Immunity, Vol. 32 No. 1, pp. 198–203.
Zarember, K.A. and Godowski, P.J. (2002), “Tissue Expression of Human Toll-Like Receptors and Differential Regulation of Toll-Like Receptor mRNAs in Leukocytes in Response to Microbes, Their Products, and Cytokines”, Journal of Immunology, Vol. 168 No. 2, pp. 554–561.
Zenobia, C., Hasturk, H., Nguyen, D., Van Dyke, T.E., Kantarci, A. and Darveaua, R.P. (2014), “Porphyromonas gingivalis lipid a phosphatase activity is critical for colonization and increasing the commensal load in the rabbit ligature model”, Infection and Immunity, Vol. 82 No. 2, pp. 650–659.
Zhang, J., Dong, H., Kashket, S. and Duncan, M.J. (1999), “IL-8 degradation by Porphyromonas gingivalis proteases”, Microbial Pathogenesis, Vol. 26 No. 5, pp. 275–280.
Zhang, Q., Zmasek, C.M., Cai, X. and Godzik, A. (2011), “TIR domain-containing adaptor SARM is a late addition to the ongoing microbe-host dialog”, Developmental and Comparative Immunology, Elsevier Ltd, Vol. 35 No. 4, pp. 461–468.
Zijnge, V., Van Leeuwen, M.B.M., Degener, J.E., Abbas, F., Thurnheer, T., Gmür, R. and Harmsen, H.J.M. (2010), “Oral biofilm architecture on natural teeth”, PLoS ONE, Vol. 5 No. 2, pp. 1–9.
Zipfel, P.F. and Skerka, C. (2009), “Complement regulators and inhibitory proteins”, Nature Reviews Immunology, Vol. 9 No. 10, pp. 729–740.
Zurawski, D. V., Mitsuhata, C., Mumy, K.L., McCormick, B.A. and Maurelli, A.T. (2006), “OspF and OspC1 are Shigella flexneri type III secretion system effectors that are required
203
for postinvasion aspects of virulence”, Infection and Immunity, Vol. 74 No. 10, pp. 5964–5976.
204
Appendix Table A2 Mammalian TIR domains used for protein sequence alignments
Protein Accession number TIR domain TLR1 Q15399 635-779 TLR2 O60603 639-784 TLR3 O15455 754-896 TLR4 O00206 672-818 TLR5 O60602 691-837 TLR6 Q9Y2C9 640-784 TLR7 Q9NTK1 878-1036 TLR8 Q9NR97 878-1025 TLR9 Q9NR96 868-1025 MAL P58753 84-221 MYD88 Q99836 159-296 TRAM Q86XR7 73-232 TRIF Q8IUC6 390-460 SARM Q6SZW1 559-657
205
Table A3 Bacterial TIR domain-containing proteins
Species Protein Accession number TIR domain Porphyromonas gingivalis PG0382 I9E6I1 341-490 Brucella melitensis TcpB Q8YF53 120-490 Escherichia coli TcpC G8Z3N0 171-307 Paracoccous dentrificans PdTIR A1AY86 168-258 Yersinia pestis YpTdp Q8CL16 130-285 Staphycoccous aureus TirS ORF020 142-245
Minerva Access is the Institutional Repository of The University of Melbourne
Author/s:Aw, Jiamin
Title:Host-pathogen interactions of porphyromonas gingivalis
Date:2018
Persistent Link:http://hdl.handle.net/11343/214396
Terms and Conditions:Terms and Conditions: Copyright in works deposited in Minerva Access is retained by thecopyright owner. The work may not be altered without permission from the copyright owner.Readers may only download, print and save electronic copies of whole works for their ownpersonal non-commercial use. Any use that exceeds these limits requires permission fromthe copyright owner. Attribution is essential when quoting or paraphrasing from these works.