type iv pilins and identification of a novel d ......diversity of pseudomonas aeruginosa type iv...

DIVERSITY OF PSEUDOMONAS AERUGINOSA TYPE IV PILINS

AND IDENTIFICATION OF A NOVEL D-ARABINOFURANOSE POST-TRANSLATIONAL

MODIFICATION

BY

JULIANNE V. KUS

A thesis submitted in conformity with the requirements for the Degree of Doctor of Philosophy

Graduate Department of Dentistry University of Toronto

© 2008 Julianne V. Kus

ii

DIVERSITY OF PSEUDOMONAS AERUGINOSA TYPE IV PILINS AND IDENTIFICATION OF A NOVEL D-ARABINOFURANOSE

POST-TRANSLATIONAL MODIFICATION

Julianne V. Kus

Doctor of Philosophy, Faculty of Dentistry, University of Toronto

2008

ABSTRACT The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili

(T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are

formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were

described previously (I and II), distinguished by protein length and sequence. PilAI was

glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded

downstream of pilAI. To determine if additional pilin variants existed, analysis of the pilin

locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three

additional pilin alleles were discovered, each of which was invariantly associated with a

unique, previously unidentified, downstream gene(s): pilAIII+tfpY, pilAIV+tfpW+tfpX,

pilAV+tfpZ. This survey also revealed that strains with group I T4P were more commonly

associated with respiratory infections than strains with other pilins, suggesting that

glycosylated T4P may confer a colonization advantage in this environment. The newly

identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA

gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff

staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen

biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these

pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic

resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be

uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-

arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop

region of the protein, appear to be the preferred, but not exclusive sites of modification, each

being modified with up to four D-Araf sugars. This region of the pilin is partially surface-

exposed in the pilus, therefore modification of these sites may influence the surface

chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand

iii

region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses

and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase

necessary for PilAIV modification. This research has increased our understanding of the

complexity of this virulence factor, and may aid in development of new therapeutics for P.

aeruginosa and mycobacterial infections.

iv

ACKOWLEDGEMENTS

I am grateful for the mentorship, support, patience, enthusiasm and friendship of my

supervisors Dr. Lori Burrows and Dr. Dennis Cvitkovitch. I truly appreciate the

opportunities, and challenges, that you have provided me with over the years. I

would also like to thank Dr. John Kelly and his colleagues at the National Research

Council for their help with my research; their contributions were invaluable. I am

also thankful for the critiques, suggestions and guidance of my supervisory

committee members, Dr. Richard Ellen and Dr. Cliff Lingwood.

I also want to thank the many people that I’ve had the privilege to work with at

Toronto General Hospital, SickKids and the Faculty of Dentistry. I’ve been lucky to

have developed some truly great friendships during this degree. There were many

times when it was these friendships that brought me into the lab, even when my

experiments weren’t working. Thank you especially: Claude, Melissa, Craig, Poney,

Marc, Selva, Kirsten, Kowthar, and Carlos.

Beyond the lab, I need to thank my friends for their support and for trying to

understand why I always seemed to be busy.

Finally, I need to thank my family, especially my parents who have always

encouraged me, and believed in me, even when I didn’t believe in myself. Thank

you for all the opportunities that your love and support have made available to me.

v

TABLE OF CONTENTS

ABSTRACT ii ACKNOWLEDGEMENTS iv TABLE OF CONTENTS v LIST OF TABLES xi LIST OF FIGURES xii ABBREVIATIONS xiv PUBLICATIONS FROM THIS DEGREE xvi AWARDS HELD DURING THIS DEGREE xvii QUOTATION xviii CHAPTER 1: LITERATURE REVIEW _1 I. PSEUDOMONAS AERUGINOSA _2

A. Pseudomonas aeruginosa _2

B. The clinical significance of P.aeruginosa _3

1. Nosocomial infections _3

2. Cystic fibrosis and other pulmonary infections _3

C. The biofilm mode of growth _6

D. Attaching to a surface _7

II. TYPE IV PILI 10

A. History and classification 10

B. Distribution 10

C. Pilin to pilus 11

1. N-terminal α-helix 14

2. αβ-loop 14

3. β-strands 15

4. Disulphide-bonded-loop 15

5. The pilus fibre 16

6. Pilus assembly complex 20

D. Functions 21

1. Twitching motility 21

2. Adherence 24

E. Diversity of PilA 25

vi

1. Group II 26

2. Group I 27

3. Group III 27

4. Horizontal gene transfer 28

5. Additional mechanisms of pilin diversity 29

III. T4P GLYCOSYLATION SYSTEMS 30

A. Introduction to Gram-negative glycoproteins 30

B. Neisserial pilin glycosylation 32

1. Site of modification 32

2. Mechanism 33

3. Role of glycosylation in T4P function 34

C. P. aeruginosa pilin glycosylation: strain 1244 36

1. Site of modification 36

2. Mechanism 37

3. Role of glycosylation in T4P function 39

IV. RESEARCH AIMS 40

A. Hypothesis 40

B. The main objectives of this dissertation 40

V. REFERENCES 42

CHAPTER 2: SIGNIFICANT DIFFERENCES IN TYPE IV PILIN ALLELE DISTRIBUTION AMONG PSEUDOMONAS AERUGINOSA ISOLATES FROM CYSTIC FIBROSIS (CF) VERSUS NON-CF PATIENTS 58 I. ABSTRACT 59

II. INTRODUCTION 59

III. MATERIALS AND METHODS 63

A. Bacterial strains and media 63

B. Twitching assays 64

C. Polymerase chain reaction (PCR), cloning and DNA sequencing 65

D. Identification of novel accessory genes 66

E. Restriction fragment length polymorphism typing of tfpOb strains 66

F. Pulsed-field gel electrophoresis 67

G. Analysis of pilin proteins by SDS-PAGE 68

H. Phylogenetic analysis 69

vii

I. Statistical analysis _69

J. Accession numbers _69

IV. RESULTS _70

A. Twitching motility in CF versus non-CF strains _70

B. PCR amplification analysis of pilA and flanking sequences _70

C. Group II pilins _71

D. Group III pilins _73

E. Group IV pilins _75

F. Group V pilins _76

G. Group I pilin genes and their predominance in CF isolates _77

H. Analysis of novel pilin proteins _79

I. Phylogenetic analysis of PilA proteins from P. aeruginosa

confirms that there are five distinct groups _80

V. DISCUSSION _81

VI. ACKNOWLEDGEMENTS _89

VII. REFERENCES _97

CHAPTER 3: THE PREDOMINANCE OF P. AERUGINOSA STRAINS WITH GROUP I T4P IN CF SPUTUM IS NOT CF-SPECIFIC 104 I. INTRODUCTION 105

II. MATERIALS AND METHODS 107

A. Bacterial Strains 107

B. Expression of pilin variants in a common background strain 107

III. RESULTS AND CONCLUSIONS 108

A. Pulsed-field gel electrophoresis 108

B. PCR amplification analysis of pilA and accessory genes 108

C. Distribution of T4P groups 109

D. Pilin variants in a common background strain 110

IV. REFERENCES 119

CHAPTER 4: GLYCOSYLATION OF PSEUDOMONAS AERUGINOSA STRAIN PA5196 TYPE IV PILINS WITH MYCOBACTERIAL-LIKE α-1,5 LINKED D-ARAf OLIGOSACCHARIDES 122 I. ABSTRACT 123

viii

II. INTRODUCTION 124

III. MATERIALS AND METHODS 126

A. Bacterial strains and serotyping 126

B. Generation of a wbpM mutant of Pa5196 127

C. Pilin isolation 128

D. Fluorescent glycoprotein detection 129

E. Mass spectrometry analysis of the intact pilin 129

F. Enzymatic digestion and nanoLC-MS/MS analysis of the pilin 129

G. Fractionation of the enzymatic digests and analysis by

nano-electrospray ionization – mass spectrometry (nESI-MS) 130

H. Glycopeptide purification for NMR analysis 131

I. NMR analysis of the pilin glycan 131

J. Determination of the enantiomeric form of the glycan by GC-MS 132

K. M. smegmatis whole cell lysate preparation and Western blot

analysis 133

IV. RESULTS 134

A. Pa5196 pilins are modified with a novel glycan that is not the

O-antigen 134

B. Intact protein mass determination by ESI-MS 136

C. Analysis of enzymatic digests by nanoLC-MS/MS 136

D. Characterization of the T55-79 tryptic glycopeptide by

nESI-feCID-MS/MS 140

E. Elucidation of the O-linked pilin glycopeptide structure 141

F. Determination of the chiral orientation of the arabinose glycan 143

G. Antigenic identity of the Pa5196 pilin glycan with M. tuberculosis

lipoarabinomannan 144

V. DISCUSSION 146

VI. ACKNOWLEDGMENTS 151

VII. REFERENCES 152

ix

CHAPTER 5: MODIFICATION OF THE TYPE IV PILINS IN PSEUDOMONAS AERUGINOSA PA5196 AT MULTIPLE SITES WITH 1,5-D-ARABINO FURANOSE AND IDENTIFICATION OF THE PUTATIVE D-ARABINOSYL-TRANSFERASE, TFPW 159 I. ABSTRACT 160

II. INTRODUCTION 161

III. EXPERIMENTAL PROCEDURES 164

A. Bacterial strains and growth conditions 164

B. Sequence analysis of PilA and TfpW 165

C. Generation of pilA and tfpW mutants 168

D. Complementation of mutants 169

E. Site-directed mutagenesis 170

F. Twitching motility assays 170

G. Surface pilin isolation, SDS-PAGE and Western blots 171

H. Mass spectrometry analysis of intact pilin 173

I. Enzymatic digestion and LC-MS analysis of the pilA protein

from Pa5196NP+pilA 174

J. Electron-transfer dissociation mass spectrometry 175

IV. RESULTS 175

A. Antibodies to arabinosylated pilins recognize mycobacterial cell

wall material 175

B. Modification sites of Pa5196 PilA 179

C. Electron-transfer dissociation analysis of Pa5196 pilins 186

D. Glycosylation of other pilins in the Pa5196 background 188

E. Characterization of the putative arabinosyltransferase, TfpW 189

V. DISCUSSION 193

A. TfpW: a novel glycosyltransferase? 198

VI. ACKNOWLEDGEMENTS 203

VII. REFERENCES 204

CHAPTER 6: SUMMARY AND CONCLUSIONS 213 I. SUMMARY OF DISSERTATION AND DISCUSSION 214

A. T4P variation and distribution 214

B. A novel P. aeruginosa T4P modification 218

x

C. Sites of PilA5196 glycosylation 220

D. TfpW: A novel arabinosyltransferase 224

E. Proposed mechanisms for PilA5196 glycosylation 226

II. FUTURE WORK 229

A. Identifcation of arabinose biosynthetic genes 229

B. Determining the biological role of arabinose-modified pilins 230

III. SIGNIFICANCE AND CONCLUSION 231

IV. REFERENCES 233

xi

LIST OF TABLES

CHAPTER 2: SIGNIFICANT DIFFERENCES IN TYPE IV PILIN ALLELE DISTRIBUTION AMONG PSEUDOMONAS AERUGINOSA ISOLATES FROM CYSTIC FIBROSIS (CF) VERSUS NON-CF PATIENTS Table 2-1. Distribution of pilin alleles among isolates from various sources _72

Supplemental Table 2-1.

Pseudomonas aeruginosa strains analyzed for this study _90

CHAPTER 3: THE PREDOMINANCE OF P. AERUGINOSA STRAINS WITH GROUP I T4P IN CF SPUTUM IS NOT CF-SPECIFIC Table 3-1. Primers to amplify pilA variants 115

Table 3-2. Primer combinations and templates used to amplify pilin genes 115

Table 3-3. Non-CF respiratory disease isolates of P. aeruginosa 116

Table 3-4. Percentage of each T4P group by source of isolation 117

CHAPTER 4: GLYCOSYLATION OF PSEUDOMONAS AERUGINOSA STRAIN PA5196 TYPE IV PILINS WITH MYCOBACTERIAL-LIKE α-1,5 LINKED D-ARAf OLIGOSACCHARIDES Table 4-1. 1H and 13C chemical shift assignments for the two αAraf

monomers that give rise to the dominant peaks in the NMR spectra

of the pilin-associated glycan from P. aeruginosa Pa5196 144

CHAPTER 5: MODIFICATION OF THE TYPE IV PILINS IN PSEUDOMONAS AERUGINOSA PA5196 AT MULTIPLE SITES WITH 1,5-D-ARABINO- FURANOSE AND IDENTIFICATION OF THE PUTATIVE D-ARABINOSYL-TRANSFERASE, TFPW Table 5-1. List of bacteria strains and plasmids used in this study 166 Table 5-2. PCR primers used in this study 172 Table 5-3. Selected BLASTP results for TfpW5196 191

xii

LIST OF FIGURES

CHAPTER 1: LITERATURE REVIEW Figure 1-1. Crystal structures of the pilin structural subunits of

N. gonorrhoeae and P. aeruginosa _13

Figure 1-2. The pole of a P. aeruginosa cell displaying T4P and flagella _18

Figure 1-3. Simplified cartoon of P. aeruginosa pilus assembly complex _20

Figure 1-4. Twitching motility assay _22

Figure 1-5. Genetic organization of the pilin locus of P. aeruginosa _26

Figure 1-6. Structure and molecular mechanism of pilin glycosylation _35

CHAPTER 2: SIGNIFICANT DIFFERENCES IN TYPE IV PILIN ALLELE DISTRIBUTION AMONG PSEUDOMONAS AERUGINOSA ISOLATES FROM CYSTIC FIBROSIS (CF) VERSUS NON-CF PATIENTS Figure 2-1. Five distinct pilin alleles in P. aeruginosa _71

Figure 2-2. Amino acid sequence alignment of the pilins’ C-terminal

DSL region _74

Figure 2-3. PFGE typing of group V strains _77

Figure 2-4. SDS-PAGE of sheared pilins _80

Figure 2-5. Kyte-Doolittle hydropathy plots of TfpO and TfpW proteins _82

Figure 2-6. Phylogenetic relationships between PilA and related proteins _83

CHAPTER 3: THE PREDOMINANCE OF P. AERUGINOSA STRAINS WITH GROUP I T4P IN CF SPUTUM IS NOT CF-SPECIFIC Figure 3-1. Twitching assays of transgenic strains 112

CHAPTER 4: GLYCOSYLATION OF PSEUDOMONAS AERUGINOSA STRAIN PA5196 TYPE IV PILINS WITH MYCOBACTERIAL-LIKE α-1,5 LINKED D-ARAf OLIGOSACCHARIDES Figure 4-1. Pa5196 pilins continue to be glycosylated in a wbpM mutant 135

Figure 4-2. Analysis of the intact Pa5196 pilin by ESI-MS 137

Figure 4-3. Identification of pilin glycopeptides by MS/MS 138

Figure 4-4. feCID-MS/MS analysis of the T55-79 glycopeptide 141

xiii

Figure 4-5. 1D 1H spectrum and 1H-13C HSQC spectrum of the O-linked

pilin glycan from P. aeruginosa 143

Figure 4-6. Recognition of P. aeruginosa Pa5196 pilins by antibodies

to lipoarabinomannan 146

CHAPTER 5: MODIFICATION OF THE TYPE IV PILINS IN PSEUDOMONAS AERUGINOSA PA5196 AT MULTIPLE SITES WITH 1,5-D-ARABINO- FURANOSE AND IDENTIFICATION OF THE PUTATIVE D-ARABINOSYL-TRANSFERASE, TFPW Figure 5-1. CLUSTALW amino acid alignment of mature pilins 177

Figure 5-2. Pilins of strains Pa5196 and PA7 are modified with D-Araf 178

Figure 5-3. Twitching motility of PilA5196 site-directed mutants 180

Figure 5-4. SDS-PAGE and Westerns of SDM surface T4P 182

Figure 5-5. Mass spectrometry of sheared intact pilins 183

Figure 5-6. LC-MS analysis of the tryptic digest of pilin protein

isolated from P. aeruginosa 5196NP + PilA5196 185

Figure 5-7. ETD-MS analysis of glycosylated peptides of 5196NP+pilA5196 187

Figure 5-8. Topology model of TfpW 190

Figure 5-9. TfpW influences twitching motility 191

Figure 5-10. TfpW participates in the glycosylation of PilA5196 194

Figure 5-11. Model of truncated Pa5196 PilA illustrating sites, and potential

sites, of D-Araf modification 196

xiv

ABBREVIATIONS

AA Amino acid

AG Arabinogalactan

Ala Alanine

AP Alkaline phosphatase

Asn Asparagine

BAL Bronchoalveolar lavage

CAD Collision assisted dissociation

Carb Carbenicillin

CF Cystic fibrosis

CFTR Cystic fibrosis transmembrane regulator

CID Collision induced dissociation

COSY Correlation Spectroscopy

Cys Cysteine

Da Daltons

D-Araf D-arabinofuranose

DATDH 2,4-diacetimido-2,4,6-trideoxyhexopyranose

DPB Diffuse panbronchiolitis

DSL Disulphide-bonded loop

ELISA Enzyme-linked immunosorbent assay

ESI-MS Electrospray ionizing mass spectrometry

ETEC Enterotoxigenic Escherichia coli

ETD-MS Electron transfer dissociation mass spectrometry

feCID Front-end collision induced dissociation

GC-MS Gas-chromatography mass spectrometry

Gg4 Gangliotetraosylceramide

Gm Gentamicin

asialo-GM1 Gangliotetraosylceramide

HMBC Heteronuclear multiple bond correlation

HPLC High proformance liquid cromatography

HSQC Heteronuclear single quantum coherence

IATS International antigenic typing scheme

ICU Intensive care unit

xv

LAM Lipoarabinomannan

LB Luria-Bertani

LC Liquid chromatography LPS Lipopolysaccharide

LTQ Linear trap quadrupole

mAb Monoclonal antibody

MDCK Madin-Darby canine kidney min minutes

MS Mass spectrometry

NIAID National institute of allergy and infectious diseases

NIH National institute of health

NMR Nuclear magnetic resonance

NOESY Nuclear overhauser effect spectroscopy

NP No pili

ODS Octadecyl silane

O-GlcNAc O-linked N-acetylglucosamine

ORF Open reading frame

PAS Periodic acid-Schiff

PCR Polymerase chain reaction

PFGE Pulsed-field gel electrophoresis

Pro Proline

Q-TOF2 Quadrupole time-of-flight mass spectrometer

rpHPLC-MS Reversed-phase high performance liquid chromatography mass

spectroscopy

s seconds

SDM Site-directed mutagenesis

SDS-PA Sodium dodecyl sulfate polyacrylamide

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

Ser Serine

T4P Type IV pili

Thr Threonine

TOCSY Total correlation spectroscopy

WT Wild-type

xvi

PUBLICATIONS FROM THIS DEGREE PUBLICATIONS FROM THE DISSERTATION CHAPTERS 1. Kus, J.V., Tullis, E., Cvitkovitch, D.G. and Burrows, L.L. (2004). Significant

differences in type IV pilin distribution among Pseudomonas aeruginosa isolates

from cystic fibrosis (CF) versus non-CF patients. Microbiology 150: 1315-26.

2. Voisin, S.*, Kus, J.V.*, Houliston, S., St-Michael, F., Watson, D., Cvitkovitch, D.G., Kelly, J., Brisson, J-R., and Burrows, L.L. (2007). Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterial-like α-

1,5 linked D-Araf oligosaccharides. Journal of Bacteriology 189:151-9. *authors

contributed equally to this work. 3. Kus, J.V., Kelly, J., Tessier, L., Harvey, H., Cvitkovitch, D.G., and Burrows, L.L.

(2008). Modification of Pseudomonas aeruginosa Pa5196 type IV pilins at

multiple sites with 1,5-D-arabinosfuranose and identification of the putative

arabinosyltransferase, TfpW. Submitted to Journal of Biological Chemistry,

February 2008.

PUBLICATIONS NOT INCLUDED IN THIS DISSERTATION 1. Moraes, T.J.,Plumb, J., Martin, R., Vachon, E., Cherepanov, V., Koh, A., Loeve,

C., Jongstra-Bilen, J., Kus, J.V., Burrows, L.L., Grinstein, S. and Downey, G.P. (2006) Abnormalities in the Pulmonary Innate Immune System in Cystic Fibrosis. American Journal of Respiratory Cell and Molecular Biology. 34: 364-74.

2. Eman, A., Yu, A.R., Park, H-J, Mahfoud, R., Kus, J.V., Burrows, L.L. and

Lingwood, C.A. (2006). Laboratory and clinical Pseudomonas aeruginosa strains do not bind glycosphingolipids in vitro or during type IV pili-mediated initial host

cell attachment. Microbiology. 152: 2789-99.

xvii

AWARDS HELD DURING THIS DEGREE

2007 Constantine Maniatopoulos Graduate Scholarship, awarded for presenting the best student poster, Basic Sciences, Faculty of Dentistry’s Annual Research Day

2006 Student Presentation Award/Travel Award, Federation of American

Societies for Experimental Biology (FASEB) Conference on Microbial Polysaccharides of Medical, Agricultural and Industrial Importance

2002-2007 Canadian Institute for Health Research (CIHR) Fellowship, “Cell

Signaling in Mucosal Inflammation and Pain” STP-53877 2006-2007 Ontario Graduate Scholarship 2002-2007 Harron Scholarship, Faculty of Dentistry, University of Toronto 2005-2006 University of Toronto Open Fellowship 2004-2006 Canadian Cystic Fibrosis Foundation 2003-2004 Seymour Bresalier Ontario Graduate Scholarship in Science and

Technology 2002 McMurrich Award (Best Basic Science Poster Presentation), 1st Annual

University of Toronto, Department of Surgery, Gallie-Bateman Research Day

xviii

"If we knew what it was we were doing,

it would not be called research, would it?”

Albert Einstein

1

CHAPTER 1:

LITERATURE REVIEW

2

I. PSEUDOMONAS AERUGINOSA

A. Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative rod-shaped bacterium that is

ubiquitous in nature, inhabiting diverse environments including soil, water, plants,

animals and humans (Green et al., 1974; Lyczak et al., 2000; Ramos, 2004). They

belong to the Gamma Proteobacteria, a group which includes several medically

significant pathogenic bacteria including Escherichia coli, Vibrio cholerae,

Salmonella spp., Haemophilus influenzae and Legionella pneumophila.

P. aeruginosa is a metabolically versatile microorganism with meagre

nutritional requirements and are capable of tolerating diverse physical conditions

including a wide range of temperatures (>42°C), high salt concentrations, weak

antiseptics, and many antimicrobial agents. These characteristics have contributed

to the success of P. aeruginosa as an opportunistic pathogen of humans, and a

dominant nosocomial pathogen capable of colonizing virtually any site within

immunocompromised individuals (Bodey et al., 1983; Canadian Antimicrobial

Resistance Alliance, 2007; Lyczak et al., 2000). P. aeruginosa’s ability to infect a

variety of organisms, including the model hosts Mus musculus, Drosophila

melanogaster, Caenorhabditis elegans and Arabidopsis thaliana has allowed for

genetic studies of this bacterium’s mechanisms of pathogenicity (Mahajan-Miklos et

al., 2000).

3

B. The clinical significance of P. aeruginosa

1. Nosocomial infections. Nosocomial P. aeruginosa infections are most

frequently associated with ventilator-associated acute pneumonia in intensive care

unit (ICU) patients, however blood, wound, eye and urinary tract isolates are also

common (Bodey et al., 1983; Canadian Antimicrobial Resistance Alliance, 2007;

Lyczak et al., 2000). P. aeruginosa possesses a wide range of virulence factors that

contribute to its success as an opportunistic pathogen, including a variety of

extracellular enzymes and toxins which damage host cells and tissues, a number of

different adhesins, and several mechanisms for antimicrobial resistance (Lyczak et

al., 2000). It is currently the most commonly isolated pathogen in Ontario ICUs, and

ranks in the top six pathogens isolated from ICUs throughout the rest of Canada,

contributing significantly to patient morbidity and length of hospital stay (Canadian

Antimicrobial Resistance Alliance, 2007; Carmeli et al., 1999). Clinical isolates are

becoming increasingly resistant to multiple classes of antibiotics, further limiting

treatment options for these infections (Pfaller et al., 2006). One study of patients in

a North American hospital setting reported that patients infected with P. aeruginosa

strains in which resistance had emerged had a 3-fold higher mortality rate than the

overall hospital mortality rate, highlighting the severity of these infections (Carmeli et

al., 1999).

2. Cystic fibrosis and other pulmonary infections. P. aeruginosa

pulmonary infections are the leading cause of morbidity and mortality amongst cystic

fibrosis (CF) patients; it is reported that respiratory failure is the primary cause of

4

death in 90% of persons with CF (Cystic Fibrosis Foundation, 2006; Davies, 2002;

Lyczak et al., 2002). Colonization of the respiratory tract by P. aeruginosa typically

occurs early in the life of a CF patient and is never resolved (Burns et al., 2001;

Murray et al., 2007). These individuals are not immunocompromised, but have a

mutation in the gene encoding the CF transmembrane conductance regulator

(CFTR) chloride ion channel which results in decreased salt transport across

mucosal membranes, causing mucosal secretions to be thick and dehydrated rather

than fluid. It is thought that mucociliary clearance of the lungs is inhibited by this

viscous mucus and as a consequence, microorganisms become trapped and persist

in the airways (Boucher, 2007a; Boucher, 2007b; Lyczak et al., 2002). This chronic

infection eventually causes irreversible lung damage to which the majority of patients

succumb.

P. aeruginosa is not normally the pioneering microorganism of the CF lung,

typically colonizing the lung after Staphylococcus aureus or Haemophilus influenzae

infections; however, over time it becomes the dominant and prevailing pathogen in

these patients (Tummler & Kiewitz, 1999). One theory of P. aeruginosa’s

prevalence in the CF lung is that its type IV pili (T4P) specifically bind to the receptor

gangliotetraosylceramide (asialo-GM1 or Gg4), which has been reported to be

present in higher amounts on epithelial cells of CF patients homozygous for the

common ∆F508 mutation of cftr (Imundo et al., 1995; Krivan et al., 1988; Saiman &

Prince, 1993). However, this issue is currently debated, as there is evidence that P.

aeruginosa does not require Gg4 to bind epithelial cells (see Section I, D) (Emam et

al., 2006; Schroeder et al., 2001).

5

Evidence that P. aeruginosa is more of a generalist, capable of exploiting a

stressed lung environment, as opposed to being a specific pathogen of CF patients,

is the fact that it is a common pathogen of other chronic lung diseases such as

diffuse panbronchiolitis (DPB) and non-CF related bronchiectasis (King et al., 2007;

Martinez-Garcia et al., 2007; Poletti et al., 2006). Again, in these diseases P.

aeruginosa colonization typically follows infection by other pathogens. As opposed

to other pathogens, DPB and bronchiectasis patients colonized with P. aeruginosa

had the worst clinical features, poorest lung function and the most extensive

disease, often leading to death (King et al., 2007; Poletti et al., 2006).

Although it remains unclear precisely why P. aeruginosa is the dominant

pathogen within the lungs of patients with CF and other pulmonary disorders,

several factors likely play significant roles in its success as a pathogen. These

factors include its widespread presence in the environment (including food, medical

instruments, sinks, drains, mops and dental water lines), the production of numerous

virulence factors, its innate resistance to antimicrobials, as well as it ability to grow

as a biofilm within the lung (Lyczak et al., 2000; Regnath et al., 2004; Singh et al.,

2000). As an environmental organism, perhaps P. aeruginosa’s greatest asset is the

ability to survive in a wide variety of niches and under continually changing and

stressful conditions. One mechanism for survival under unfavourable conditions is

the biofilm mode of growth.

6

C. The biofilm mode of growth.

Biofilms are organized communities of microorganisms that are attached to

biotic or abiotic surfaces encased in a self-produced polymeric matrix (Costerton et

al., 1999). This mode of growth is clinically significant as it is predicted that biofilms

account for >65% of all bacterial infections, including chronic and device-related

infections (Lewis, 2001). Unlike free-swimming bacteria, when living as part of a

biofilm bacteria are resistant to high levels of antibiotics and are able to evade the

host immune system (Drenkard, 2003; Hoyle & Costerton, 1991; Leid et al., 2005;

Lyczak et al., 2000). This resistance pattern is not due to a classic genetic

mechanism for antibiotic resistance, but instead appears to be an intrinsic and

complex property of the biofilm mode of growth (Drenkard, 2003). It is thought that

in addition to being encased in a polymeric matrix which affords protection from the

immune system, many of the bacteria within the biofilm enter a stationary state of

growth and are therefore not susceptible to antibiotic agents that target actively

dividing cells (del Pozo & Patel, 2007; Drenkard, 2003; Leid et al., 2005). These

properties of the biofilm contribute to the persistence of bacteria in chronic

infections.

Biofilm development occurs in several stages: the first step is bacterial

attachment to a surface, followed by microcolony formation, expansion, and finally

detachment, where bacteria return to the planktonic phase of growth to leave the

biofilm and establish a new community (O'Toole et al., 2000). Given that it is only

after the bacteria switch from the planktonic to sessile mode of growth that traditional

7

treatment regimes are no longer effective, strategies aimed at preventing initial

bacterial attachment, and subsequent biofilm formation, must be examined.

D. Attaching to a surface

In an effort to understand the processes involved in the initiation of biofilm

development, O’Toole and Kolter (1998) examined the roles of flagella and T4P in

the early stages of P. aeruginosa biofilm formation in a simple experiment examining

a series of surface attachment defective (sad) mutants and their ability to colonize

polyvinylchloride (PVC) plastic (O'Toole & Kolter, 1998). Flagellum-mediated

swimming motility was found to be required for bacterial translocation to the surface

for initial attachment to occur, however the role of T4P was less clear. Under their

experimental conditions, T4P did not appear to be necessary for initial attachment to

the surface as non-piliated (pilA-) strains became associated with the surface but

failed to form microcolonies (O'Toole & Kolter, 1998). They speculated that T4P

mediated twitching motility resulted in bacterial aggregation and that T4P

participated in stabilizing interactions with the surface following attachment.

However, work from our group and others has demonstrated that T4P function as

important bacterial adhesins to both biotic and abiotic surfaces and are essential in

the first step of biofilm formation when shear forces are present (Chi et al., 1991;

Chiang & Burrows, 2003; Emam et al., 2006; Giltner et al., 2006; Hahn, 1997).

Chiang and Burrows (2003) demonstrated that pilus-mediated initial

attachment to glass was required for biofilm formation, as non-piliated (pilA-)

bacteria failed to adhere to the surface. Interestingly, hyperpiliated, but twitching

8

deficient (pilT-), strains were capable of adhering to the surface and forming dense

biofilms, indicating that twitching motility is not required for attachment or biofilm

formation (Chiang & Burrows, 2003). Twitching motility however, did appear to be

required for “normal” biofilm development as the pilT- strains failed to produce

differentiated structures like those of the wild-type strains, pointing to T4P’s

importance in both the establishment and development of P. aeruginosa biofilms

(Chiang & Burrows, 2003). T4P clearly play an important role in bacterial

attachment to abiotic surfaces, which can contribute to disease in the case of

implanted medical devices, or act as a reservoir and potential source of infection.

The role of T4P in attachment to eukaryotic cells has been widely reported

(Chi et al., 1991; Emam et al., 2006; Lee et al., 1989; Ramphal et al., 1984; Sato et

al., 1988; Tang et al., 1995; Woods et al., 1980). Significantly, P. aeruginosa T4P

have been demonstrated to be essential virulence factors and adhesins to the Gg4

(commonly termed asialoGM1) receptor on CF-epithelial cells (Comolli et al., 1999;

Gupta et al., 1994; Hahn, 1997; Krivan et al., 1988; Lee et al., 1994; Saiman &

Prince, 1993; Schweizer et al., 1998; Sheth et al., 1994). In one study Comolli et al.

(1999) demonstrated in vitro that P. aeruginosa lab strain PA103 showed increased

binding to Gg4-supplemented Madin-Darby canine kidney (MDCK) cells compared

to those cells not treated with Gg4, and that this binding was inhibited when cells

were treated with a commercially available antibody to Gg4. Additionally, they

showed that this binding was T4P specific as a non-piliated strain showed little

binding to the cells or to Gg4 (Comolli et al., 1999).

9

Despite this evidence supporting its role in attachment, there are

contradictory reports on the specificity of Gg4 as a receptor for P. aeruginosa

(Emam et al., 2006; Schroeder et al., 2001). One study examined the binding of

PA103 as well as eight minimally-passaged clinical isolates to Gg4-supplemented

MDCK cells and found that while PA103 appeared to demonstrate increased

adherence to these cells, none of the clinical isolates displayed similarly enhanced

binding (Schroeder et al., 2001). This study was also critical of the use of the

commercially available Gg4 antibody in the cell-binding-inhibitory studies, as they

determined that this antibody preparation was contaminated with high titres of

antibodies to several P. aeruginosa antigens, including pili, thus bringing into

question the role of Gg4 as a pilus, or P. aeruginosa, receptor (Schroeder et al.,

2001). These findings were supported by work by members of our group that

demonstrated failure of eleven clinical and laboratory strains to bind to Gg4 or other

glycosphingolipids in a thin-layer chromatography overlay experiment, as well as a

glycosphingolipid ELISA, while the control strain enterohaemorrhagic E. coli strain

CL56 displayed binding (Emam et al., 2006). Additionally, P. aeruginosa were found

to bind to cultured lung epithelial cells in a pilus-dependent manner and the

frequency of binding was not altered when Gg4 was chemically depleted from

epithelial cells indicating that Gg4 is not a specific P. aeruginosa receptor (Emam et

al., 2006). It seems more likely that as a primarily environmental organism, P.

aeruginosa has evolved to utilize T4P to attach to diverse surfaces in a receptor-

independent manner.

10

II. TYPE IV PILI

A. History and Classification

Some of the earliest recorded observations of bacterial pili were micrographs

taken at the turn of the 20th century by Hinterberger and Reitman who observed

numerous fine threads on cultures of Pseudomonas pyocyanea (aeruginosa) on old

media; flagella however were observed more frequently on bacteria on moist agar

and in liquid culture (described in Houwink & van Iterson, 1950). Years later, while

studying flagella of E. coli and P. pyocyanea, Houwink and van Iterson made the first

detailed report of “non-flagellar appendages”, noting that these long thread-like

structures were apparent on bacteria associated with surfaces, however were not

observed on swimming bacteria (Houwink & van Iterson, 1950). In 1955 the terms

“fimbriae” (Latin for threads or fibres) and “pili” (Latin for hair or fur) were introduced

to describe these structures (Brinton, 1959; Duguid et al., 1955) thus establishing

the divisive nomenclature of these bacterial structures. In 1975, a standardized

classification system was proposed and the flexible pili that contribute to twitching

motility were placed into Group IV (Ottow, 1975). With the advent of molecular

biology and structural biochemistry, T4P have been further divided into two sub-

types, T4Pa and T4Pb based on protein size, sequence and structure (described

below) (Craig et al., 2004). The T4Pa of P. aeruginosa are the focus of this thesis.

B. Distribution

T4P are primarily found on Gram negative bacteria of the Beta, Gamma and

Delta Proteobacteria, including Haemophilus influenzae, Moraxella spp., Neisseria

11

gonorrhoeae, N. meningitidis, Pseudomonas spp., Eikenella corrodens, Myxococcus

xanthus, Dichelobacter nodosus, enteropathogenic E. coli and Vibrio chloerae

(Bakaletz et al., 2005; Fullner & Mekalanos, 1999; Henrichsen, 1975; Mattick, J.,

Hobbs, M., Cox, PT., Dalrymple, BP, 1993). As well, there is a report of twitching

motility and polar fimbriae in the Gram positive bacterium Streptococcus sanguis

(Henrichsen, 1975), and the recently published genome of this opportunistic

pathogen contains PilB and PilT-like proteins, as well as an open reading frame with

weak homology to the pilin structural subunit, suggesting that these structures may

be more widely distributed than once thought (Xu et al., 2007).

Interestingly, T4Pb seem to be strictly associated with human pathogens that

colonize the intestines, including V. cholerae, S. enterica serovar Typhi, EPEC and

ETEC (Donnenberg et al., 1992; Giron et al., 1991; Giron et al., 1994; Shaw &

Taylor, 1990; Taniguchi et al., 1995; Zhang et al., 2000). In contrast, bacteria

possessing T4Pa are found in a variety of different environments (Craig et al., 2004).

Regardless, both types of T4P are considered important colonization factors.

C. Pilin to Pilus

The T4P of P. aeruginosa are composed of thousands of monomeric subunits

of the protein PilA, or pilin, encoded by the gene pilA (Pasloske et al., 1985). The

pilins are approximately 150 amino acids in length and ~15 kDa (Frost &

Paranchych, 1977; Pasloske et al., 1985). PilA is synthesized as a pre-pilin with a

short (5 - 6 amino acids), positively charged leader sequence which is highly

conserved amongst species (Alm & Mattick, 1997; Mattick, 2002; Watson et al.,

12

1996). This sequence is followed by an N-terminal hydrophobic α helix, and a region

of anti-parallel β-strands which end in a C-terminal disulphide-bonded loop (DSL),

creating overall a ladle- or lollipop-like structure (Figure 1-1) (Craig et al., 2003;

Craig et al., 2004; Craig et al., 2006; Dalrymple & Mattick, 1987; Hazes et al., 2000;

Parge et al., 1995).

Pseudopilins are pilin-like proteins which are thought to combine to form a

short dynamic fibre (the pseudopilus) that acts as a piston in the type II secretion

system of many Gram-negative bacteria (Durand et al., 2003; Filloux, 2004). In the

event of over-expression of the major subunit of the pseudopilus, an extracellular

pilin-like fibre is formed (Durand et al., 2003; Vignon et al., 2003). The pseudopilin

subunits, like T4Pa subunits are composed of a similar and conserved positively

charged N-terminal leader sequence followed by a 20 – 30 amino acid α-helix, and a

globular head domain composed of four β-strands, however, the C-terminal DSL is

not present (Kohler et al., 2004; Nunn & Lory, 1993). Through the study of the

pseudopili of Klebsiella oxytoca and P. aeruginosa information on the early events of

pseudopilus, and ostensibly pilus biogenesis, have been generated. The pre-

pseudopilins are translated in the cytoplasm and translocated across the inner

membrane in a Sec-dependent, SRP-dependent process, in which the N-terminal

leader sequence acts as a Sec-signal sequence (Arts et al., 2007; Francetic et al.,

2007). The hydrophobic α-helix portion of the protein becomes embedded in the

membrane, with the C-terminus on the periplasmic side. Once in the membrane, the

signal-sequence is cleaved by the bi-functional enzyme XcpA/PilD, which also N-

methylates the new N-terminal residue, which is most often phenylalanine (Arts et

13

al., 2007; Strom et al., 1991; Strom & Lory, 1992; Strom et al., 1994). This

processing step is essential to make the pilins competent for assembly, as mutants

lacking XcpA/PilD are unable to express pili on the cell surface.

A B

Figure 1-1. Crystal structures of the pilin structural subunits of N. gonorrhoeae and P.

aeruginosa. A. N. gonorrhoeae pilin PilE, displaying the different regions of the protein and the

covalently attached glycan at Ser63 (orange) and a phosphate at Ser68 (red). B. P. aeruginosa

group II PAK pilin, PilA. The α-helix and β- strands are in grey, the αβ-loop is in green, the D-region

(disulphide-bonded loop region) is purple, the disulphide bond is cyan. Reproduced from Craig et

al., 2004, with the permission of Nature Reviews Microbiology, Nature Publishing Group.

A BA B

Figure 1-1. Crystal structures of the pilin structural subunits of N. gonorrhoeae and P.

aeruginosa. A. N. gonorrhoeae pilin PilE, displaying the different regions of the protein and the

covalently attached glycan at Ser63 (orange) and a phosphate at Ser68 (red). B. P. aeruginosa

group II PAK pilin, PilA. The α-helix and β- strands are in grey, the αβ-loop is in green, the D-region

(disulphide-bonded loop region) is purple, the disulphide bond is cyan. Reproduced from Craig et

al., 2004, with the permission of Nature Reviews Microbiology, Nature Publishing Group.

14

1. N-terminal α-helix. The N-terminal α-helix portion of the mature pilin is

highly conserved among all T4P, including T4Pa and T4Pb and can be divided into

two regions, α1-N (residues 1 - 28) and α1-C, (residues 29 - ~53) (Figure 1-1) (Craig

et al., 2003; Craig et al., 2004). The α1-N portion of the α-helix is hydrophobic and

retains the pilins in the inner membrane for additional processing (i.e. post

translational modifications) and as a reservoir for rapid pilus assembly, and is also

involved in mediating pilus assembly through hydrophobic interactions between

subunits (Castric, 1995; Craig et al., 2004; Dalrymple & Mattick, 1987; Hegge et al.,

2004; Stimson et al., 1995; Stimson et al., 1996). The α1-C portion of the α-helix is

amphipathic in nature, is embedded in the C-terminal region of the protein and

contributes to pilus stability (Craig et al., 2004). The α-helix, in part, drives pilus

assembly; the α-helices of the pilin subunits form a strong but flexible hydrophobic

helical bundle that composes the core of the pilus fibre, while the β-strands of the C-

terminal region cover the surface of the fibre (Aas et al., 2007b; Craig et al., 2003;

Craig et al., 2004; Craig et al., 2006; Parge et al., 1995).

2. αβ-loop. Joining the α-helix and the β-strands of the pilin is a short region

called the αβ-loop (Figure 1-1). Crystal structure studies have demonstrated that

this region is structurally variable among all T4Pa examined to date; in P.

aeruginosa strain PAK this region is composed of a minor β-sheet made up of three

short β-strands, while in the related P. aeruginosa strain K122-4 this region is poorly

organized, with a short α-helix (Craig et al., 2004; Hazes et al., 2000; Keizer et al.,

2001). In N. gonorrhoeae the αβ-loop is extended and forms a ridge containing

15

residues which are post-translationally modified (Craig et al., 2006; Forest et al.,

1999; Marceau et al., 1998; Parge et al., 1995). In all assembled T4P examined to

date, this region is partially buried within the pilus fibre and therefore participates in

subunit-subunit interactions, but it is also partially exposed on the pilus surface thus

influencing the fibres’ interaction with the environment (Craig et al., 2003; Craig et

al., 2004; Craig et al., 2006).

3. β-strands. Following the αβ-loop, and comprising the bulk of the globular

head domain, is a short series of anti-parallel β-strands which form the outer face of

the pilus fibre (Figure 1-1) (Craig et al., 2003; Craig et al., 2004; Craig et al., 2006).

Despite substantial sequence differences in this part of the protein, all T4P contain

this β-sheet region which acts as the structural core of the globular head. Within the

T4Pa this region is composed of 4 strands, while in T4Pb it is made up of 5 strands

(Craig et al., 2003; Craig et al., 2004). Interestingly, this portion of the T4Pb protein

contains a novel protein fold (Craig et al., 2003) which has led some researchers to

suggest that the evolutionary relationship between T4Pa and T4Pb may be indirect

(Craig et al., 2004).

4. Disulphide-bonded-loop. At the C-terminal portion of the protein there

are 2 conserved cysteine residues that form a disulphide-bonded loop (DSL) which

has been shown to be essential for pilus assembly (Figure 1-1) (Harvey et al.,

submitted 2007). However, there are studies in which the Cys residues were

mutated or replaced, or in which the disulphide-bond isomerase DsbA was mutated,

16

which have shown that the DSL is not necessary for fibre assembly, but did appear

to play a role in pilus function (Burrows, 2005; Farinha et al., 1994; Ha et al., 2003).

The length of the DSL varies between species with the most dramatic

differences occurring between those of T4Pa and T4Pb, with average lengths of 22

and 55 amino acids respectively (Craig et al., 2004). These differences in size and

sequence result in distinct folding patterns and influence subunit-subunit

interactions. However, in all pilins, this region is predicted to form a protruding edge

on the opposite side of the globular head from the αβ-loop ridge, thereby influencing

the fibres’ surface chemistry (Craig et al., 2004; Craig et al., 2006). The P.

aeruginosa pilins that have been crystallized (PAK and K122-4) have DSLs of 12

amino acids which are predicted to be exposed only at the tip of the pilus, and buried

along the length of the pilus fibre (Craig et al., 2003; Craig et al., 2004; Hazes et al.,

2000; Keizer et al., 2001; Lee et al., 1994). This region is of particular interest in P.

aeruginosa, as it is implicated as the adhesive part of the pilus that mediates

attachment to host cells and abiotic surfaces, and in some strains has been shown

to be glycosylated (Comer et al., 2002; Giltner et al., 2006; Lee et al., 1994; Sheth et

al., 1994). The DSL is also considered a good target for the development of

vaccines against P. aeruginosa infections (Audette et al., 2004; Cachia et al., 1998;

Sheth et al., 1995; Suh et al., 2001).

5. The pilus fibre. Mature pili are long (> 1 µm), thin (~ 6 – 8 nm) flexible

fibres that are extremely strong, capable of withstanding > 100 pN of force (Figure 1-

2) (Maier et al., 2002; Maier, 2005; Mattick, 2002; Parge et al., 1987; Strom & Lory,

17

1993). There is much interest in how the pilin subunits combine to form such

remarkable filaments. Recently Craig et al. (2006) developed a high resolution

model of assembled T4P from N. gonorrhoeae, using both cryo-electron microscopy

and crystallography. The model predicts that the subunits pack in a twisted three-

helix bundle with the N-terminal α helices pointed towards the pilus base (Craig et

al., 2006). Like previous models, this current model places the first half of the N-

terminal α helix (α1-N) of the subunits inside the central core of the fibre in a

staggered helix of interlocking subunits, stabilized through hydrophobic interactions

(Craig et al., 2004; Craig et al., 2006; Parge et al., 1995). Contrary to previous

models that predicted the β-sheets of the globular heads were wrapped tightly

around the fibre (Forest & Tainer, 1997; Parge et al., 1995), this newer model

predicts that there are deep grooves separating the heads resulting in a “corrugated”

surface (Craig et al., 2006). The αβ loop and the C-terminal domain are located at

the base of the grooves and polar interactions are observed between the αβ loop of

one subunit and the C-terminal region of the adjacent subunit. Post-translational

modifications of the N. gonorrhoeae pilin are predicted to protrude from exterior of

the filament towards the solvent (Craig et al., 2004; Craig et al., 2006). The outer

diameter of the fibre is ~ 60 Å while the central core is of variable diameter (~6 – 11

Å). The authors speculated that this channel is not solvent filled but is a

compressible space which may allow for further filament flexibility (Craig et al.,

2006).

18

This model helps to explain the strength and flexibility of the thin T4P filament: the

tight hydrophobic interactions between the α-helices provide strength, while the gaps

and grooves between the pilin heads provide flexibility and additional stability via the

αβ loop and C-terminal interactions between subunits. This model also helps

explain how pilin architecture can be conserved amongst bacteria, yet have

functional and antigenic differences. The conserved structural core supports the

diverse αβ loop and C-terminal regions line the grooves of the fibre which are

Figure 1-2. The pole of a P. aeruginosa cell displaying T4P and flagella. This image (100,000x

magnification) demonstrates the single polar flagellum of P. aeruginosa and multiple, thin, long type

IV pili. This is a pilD- mutant strain, unable to retract the T4P. Courtesy of P. Chiang.

Figure 1-2. The pole of a P. aeruginosa cell displaying T4P and flagella. This image (100,000x

magnification) demonstrates the single polar flagellum of P. aeruginosa and multiple, thin, long type

IV pili. This is a pilD- mutant strain, unable to retract the T4P. Courtesy of P. Chiang.

19

exposed to the environment and thereby subject to host-immune or other selective

pressures.

Although the N. gonorrhoeae pilus represents an excellent model, the distinct

differences between Neisseria and P. aeruginosa pili suggest that other models

must be considered in order to understand these structures. Although less detailed

than that of N. gonorrhoeae, X-ray diffraction analysis of the PAK fibres reveals that

these fibres have a 52 Å outer diameter and a 12 Å inner diameter, with the subunits

arranged in either a right-handed one-start helix with four subunits per turn or a left-

handed three-start helix with four subunits per turn (Craig et al., 2004). The most

significant structural and biological difference between the pili of these two species

involves the putative binding region. Neisseria pili are thought to be capped with a

separate PilC protein, a component involved in pilus-mediated adhesion to host cells

(Merz & So, 2000; Nassif et al., 1997; Scheuerpflug et al., 1999) whereas in P.

aeruginosa, PilA is thought to be used as both the structural component of the fibre

and the adhesive tip (Irvin et al., 1989; Lee et al., 1989; Lee et al., 1994). As

mentioned above, the C-terminal DSL portion of PilA has been demonstrated to be

the adhesive component of the pilus (Lee et al., 1994). While it appears to be at

least partially buried along the length of the fibre, antibody studies have

demonstrated its exposure at the pilus tip (Lee et al., 1994). Structural studies

support the contention that the DSL is obscured along the length of the fibre and

exposed only at its tip (Craig et al., 2004). Based on the N. gonorrhoeae pilin

packing model, up to three DSL sub-domains may be available for binding at the tip

of the P. aeruginosa T4P (Burrows, 2005; Craig et al., 2006)

20

PilC

PilT PilU

PilD/XcpA

PilQ

Figure 1-3. Simplified cartoon of P. aeruginosa pilus assembly complex. Pre-pilins (PilA) are

produced in the cytoplasm, targeted to the inner membrane (IM) via the N-terminal signal peptide.

PilD/XcpA cleaves off the signal sequence and N-methylates the N-terminal phenylalanine. The

mature pilins are polymerized into the pilus fibre through the action of PilB. The pilus crosses the

outer membrane (OM) through the PilQ pore. Retraction of the pilus occurs via disassembly via the

depolymerase(s) PilT and/or PilU. See text for references and details. PG = peptidoglycan.

IM

OM

PG

PilA

Pilus

PilQ

PilB

PilC

PilT PilU

PilD/XcpA

PilQ

Figure 1-3. Simplified cartoon of P. aeruginosa pilus assembly complex. Pre-pilins (PilA) are

produced in the cytoplasm, targeted to the inner membrane (IM) via the N-terminal signal peptide.

PilD/XcpA cleaves off the signal sequence and N-methylates the N-terminal phenylalanine. The

mature pilins are polymerized into the pilus fibre through the action of PilB. The pilus crosses the

outer membrane (OM) through the PilQ pore. Retraction of the pilus occurs via disassembly via the

depolymerase(s) PilT and/or PilU. See text for references and details. PG = peptidoglycan.

IM

OM

PG

PilA

Pilus

PilQ

PilB

6. Pilus assembly complex. The regulation, assembly and disassembly of

the mature pilus involves the products of over 50 genes (Alm & Mattick, 1997;

Mattick, 2002). A model of pilus assembly which highlights some of the proteins

relevant to this thesis, including the motor proteins PilB, the pilin polymerase, and

PilT the pilin depolymerase, is presented in Figure 1-3. PilB and PilT are predicted

21

to be peripherally associated with the inner membrane on the cytoplasmic face at

the base of the pilus in close proximity to the multi-protein complex that forms the

base from which the pilus extends (Chiang et al., 2005; Mattick, 2002).

D. Functions

T4P participate in many functions including bacteriophage infection (Bradley,

1974; Whitchurch & Mattick, 1994), transformation (Wolfgang et al., 1998), DNA

uptake (Aas et al., 2002; Fussenegger et al., 1997; Hamilton & Dillard, 2006;

Mattick, 2002), adhesion and twitching motility (Burrows, 2005; Mattick, 2002).

Unlike some T4P-possessing bacteria such as N. gonorrhoeae and P. stutzeri, P.

aeruginosa are not naturally competent and there is little evidence that the T4P in

this system are involved in DNA binding and uptake (Graupner et al., 2000; van

Schaik et al., 2005; Wolfgang et al., 1998). For this reason, I will focus on the

functions of twitching motility and adherence.

1. Twitching motility. Twitching motility is the hallmark of T4P. It is a form

of flagellar-independent motility that can occur on moist surfaces (Bradley, 1980;

Henrichsen, 1983; Kaiser, 1979; Lautrop, 1961; Ottow, 1975). Twitching bacteria

typically move as groups, or “rafts”, and use this motility as an important means of

rapidly colonizing new environments, and for biofilm establishment and

differentiation (Klausen et al., 2003a; Klausen et al., 2003b; Mattick, 2002; O'Toole &

Kolter, 1998; Semmler et al., 1999). The term “twitching” refers to the jerky fashion

in which bacteria move during this type of motility, which is achieved by the

22

extension of the T4P, attachment of the pilus tip to a surface and subsequent

retraction of the fibre, effectively moving the bacterial cell body towards the point of

pilus attachment (Bradley, 1980; Henrichsen, 1972; Mattick, 2002; Skerker & Berg,

2001). Pilus extension is achieved through the polymerization of the pilin subunits

via the activity of the ATPase PilB, while retraction is due to disassembly of the fibre

by action of the ATPases PilT and/or the PilT homologue PilU (Hobbs & Mattick,

1993; Kaiser, 2000; Merz et al., 2000; Whitchurch et al., 1991; Whitchurch & Mattick,

1994).

Figure 1-4. Twitching motility assay. A simple assay for twitching motility. A. Bacteria are stab

inoculated to the bottom of the agar plat. B. Bacteria migrate away from the point of inoculation via

twitching motility between agar and plastic of the plate forming a “halo” or twitching zone around the

colony. C. The twitching zone can be more easily visualized by removing the agar and staining the

bacteria attached to the plastic with crystal violet dye.

A

CB

Figure 1-4. Twitching motility assay. A simple assay for twitching motility. A. Bacteria are stab

inoculated to the bottom of the agar plat. B. Bacteria migrate away from the point of inoculation via

twitching motility between agar and plastic of the plate forming a “halo” or twitching zone around the

colony. C. The twitching zone can be more easily visualized by removing the agar and staining the

bacteria attached to the plastic with crystal violet dye.

A

CB

23

Macroscopically, twitching motility can be observed as a thin opaque

monolayer of cells surrounding the edge of a colony growing on an agar plate. This

zone can be enhanced when bacteria are stab inoculated to the bottom of an agar

plate where the bacteria grow at the interstitial surface between the agar and the

plastic (Figure 1-4) (Semmler et al., 1999). Twitching zones can reach several

centimetres in diameter after overnight growth. It is thought that the smooth surface

of the agar and the moist conditions at the bottom of the agar are ideal for twitching

motility, whereas on the surface of agar, minor surface variations and variations in

moisture levels retard twitching motility (Semmler et al., 1999). At the microscopic

level, twitching motility is seen at the outermost edge of the bacterial colony where a

lattice-like pattern of cells moving away from the colony is formed (Mattick, 2002).

The motive force of twitching motility is achieved by the retraction of the T4P;

the first evidence for this was the inhibition of twitching by pilus-specific phage and

antibodies which interfered with the retraction of the pili (Bradley, 1972a; Bradley,

1972b). Additional evidence for this action was the lack of twitching motility in hyper-

piliated strains, which were found to be retraction deficient (Merz et al., 2000;

Skerker & Berg, 2001; Whitchurch et al., 1991). The hyper-piliated phenotype is due

to mutations within PilT or PilU, the ATPases involved in pilus disassembly (Chiang

& Burrows, 2003; Mattick, 2002; Whitchurch & Mattick, 1994). Direct observational

evidence for pilus retraction and measurement of the forces generated by this action

has been obtained using N. gonorrhoeae and P. aeruginosa (Merz et al., 2000; Merz

& Forest, 2002; Skerker & Berg, 2001). These studies demonstrated that individual

24

pili retract independently of one another, and that retraction forces can exceed 140

pN, two orders of magnitude greater than a eukaryotic kinesin motor.

In order for twitching motility to occur, the tips of the T4P must be firmly

attached to a surface in order to anchor the bacteria; without attachment, the

bacterial cells would not be translocated upon pilus retraction.

2. Adherence. In P. aeruginosa T4P are considered to be the dominant

adhesins responsible for initiating attachment and subsequent infections (Hahn,

1997; Irvin et al., 1989). As described earlier (Sections I, B2 & D) there is evidence

that P. aeruginosa T4P bind to the epithelial cell receptor Gg4 (Comolli et al., 1999;

Gupta et al., 1994; Hahn, 1997; Krivan et al., 1988; Lee et al., 1994; Saiman &

Prince, 1993; Schweizer et al., 1998; Sheth et al., 1994). However, unlike obligate

pathogens such as N. gonorrhoeae, P. aeruginosa’s ability to exploit many different

niches, and to be an opportunistic pathogen of a variety of organisms is likely due in

part to the general rather than specific adhesive properties of its T4P. P. aeruginosa

is known to be associated with device-related infections, particularly nosocomial

infections involving catheters and ventilators (Pierce, 2005; Rosenthal et al., 2006)

and has long been known to colonize contact lenses, leading to microbial keratitis

(Willcox, 2007). Also, P. aeruginosa has been shown to be a problem in industrial

settings and on medical equipment, as it binds readily to stainless steel (Giltner et

al., 2006; Vanhaecke et al., 1990). Giltner et al. (2006) have demonstrated a role for

T4P in P. aeruginosa adherence to stainless steel, as wild-type and flagella-deficient

strains were capable of binding to stainless steel while pilin-deficient strains did not

25

bind. Further, the DSL of the pilin was implicated as the adhesive domain since

purified T4P were shown to bind to stainless steel, polystyrene and PVC, and this

binding was inhibited with the addition of an antibody against the pilin DSL (Giltner et

al., 2006). The authors point out that the sequence of the DSL varies greatly

amongst P. aeruginosa strains, however all variants tested have retained the ability

to bind to biotic and abiotic surfaces, albeit with different affinities.

The strains examined by Giltner et al. (2006) varied in amino acid sequence

in the C-terminal region, however all strains tested had DSLs of 12 amino acids.

There have been reports of strains with DSLs of different lengths among P.

aeruginosa T4P, for example the DSL of strain 1244 is 17 amino acids and that of

strain G7 is 31 amino acids (Castric et al., 1989; Castric & Deal, 1994; Spangenberg

et al., 1995). This range in size and sequence diversity of the adhesive region of the

protein raises questions regarding binding specificities of the T4P of this organism.

E. Diversity of PilA

In the PAO1 chromosome, pilA is located at approximately 72 minutes on the

genetic map, and lies in a region that contains several pilin-associated genes,

(Farinha et al., 1993; Mattick et al., 1996). The pilA gene is divergently transcribed

from the other pilin-associated genes in this region, with pilBCD positioned upstream

and tRNA-Thr situated downstream (Figure 1-5) (Hobbs et al., 1988; Mattick et al.,

1996; Stover et al., 2000). Genetic mapping and partial sequencing of the

chromosomes of small groups of P. aeruginosa strains has revealed that this area is

a region of extensive inter-strain variability (Pasloske et al., 1988; Romling et al.,

26

1995; Schmidt et al., 1996; Spangenberg et al., 1995). As mentioned above

(Section II, C4) there have been reports of several pilin variants in P. aeruginosa,

which form several distinct groups based on sequence, pilin length and DSL length

(Figure 1-5) (Castric & Deal, 1994; Pasloske et al., 1988; Spangenberg et al., 1995;

Spangenberg et al., 1997).

1. Group II. Group II pilins are represented by those of the well-described

laboratory strains PAK and PAO1, which have mature T4Ps of 143-144 amino acids

long, DSLs of 12 amino acids, a pilin gene %G+C content of 48% (versus 67%

pilB

pilB

pilB

pilA

pilA

pilA

ORF1

pilO/tfpO

tRNA-Thr

tRNA-Thr

tRNA-Thr

Group II (PAO1, PAK)

Group I (P1, 1244)

Group III (G7)

Figure 1-5. Genetic organization of the pilin locus of P. aeruginosa. Three different pilin

variants have been described, two of which are associated with novel open-reading frames directly

downstream. The group I ORFhas been identified as pilO/tfpO, which encodes a protein involved in

the glycosylation of pilin (Castric, 1989; Castric & Deal, 1994; Spangenberg et al., 1995; Castric,

1995). Figure adapted from Spangenberg et al. (1995). Not to scale.

pilB

pilB

pilB

pilA

pilA

pilA

ORF1

pilO/tfpO

tRNA-Thr

tRNA-Thr

tRNA-Thr


Group I (P1, 1244)

Group III (G7)

pilB

pilB

pilB

pilA

pilA

pilA

ORF1

pilO/tfpO

tRNA-Thr

tRNA-Thr

tRNA-Thr


Group I (P1, 1244)

Group III (G7)

Figure 1-5. Genetic organization of the pilin locus of P. aeruginosa. Three different pilin

variants have been described, two of which are associated with novel open-reading frames directly

downstream. The group I ORFhas been identified as pilO/tfpO, which encodes a protein involved in

the glycosylation of pilin (Castric, 1989; Castric & Deal, 1994; Spangenberg et al., 1995; Castric,

1995). Figure adapted from Spangenberg et al. (1995). Not to scale.

27

overall for PAO1) and a tRNA-Thr gene immediately downstream of the 3’ end of

pilA (Kiewitz & Tummler, 2000; Sastry et al., 1985; Spangenberg et al., 1995;

Wolfgang et al., 2003). Pili from these strains have been shown to specifically

interact with the Gg4 glycolipid in binding studies (Comolli et al., 1999; Gupta et al.,

1994; Hahn, 1997; Krivan et al., 1988; Lee et al., 1994; Saiman & Prince, 1993;

Schweizer et al., 1998; Sheth et al., 1994).

2. Group I. Group I pilins are related to those from the P1 strain, a CF

isolate, which was originally identified as having an unusual pilin sequence over the

last three quarters of the protein, with the most notable features being its mature

length of 148 amino acids, a 17 amino acid DSL, and a pilin gene with a %G+C

content of 52% (Pasloske et al., 1988). Additional strains with group I pili, including

strain 1244, were characterized, and were found to contain an additional 1.4 kb open

reading frame between pilA and tRNA-Thr, later named pilO (Figure 1-5) (Castric,

1995; Castric et al., 1989; Castric & Deal, 1994; Spangenberg et al., 1995).

3. Group III. Group III T4P were first identified in a single report of a strain,

G7, with another unusual pilin sequence. Group III pilins have a %G+C content of

54.8%, and are the longest mature pilin, composed of of 173 amino acids, with a

DSL of 31 residues (Spangenberg et al., 1995). Similar to group I strains, additional

DNA was found between the pilA and tRNA-Thr genes; however it was only 798

base pairs long, and failed to react with a probe made from the corresponding region

from group I strains; it was designated ORF1 (Spangenberg et al., 1995).

28

Interestingly, this additional ORF had a %G+C content of 48.4%, different from both

that of the pilin gene and average chromosomal content (Spangenberg et al., 1995).

4. Horizontal gene transfer. There is less than 30% sequence identity

between the P. aeruginosa pilin groups; in phylogenetic analyses, each group is

more closely related to pilins from other species than to one another (Castric & Deal,

1994; Hazes et al., 2000; Spangenberg et al., 1995; Spangenberg et al., 1997). A

phylogenetic analysis of pilins from 19 P. aeruginosa strains, as well as pilins from

other common T4P-possessing organisms, demonstrated that group I T4P form a

tight independent cluster, while the group II pilins were more closely related to pilins

from D. nodosus and Myxococcus xanthus. The single group III pilin grouped with

E. corrodens, D. nodosus, N. gonorrhoeae and N. meningitidis, though not closely

(Spangenberg et al., 1997). The pilins of P. aeruginosa, E. corrodens and D.

nodosus vary greatly in their %G+C content relative to the rest of their genomes, in

contrast to the pilins of Neisseria and Moraxella which form tight phylogenetic

clusters and have pilins of the same %G+C content and codon usage as their

chromosomes (Spangenberg et al., 1997). The pilins of P. aeruginosa, E. corrodens

and D. nodosus have a similar %G+C content to Moraxella suggesting that this locus

was acquired from other species by horizontal gene transfer (Castric & Deal, 1994;

Hazes et al., 2000; Kiewitz & Tummler, 2000; Mattick, 2002; Spangenberg et al.,

1995).

Several factors likely contribute to the diversity of pilA, including its

chromosomal organization (adjacent to a tRNA gene) and selective pressures due to

29

interaction of pili with the environment and host immune systems. tRNA genes are

common sites of bacteriophage and plasmid integration, possibly due to their

conserved structure and the presence of multiple copies on bacterial genomes that

affords functional redundancy (Cheetham & Katz, 1995; Reiter et al., 1989). There

is evidence in D. nodosus that virulence-associated regions are often adjacent to

tRNA genes, and these regions contain genes with homology to N. gonorrhoeae

plasmids and bacteriophages (Cheetham & Katz, 1995). Upstream of pilA are the

conserved pilBCD genes; pilBC share similarity to genes involved in type II secretion

(T2S) in P. aeruginosa, and pilD encodes PilD/XcpA that acts in both the pilin and

T2S systems (Nunn & Lory, 1993; Strom et al., 1993). Because of the proximity to

the known site of recombination tRNA-Thr and the conserved upstream genes

pilBCD, this area of the P. aeruginosa chromosome is a likely region of genetic

integration and recombination that has resulted in diversity within PilA. It is

interesting to consider how the size and sequence variations of different pilins

influence the functions and binding specificities of T4P.

5. Additional mechanisms of pilin diversity. Group I and III pilins were

found to be associated with additional genetic material inserted between pilA and

tRNA-Thr (Castric & Deal, 1994). In the case of group I strains this ORF was named

pilO and was demonstrated to encode a glycosyltransferase involved in the post-

translational modification of PilA (Castric, 1995; DiGiandomenico et al., 2002).

Castric’s group have demonstrated that PilO is a glycosyltransferase that mediates

the transfer of the O-antigen to the terminal Ser148 residue of the strain 1244 pilin

30

(Comer et al., 2002; DiGiandomenico et al., 2002). This region is adjacent to the

DSL, and possibly influences the T4P’s adhesive function. The unknown ORF

downstream of the group III pilA has no known function, however due to its proximity

to the pilin structural gene may encode a product that participates in pilin modulation

or assembly.

III. T4P GLYCOSYLATION SYSTEMS

A. Introduction to Gram-negative glycoproteins

Protein glycosylation is an additional means of modulating a protein’s

structure and/or function and can contribute to diversity (Benz & Schmidt, 2002;

Upreti et al., 2003). In recent years, many prokaryotes have been found to produce

N- and O-linked glycosylated proteins, including the Gram-negative pathogens

Campylobacter jejuni, N. meningitidis, N. gonorrhoeae and P. aeruginosa (Benz &

Schmidt, 2002; Power & Jennings, 2003; Schmidt et al., 2003; Upreti et al., 2003).

Many of the glycosylated proteins of pathogenic bacteria described to date are

associated with the surface of the organism and include flagellins of P. aeruginosa,

(Brimer & Montie, 1998), and C. jejuni (Doig et al., 1996), the E. coli adhesins TibA

and AIDA-1 (Benz & Schmidt, 2002; Lindenthal & Elsinghorst, 2001), and the type IV

pilins of some strains of P. aeruginosa (Castric et al., 2001), N. meningitidis and N.

gonorrhoeae (Parge et al., 1995; Stimson et al., 1995).

The glycosylation of bacterial proteins appears to be strain-dependent in

many cases; however for such an energetically expensive process, there must be a

fitness benefit for the strains that produce the modified proteins. The exact

31

biological function, or benefit, of glycosylation in many cases remains unclear.

Some functions that have been described include the maintenance of protein

structure or conformation, increased resistance to proteolytic enzymes, alteration of

the physicochemical properties of the protein/bacterial surface (e.g. solubility,

surface charge, hydrophilicity), alteration of immune detection and cell adhesion

(Banerjee et al., 2002; Benz & Schmidt, 2002; Moens & Vanderleyden, 1997;

Schmidt et al., 2003; Szymanski et al., 2002; Upreti et al., 2003).

In several cases the glycosylation of surface proteins of pathogenic bacteria

has been shown to increase adherence and colonization of host tissue and increase

pathogen virulence; examples include C. jejuni, in which the general glycosylation

pathway, which modifies several proteins, has been shown to increase bacterial

adherence, the outer membrane protein TibA of ETEC E. coli, and the P. aeruginosa

strain 1244 T4P (Elsinghorst & Weitz, 1994; Lindenthal & Elsinghorst, 2001;

Smedley et al., 2005; Szymanski et al., 2002). However, the adhesive function of

other surface proteins does not appear to be modulated by the presence/absence of

a glycan; examples include the pili of N. meningitidis, and the flagellin of C. jejuni

(Doig et al., 1996; Stimson et al., 1995).

There is significant heterogeneity in bacterial glycosylation systems with

respect to the nature of the target proteins and the sugars that modify them. In order

to understand the function and significance of glycosylation of bacterial proteins,

comparison between the wild-type strain and a non-glycosylated, or de-glycosylated,

version of the same strain must be performed, as generalizations regarding function

cannot readily be made.

32

Amongst Gram-negative bacteria, two systems involved in the biosynthesis of

glycan used for protein glycosylation have been described. The first involves

dedicated machinery that is specific for protein glycosylation, where most of the

genes required for glycan synthesis and transfer are grouped together on a genetic

island. Examples include the well-described systems for glycosylation of C. jejuni

surface proteins, Neisseria pilins, and P. aeruginosa flagellins (Arora et al., 2001;

Power et al., 2000; Power et al., 2003; Szymanski et al., 1999; Verma et al., 2006;

Wacker et al., 2002). The second type of system appropriates glycans that have

been synthesized and transported to the periplasm by the lipopolysaccharide (LPS)

machinery; an example of this type is the P. aeruginosa 1244 pilin glycosylation

system (DiGiandomenico et al., 2002).

B. Neisseria pilin glycosylation

1. Site of Modification. The T4P of N. meningitidis are essential virulence

factors for this obligate pathogen, mediating attachment to host epithelial cells and

subsequent colonization (Power & Jennings, 2003; Virji et al., 1992). Studies of the

pilin structural subunit, PilE, of strain C311 have revealed that these proteins are

glycosylated at Ser63 with the trisaccharide Gal(β1,4)Gal(α1-3)2,4-diacetamido-

2,4,6-trideoxyhexose (DATDH) (Power & Jennings, 2003; Stimson et al., 1995). The

N. gonorrhoeae MS11 pilin is also glycosylated at this site with a similar glycan, a

disaccharide of hexose linked to 2,4-diacetamido-2,4,6-trideoxyhexose (HexDATDH)

(Figure 1-1) (Parge et al., 1995). Recently, intact pili from N400 were analysed by

mass spectrometry, showing that this pilin glycan is also O-acetylated, revealing a

33

complex post-translational modification (Aas et al., 2007a). Significantly, based on

crystallographic models, Ser63 is located on the αβ-loop region of the mature pilin

subunit, which is thought to be solvent exposed on the assembled fibre (Craig et al.,

2006; Parge et al., 1995).

2. Mechanism. The pilin glycosylation systems of N. meningitidis and N.

gonorrhoeae are representative of those dedicated to this process. Many of the

genes that encode the proteins involved in the biosynthesis, assembly and transfer

of the glycans have been identified (Aas et al., 2007a; Power & Jennings, 2003;

Power et al., 2006). The genes identified to date are found in several locations on

the chromosome, some on “pilin glycosylation loci” (pgl); however, none of the

genes found were linked to pilE, which encodes the pilin subunit (Power & Jennings,

2003).

The molecular mechanism for pilin glycosylation has been well established for

N. meningitidis and a model is presented in Figure 1-6 (Power et al., 2006). On the

cytoplasmic face of the inner membrane, PglB is involved in the biosynthesis of

DATDH and is thought to transfer this sugar to an undecaprenol carrier, although

there is currently no evidence that this precursor exists (Aas et al., 2007a; Kahler et

al., 2001; Power et al., 2000). The trisaccharide is then assembled by the addition of

two galactose residues by the galactosyltransferases PglA and PglE (Banerjee et al.,

2002; Jennings et al., 1998; Power & Jennings, 2003). Once fully assembled, the

mature glycan is translocated to the periplasmic face of the membrane by the

flippase PglF (Kahler et al., 2001; Power et al., 2000). PglL in N. meningitidis and

34

PglO in N. gonorrhoeae have been identified as the oligosaccharyltransferases that

transfer the completed glycan to the PilE subunits prior to their incorporation into the

pilus fibre (Aas et al., 2007a; Power et al., 2006).

PglLNM and PlgONG were identified as the pilin oligosaccharyltransferases

based on their similarities to the P. aeruginosa 1244 pilin oligosaccharyltransferase

PilO, and the O-antigen ligase WaaL, which transfers preformed O-antigen polymers

from a lipid-linked precursor to the separately synthesized core-lipid A portion of LPS

(Aas et al., 2007a; Abeyrathne & Lam, 2007; Power et al., 2006). PglLNM is

predicted to have 13 transmembrane spanning domains, with a short cytoplasmic N-

terminal segment and a longer periplasmic C-terminal domain. A Wzy_C (PFAM

PF04932) domain, present in PilO1244 and WaaL, is predicted in the large

periplasmic loop between transmembrane segments 9 and 10 and is thought to be

the site at which ligation of the O-antigen to lipid A-core takes place (Mulford &

Osborn, 1983; Power et al., 2006). Mutants that lacked PglL produced non-

glycosylated T4P, demonstrating this protein has an essential role in pilin

glycosylation (Power et al., 2006).

3. Role of Glycosylation in T4P function. The role of glycosylation of

Neisseria pili is still not well understood. Due to the location of Ser63 on the αβ-loop

ridge of the pilin, the glycans are predicted to be exposed to the environment and

35

Figure. 1-6. Structure and molecular mechanism of pilin glycosylation. A. Structure of the N.

meningitidis pilin glycan and enzymes responsible for its biosynthesis. B. Model of ligase-

dependent pilin glycosylation. Beginning on the left-hand side the initial UDP-linked sugar is

synthesized and transferred to an undecaprenol carrier via PglB. Additional sugars are added via

the glycosyltransferases PglA and PglE. The completed undecaprenol-linked trisaccharide is

translocated across the inner membrane via the ‘flippase’ PglF to the periplasmic face where the

oligosaccaryltransferase PglL transfers it to the pilin subunit. Reproduced from Power et al. (2006),

with the permission of Biochemical and Biophysical Research Communications, Elsevier Limited.

A

BGal(β1-4)Gal(α1-3) 2,4-diacetimido-2,4,6-trideoxyhexose-Ser

Periplasm

Cytoplasm

Membrane

















A

BGal(β1-4)Gal(α1-3) 2,4-diacetimido-2,4,6-trideoxyhexose-Ser

Periplasm

Cytoplasm

Membrane

therefore able to influence the pili’s surface chemistry and interaction with its

surroundings (Craig et al., 2006; Parge et al., 1995). Experiments in which Ser63 of

PilENG was altered to Ala revealed that non-glycosylated pili are more bundled than

36

the glycosylated pili, suggesting that the surface chemistry of the fibres is altered,

influencing the fibres’ interactions with one another (Craig et al., 2006; Marceau et

al., 1998; Stimson et al., 1995). The modification at Ser63 was found to be essential

for truncated soluble pilin in N. meningitidis, but not N. gonorrhoeae, but did not

appear to influence adhesion as there was no significant difference in binding to

cells between strains expressing glycosylated or non-glycosylated T4P (Marceau &

Nassif, 1999).

Many of the pgl genes have been found to contain sequence repeats that

mediate phase variation, resulting in production of different versions of the glycans,

and leading to further diversity amongst these bacteria (Kahler et al., 2001; Power et

al., 2003). Neisseria are capable of further modifying the pilins with

phosphoethanolamine and/or phosphocholine at Ser68, and these modifications can

also undergo phase variation (Craig et al., 2006; Hegge et al., 2004; Weiser et al.,

1998). It is possible that the main function of T4P glycosylation in Neisseria is host

immune evasion. The diversity of post-translational modifications observed in these

systems is likely influenced by the fact that N. meningitidis and N. gonorrhoeae are

obligate human pathogens which are subject to strong pressures from the host

immune system, and must escape detection in order to survive.

C. P. aeruginosa pilin glycosylation: strain 1244

type iv pilins and identification of a novel d ......diversity of pseudomonas aeruginosa type iv...

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