antiphagocytosis by yersinia pseudotuberculosis
TRANSCRIPT
UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New Series No. 1072 ISSN: 0346-6612 ISBN: 91-7264-219-X
Antiphagocytosis by Yersinia pseudotuberculosis −Role of the YopH Target Proteins
Ming Yuan
Department of Molecular Biology
Umeå University Umeå, Sweden
2006
Copyright © 2006 by Ming Yuan
ISBN 91-7264-219-X
Printed by Hemströms Offset-Boktryck AB
Härnösand, Sweden
2
TABLE OF CONTENTS
ABSTRACT............................................................................................................ 5
PAPERS INCLUDED IN THIS THESIS.......................................................... 6
ABBREVIATIONS.................................................................................................. 7
INTRODUCTION……………………………………………............................ 8
1. Pathogenic Yersinia species........................................................................................ 8
1.1 Overview............................................................................................................. 8
1.2 Virulence mechanisms.......................................................................................... 8
1.2.1 The virulence plasmid.................................................................................. 8
1.2.2 Bacterial adhesion and invasion.................................................................... 9
Invasin....................................................................................................... 9
YadA.......................................................................................................... 10
1.2.3 Yop effectors............................................................................................... 11
1.2.3.1 Antiphagocytic Yop effectors............................................................... 11
YopH............................................................................................... 11
YopE............................................................................................... 13
YopT…………………………………………………………………... 13
YpkA…………………………………………………………… 13
1.2.3.2 Other intracellular Yop effectors………………………………….. 15
YopJ…………………………………………………………………… 15
YopM…………………………………………………………………. 15
2. Targets of YopH in host cells……………………………………………………………. 16
2.1 Epithelial cells………………………………………………………………………. 16
Cas………………………………………………………………………………….. 16
FAK…………………………………………………………………………………. 17
2.2 Professional phagocytes…………………………………………………………….. 18
Fyb………………………………………………………………………………….. 19
SKAP-HOM………………………………………………………………………… 21
3. Phagocytosis…………………………………………………………………………….. 22
3.1 Background………………………………………………………………………….. 22
3.1.1 FcγR-mediated phagocytosis………………………………………………….. 22
3
3.1.2 CR3-mediated phagocytosis…………………………………………………... 23
3.1.3 Bacterial uptake mediated by integrin-invasin interaction..……………............. 23
3.2 Actin dynamics during phagocytosis………………………………………………... 24
3.2.1 Actin-based complex………………………………………………………….. 25
3.2.2 Regulators of the actin cytoskeleton…………………………………………... 25
3.3 Endocytic fusion and recycling during phagocytosis……………………………….. 27
AIM………………………………………………………………………….. 30
RESULTS and DISCUSSION………………………………………………... 31
Interaction between YopH and Cas is required for YopH-mediated disruption of focal
adhesion ……………………………………………………………………………………
31
YopH N terminus binds Fyb and it is important for YopH-mediated effects in
macrophages………………………………………………………………………………..
34
mAbp1 is a novel interactor of Fyb………………………………………………………... 35
mAbp1 influences spreading of macrophages and antiphagocytosis mediated by
pathogenic Yersinia ………………………………………………………………………..
38
Role of the FYB/SKAP-HOM complex in macrophages………………………………….. 40
CONCLUSIONS……………………………………………………………. 46
ACKNOWLEDGEMENTS………………………………………………... 47
REFERENCES……………………………………………………………... 49
PAPERS I–IV
4
ABSTRACT The enteropathogenic bacterium Yersinia pseudotuberculosis binds to β1 integrins
on a host cell via its surface protein invasin. This event stimulates signal transduction to the actin cytoskeleton of the eukaryotic cell, which allows the cell to engulf the bacterium that is attached to its surface. However, the pathogen Y. pseudotuberculosis can evade such phagocytosis by injecting virulence effectors that interfere with the antipathogenic machinery of the host cells. One of these virulence effectors is the tyrosine phosphatase YopH. Through its enzymatic activity, YopH blocks phagocytosis by affecting the signalling that is associated with cytoskeletal rearrangements.
Cas is a substrate of YopH in both professional and non-professional phagocytes. We showed that YopH binds to the central substrate domain of Cas and that this interaction is required for YopH to target focal adhesion structures in host cells. We also demonstrated that YopH binds another substrate, FAK, through Cas. Moreover, we suggested that targeting of Cas is necessary for the cytotoxic effects mediated by YopH.
The protein Fyb is specific to immune cells, and it has been identified as a substrate of YopH in macrophages. We discovered that both the N-terminal substrate-binding domain and the C-terminal catalytic region of YopH bind Fyb in a phosphotyrosine-dependent manner. Moreover, we observed that both the substrate-binding domain and the phosphatase activity of YopH are essential for the effects of this protein on macrophages, which include dephosphorylation of Fyb, blocking of phagocytosis, and cytotoxicity.
The role of Fyb in macrophages is largely unknown, although there is evidence that this protein is involved in integrin-linked actin organization. We identified a novel interaction partner of Fyb, mAbp1, which is a protein that binds to F-actin. Studies in vitro indicated that mAbp1 binds to the N terminus of Fyb via a C-terminal SH3 domain. We also found that both Fyb and mAbp1 co-localize with F-actin at the leading edges of macrophages. Further studies suggested that mAbp1 influences the spreading of macrophages and the antiphagocytosis mediated by pathogenic Yersinia. These results support a role for Fyb in signalling that affects F-actin dynamics, and they also provide additional insight into the mechanisms involved. Fyb has been shown to form a complex with SKAP-HOM, another substrate of YopH in macrophages. Our data implied that the level of SKAP-HOM protein depends on the presence of Fyb, but the function of the Fyb/SKAP-HOM complex in macrophages has not been determined. However, since Fyb is the only known haematopoietic-specific substrate of YopH, it is possible that Fyb is involved in other antimicrobial functions.
Key words: Cas, Fyb, mAbp1, SKAP-HOM, Yersinia pseudotuberculosis, YopH.
5
PAPERS INCLUDED IN THIS THESIS This thesis is based on the following publications, which are referred to in the text by
their roman numerals (I–IV).
I. Mogemark L, McGee K, Yuan M, Deleuil F and Fällman M (2005). Disruption of
target cell adhesion structures by the Yersinia effector YopH requires interaction
with the substrate domain of p130Cas. Eur J Cell Biol. 84:477-89. Reprinted with
permission from Elsevier.
II. Yuan M, Deleuil F and Fällman M (2005). Interaction between the Yersinia
tyrosine phosphatase YopH and its macrophage substrate, Fyn-binding protein,
Fyb. J Mol Microbiol Biotechnol. 9:214–23. Reprinted with permission from Karger.
III. Yuan M, Mogemark L and Fällman M (2005). Fyn binding protein, Fyb,
interacts with mammalian actin binding protein, mAbp1. FEBS Lett. 25:2339–47.
Reprinted with permission from Elsevier.
IV. Yuan M, Carlsson SE, Fahlgren A and Fällman M (2006). Mammalian
actin-binding protein 1 influences spreading of macrophages. Manuscript.
The following paper also concerns part of the present research, but it is not discussed
extensively in this thesis.
Gustavsson A, Yuan M and Fällman M (2004). Temporal dissection of β1-integrin
signaling indicates a role of p130Cas in filopodia formation. J Bio. Chem. 279:
22893–22901.
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ABBREVIATIONS
ADAP adhesion- and degranulation-promoting adapter protein
ADF actin-depolymerizing factor
Arp2/3 actin-related protein 2/3
Cas Crk-associated substrate
CR3 complement receptor 3
Ena/VASP enabled/vasodilator stimulated phosphoprotein
FAK Focal adhesion kinase
F-actin filamentous actin
FcγR Fcγ receptor
Fyb Fyn-T-binding protein
G-actin globular actin
GST glutathione S-transferase
HIP-55 haematopoietic progenitor kinase 1 interacting protein of 55 kDa
HPK1 haematopoietic progenitor kinase 1
mAbp1 mammalian actin-binding protein 1
MEF mouse embryonic fibroblast
MYM multiple Yop mutant
PRAM-1 promyelocytic leukaemia RARα target gene encoding an adapter
molecule-1
PTPase protein tyrosine phosphatase
SH2 Src homology 2
SH3 Src homology 3
SKAP55 Src kinase-associated protein of 55kDa
SKAP-HOM Src kinase-associated protein of 55kDa homologue
SHP-1 SH2 domain-containing protein-tyrosine phosphatase1
SHPS-1 SH2 domain-containing protein tyrosine phosphatase substrate 1
SLP-76 SH2 domain-containing leukocyte phosphoprotein of 76 kDa
SLAP-130 SLP-76-associated protein of 130 kDa
TCR T cell receptor
TTSS type III secretion system
WASP Wiskott-Aldrich syndrome protein
Yop Yersinia outer protein
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INTRODUCTION
1. Pathogenic Yersinia species
1.1. Overview
The Yersinia species belong to the family Enterobacteriaceae, and they are
gram-negative, catalase-positive, oxidase-negative facultative anaerobic rods that can
grow at temperatures of 4 to 40 ºC (Nilehn, 1969; Smego et al., 1999). Eleven Yersinia
species are known, three of which are human pathogens: Y. pestis, Y. enterocolitica,
and Y. pseudotuberculosis (Sulakvelidze, 2000). Y. pestis is non-motile, whereas Y.
enterocolitica and Y. pseudotuberculosis are flagellated and motile at 25 ºC but no
longer motile at 37 ºC in a mammalian host (Boyce, 1985; Hanna et al., 1979).
Y. pestis is the causative agent of bubonic and pneumonic plague and is thereby
one of the most pathogenic bacterial species in human history. Y. pestis is normally
maintained in infected rodents, and it can be transmitted by infected fleas to humans,
where it invades lymphatic tissue and proliferates in the lymph nodes. It can also be
transmitted by air and blood (Perry and Fetherston, 1997). Y. enterocolitica and Y.
pseudotuberculosis cause a self-limited enteric disease called yersiniosis. These
pathogens are orally transmitted to humans in contaminated food or water (Bottone,
1999; Smego et al., 1999), and, similar to Y. pestis, they proliferate in lymphatic tissue,
which they enter though the M cells overlying the surface of Peyer’s patches in the
small intestine (Autenrieth and Firsching, 1996; Clark et al., 1998; Fallman et al.,
1997).
1.2. Virulence mechanisms
1.2.1 The virulence plasmid
8
Figure 1. The 70-kb virulence plasmid of Y.
pseudotuberculosis (adapted from Cornelis et al.,
1998).
Common to all three human
pathogenic Yersinia species is that they
can invade and persist in lymphatic
tissue and thereby evade the primary
immune defence, and this feature is due
to a 70-kb plasmid that is essential for
virulence (Gemski et al., 1980a;
Gemski et al., 1980b; Portnoy and
Falkow, 1981; Portnoy et al., 1981;
Zink et al., 1980). This plasmid
encodes the following: (i) a type
three secretion system (TTSS); (ii) the virulence effectors called Yersinia outer
proteins (Yops); (iii) a translocation system for delivery of Yops; (iv) regulators of
secretion and translocation (Cornelis, 1998; Cornelis et al., 1998). Y. pestis has two
additional plasmids of 100- and 9.5-kb, respectively, which contribute to the virulence
effect of this pathogen (Ferber and Brubaker, 1981).
1.2.2 Bacterial adhesion and invasion
Enteropathogenic Yersinia species must adhere to host cells in order to cause
infection, and the two main adhesins expressed by these bacteria are invasin and
Yersinia adhesin A (YadA) (Isberg and Barnes, 2001; Isberg et al., 2000; Isberg et al.,
1987).
Invasin
Invasin is a 103-kDa outer membrane protein that is chromosomally encoded by
the gene designated inv (Isberg et al., 1987). Maximal expression of invasin in Y.
enterocolitica and Y. pseudotuberculosis occurs at the low temperature of 26 °C, but
not at 37 °C (Isberg et al., 1987; Pepe and Miller, 1993), whereas invasin is not
expressed in Y. pestis (Rosqvist et al., 1988b; Simonet et al., 1996). Recently, two
regulators of invasin expression were described: RovA, which is required for
expression of invasin (Nagel et al., 2001; Revell and Miller, 2000); and YmoA, which
9
is involved in the negative regulation of inv (Ellison et al., 2003).
Invasin binds to β1 integrins (α3β1, α5β1, and α6β1) in host cells through the 192
amino acids of its C terminus and thereby triggers bacterial uptake (Isberg and Leong,
1990; Leong et al., 1990). A central region in the invasin of Y. pseudotuberculosis
causes self-association and enhances uptake of the bacteria (Dersch and Isberg, 1999).
However, invasin is not required for the virulence of Yersinia (Mecsas et al., 2001;
Rosqvist et al., 1988b). The vital effects of invasin in Yersinia infection are exerted at
an early stage of the process, and they include mediating both the attachment and entry
of the bacteria into host cells, such as the M cells in mouse Peyer’s patches (Clark et
al., 1998; Isberg et al., 2000; Isberg and Tran Van Nhieu, 1994; Rankin et al., 1992;
Wiedemann et al., 2001; Young et al., 1990). In agreement with this, a Y.
pseudotuberculosis strain that expresses a mutated variant of invasin has been reported
to have a slower rate of oral infection in mice (Rosqvist et al., 1988b). The
invasion-integrin interaction has also been shown to stimulate T cell activity (Brett et
al., 1993), activate human peripheral B cells (Lundgren et al., 1996), trigger platelet
aggregation (Simonet et al., 1992), and mediate production of the proinflammatory
chemokines interleukin (IL)-8 in intestinal epithelial cells (Grassl et al., 2003).
YadA
YadA is a 44–47-kDa protein, which, along with Yops, is encoded by the
virulence plasmid (El Tahir and Skurnik, 2001). Unlike invasin, YadA expression is
induced at 37 °C (Kapperud et al., 1987). YadA is essential for the virulence of Y.
enterocolitica, but not Y. pseudotuberculosis, and it is not present in Y. pestis (El Tahir
and Skurnik, 2001).
YadA binds indirectly to β1 integrin in host cells through extracellular matrix
(ECM) components such as collagen, fibronectin, and laminin (Bliska et al., 1993; El
Tahir and Skurnik, 2001). YadA has been implicated in the formation of fibrillae
(Kapperud and Namork, 1987) and in bacterial adhesion and invasion during Yersinia
infection (Eitel et al., 2005; Heesemann et al., 1987; Kapperud et al., 1987). The YadA
of Y. pseudotuberculosis has a unique N-terminal amino acid sequence that mediates
tight anchoring of fibronectin to α5β1 integrins (Heise and Dersch, 2006), and, in the
absence of invasin, it can promote adherence of the bacteria to, and to some degree
10
uptake of the bacteria by, cultured cells (Eitel and Dersch, 2002; Grosdent et al., 2002;
Roggenkamp et al., 1995; Roggenkamp et al., 1996).
Two other adhesins are pH6 antigen and attachment invasion locus (Ail), both of
which are expressed at 37 °C (Bliska and Falkow, 1992; Iriarte et al., 1993; Lindler
and Tall, 1993). Studies of murine models have indicated that Ail is not required for
establishing systemic infection (Miller et al., 1989; Wachtel and Miller, 1995). It is
also known that pH6 antigen contributes to the virulence of Y. pestis (Lindler et al.,
1990), although the roles of this protein in Y. enterocolitica and Y. pseudotuberculosis
are still unclear.
1.2.3 Yop effectors
When Yersinia comes in contact with a target cell, the Yop effectors are delivered
from the extracellularly located bacterium into the eukaryotic cell via the type III
secretion/translocation mechanism (Cornelis, 1997; Cornelis et al., 1998; Francis et al.,
2002). The six Yop effectors that have been identified are YopH, YopE, YopJ/P,
YpkA/YopO, YopT, and YopM, and four of those (YopH, YopE, YopJ/P, and
YpkA/YopO) are responsible for the disruption of actin cytoskeleton rearrangement
(Barbieri et al., 2002; Viboud and Bliska, 2005).
1.2.3.1 Antiphagocytic Yop effectors
YopH: a protein tyrosine phosphatase
YopH is 51-kDa protein tyrosine phosphatase (Guan and Dixon, 1990) that is
essential for Yersinia virulence (Andersson et al., 1996; Bliska et al., 1991; Rosqvist et
al., 1988a). This protein contains 468 amino acids arranged in an N-terminal
non-catalytic domain (residues 1–130), followed by a proline-rich region and a
C-terminal phosphatase domain (residues 201–468). Previous studies have shown that
the 130 N-terminal amino acids of YopH form a multifunctional domain that is
essential for secretion (residues 1–17) and translocation into host cells (residues
18–71), and also binds to the chaperone SycH in the bacteria to deliver the YopH to
11
S PTPaseT
FCT P-loop
YopH Pro
tyrosine-phosphorylated target proteins in the eukaryotic cells (Cornelis, 2002;
Montagna et al., 2001; Wattiau et al., 1994; Woestyn et al., 1996). A region comprising
residues 223–226 has been shown to be important for immediate targeting of YopH to
host cell peripheral focal adhesion complexes (Persson et al., 1999). The C-terminal
phosphatase domain of YopH contains a phosphate-binding loop (P-loop; residues
403–410), and substitution of the Cys at position 403 with either Ser or Ala inactivates
the phosphatase (Guan and Dixon, 1990). This catalytically inactive variant of YopH
can form stable complexes with its substrates in vivo, and it has been used as a
substrate trap to identify the host cell proteins targeted by this effector. In HeLa cells,
YopH dephosphorylates focal adhesion kinase (FAK) and Crk-associated substrate
(Cas) (Black and Bliska, 1997; Bliska et al., 1992; Persson et al., 1997), which disrupts
the peripheral focal adhesion complexes. In macrophages, besides Cas, YopH targets
Fyn-T-binding protein (Fyb) and Src-kinase-associated protein of 55kDa homology
(SKAP-HOM) (Black et al., 2000; Hamid et al., 1999).
Figure 2. Schematic representation of YopH. S, secretion; T, translocation; Pro, proline-rich region;
PTPase, protein tyrosine phosphatase domain; FCT, focal complex targeting; P-loop, phosphatase-binding
loop.
It is assumed that the primary function of YopH is to block phagocytosis in
macrophages and neutrophils, and, through its PTPase activity, also to inhibit the
oxidative burst in these cells (Bliska and Black, 1995; Ruckdeschel et al., 1996). The
results of infection studies in a murine model have suggested that YopH counteracts
innate immunity, as indicated by the observation that a yopH mutant failed to pass the
stage of Peyer’s patches (Logsdon and Mecsas, 2003; Trulzsch et al., 2004; Viboud and
Bliska, 2005). YopH has also been implicated in the signalling cascades associated
with antigen-induced activation of T or B cells (Yao et al., 1999).
12
YopE: a GTPase-activating protein
YopE is a 23-kDa protein that is essential for Yersinia virulence (Forsberg and
Wolf-Watz, 1988; Viboud et al., 2006). It contains N-terminal secretion and
translocation sequences, and a C-terminal Rho GTPase-activating protein (GAP)
domain (Schesser et al., 1996; Sory et al., 1995) that is necessary for the function of
the protein (Aili et al., 2002; Black and Bliska, 2000). YopE can deactivate the Rho
family GTPases Rac1 and RhoA, but not Cdc42 (Aepfelbacher, 2004; Aili et al., 2006;
Black and Bliska, 2000), and it thereby disrupts the actin cytoskeleton, which causes
rounding and detachment of infected cells, a phenomenon known as cytotoxicity.
Besides blocking phagocytosis, YopE inhibits activation of caspase-1 by deactivating
Rac1, and it thereby prevents the maturation and secretion of IL-1β and IL-18 as a
means of regulating the inflammatory response to the Yersinia infection (Schotte et al.,
2004).
YopT: a cysteine protease
YopT is a 35.5-kDa protein, which, similar to YopE, affects the cytoskeleton of
eukaryotic cells (Iriarte and Cornelis, 1998). YopT acts as a cysteine protease to cleave
the prenyl groups of Rho GTPases and in that way inactivate those proteins, and
subsequently remove them from membranes (Aepfelbacher et al., 2005; Shao et al.,
2002; Shao et al., 2003; Sorg et al., 2001; Zumbihl et al., 1999). In vivo, YopT targets
RhoA but not Rac or Cdc 42 (Aepfelbacher et al., 2003; Aepfelbacher et al., 2005;
Zumbihl et al., 1999). The polybasic sequence in the C terminus of RhoA is essential
for substrate recognition and cleavage by YopT (Shao et al., 2003). The role of YopT in
Yersinia pathogenesis is still unclear, since this protein is not essential for the virulence
of Y. enterocolitica, and it is not expressed in Y. pestis (Iriarte and Cornelis, 1998).
Recently, in a study of mouse infection and cell culture models (Viboud et al., 2006), it
was found that YopE has stronger antiphagocytic activity than YopT, and the latter
protein can only partially compensate for loss of the former.
YpkA: a serine/threonine kinase
YpkA (YopO in Y. enterocolitica) is an 81.7-kDa autophosphorylating protein
with a serine/threonine kinase catalytic domain in the N terminus (Galyov et al., 1993;
13
CasFAK
PP
βα βα
InvasinIntegrinvir
Galyov et al., 1994; Hakansson et al., 1996). The C-terminal region of YpkA has been
shown to bind to RhoA and Rac1 (Barz et al., 2000), however, the function of such
attachment is still unknown. YpkA can disrupt the actin cytoskeleton of cultured cells
through both kinase-dependent and kinase-independent mechanisms when it is
overexpressed and in the absence of other Yop effectors (Hakansson et al., 1996; Juris
et al., 2002; Wiley et al., 2006). YpkA-mediated morphological phenotypes differ from
the YopE-mediated counterparts, as demonstrated by an investigation showing that
infected HeLa cells do not detach from the substrate (Hakansson et al., 1996). YpkA is
also essential for Yersinia virulence (Galyov et al., 1993; Wiley et al., 2006), as
suggested by a previous study indicating that this protein is involved in the
antiphagocytic effect of Y. enterocolitica on macrophages and polymorphonuclear
neutrophils (Grosdent et al., 2002).
Yersinia
Yops
14
Rho
Rac
YpkA
?
RhoGDP
Rho
GTP
YopT
YopE
SKAP-HOMFyb P
P
YopH
YopM
?nucleus
YopJ
P P
PP
MKK
Survival
IKK
Iκ BNFκ B
Cytokine productionPhagocytosis
Yops
Cas P
P
P
Figure 3. The Yersinia effectors target multiple signalling pathways. The Yops are delivered into the host
cells via a type III secretion system. YopT modifies the Rho family GTPases; YopE inactivates the Rho
family of GTPases; YpkA binds to Rac and Rho (function unknown); YopH dephosphorylates Cas and FAK
in epithelial cells, and Cas, Fyb and SKAP-HOM in macrophages. These four Yops alter or disrupt the actin
cytoskeleton and thereby block phagocytosis. YopJ impairs activation of MAPKKs and NF-κB, which
induces apoptosis and inhibits cytokine production. YopM is translocated into the nucleus (function
unknown).
1.2.3.2. Other intracellular Yop effectors
YopJ: a cysteine protease
YopJ (YopP in Y. enterocolitica) is a 33-kDa ubiquitin-like cysteine protease. It
has been reported to act downstream of the small GTPase Rac1 to inhibit the signalling
of mitogen-activated protein kinases (MAPKs), such as c-Jun N-terminal kinase (JNK),
p38, and extracellular signal-regulated kinases (ERK) 1 and 2, and that effect is
achieved by preventing activation of the family of MAPK kinases (MAPKKs)
(Mukherjee et al., 2006; Orth, 2002; Palmer et al., 1999). YopJ also inhibits the nuclear
factor κB (NFκB) signalling pathway by targeting I κB kinase β (IKKβ) in the host
cells (Aepfelbacher et al., 1999; Orth, 2002; Ruckdeschel, 2002). YopJ is the only Yop
effector that is required to induce Yersinia-mediated apoptosis in macrophages (Mills
et al., 1997; Monack et al., 1997). An early study indicated that YopJ is dispensable for
the virulence of Y. pseudotuberculosis in a murine model (Galyov et al., 1994),
whereas later experiments demonstrated that this protein participates in the
establishment of systemic infection (Monack et al., 1998). A recent study suggested
that YopJ inhibits MAPK-mediated uptake of Y. enterocolitica in dendritic cells
(Autenrieth et al., 2006).
YopM: a leucine-rich protein
YopM is a 41-kDa acidic protein that contains leucine-rich repeats (Leung and
Straley, 1989) and C-terminal nuclear localization sequences (NLSs) (Benabdillah et
al., 2004). During Yersinia infection of HeLa cells, YopM can translocate to the
nucleus via a vesicle-associated pathway (Skrzypek et al., 1998). This Yop forms a
complex with two cytoplasmic kinases called ribosomal protein S6 kinase 1 (RSK1)
and protein kinase C-like2 (PRK2) (McDonald et al., 2003). The function of YopM has
not yet been fully elucidated, although it is known that this molecule is an important
virulence factor in Yersinia. YopM mutants of both Y. pestis and Y. enterocolitica have
been found to exhibit altered virulence in a murine model (Kerschen et al., 2004;
Trulzsch et al., 2004), and it has also been demonstrated that YopM can interfere with
innate immunity by causing depletion of NK cells (Kerschen et al., 2004).
15
2. Targets of YopH in host cells
2.1. Epithelial cells
YopH is an important virulence effector of Yersinia that can block host cell
signalling through its PTPase activity (Andersson et al., 1996; Bliska et al., 1991;
Guan and Dixon, 1990; Rosqvist et al., 1988a). In many types of cultured cells, such as
HeLa cells, the targets of YopH are Cas and FAK (Black and Bliska, 1997; Bliska et al.,
1992; Persson et al., 1997). YopH has also been found to bind and dephosphorylate
paxillin, but that effect has only been seen in vitro (Black and Bliska, 1997).
Cas
Cas was originally identified as a 130-kDa highly tyrosine-phosphorylated protein
in v-Src- and v-Crk-transformed cells, and it forms a complex with the mentioned
molecules (Sakai et al., 1994). As an adaptor, Cas promotes protein-protein
interactions. An N-terminal SH3 domain in Cas interacts with FAK and protein
tyrosine kinase 2 (Pyk2), and the C terminus of Cas contains Src-binding sequences.
Moreover, a central substrate-binding domain containing 15 potential tyrosine
phosphorylation sites mediates association of Cas with the SH2 domain of Crk
(Kanner et al., 1991; Nakamoto et al., 1996; Sakai et al., 1994). The phosphorylation
of Cas can be regulated via interaction with tyrosine-phosphatases such as PTP-1B and
PTP-PEST, and serine-phosphorylated Cas has been found to bind 14-3-3 proteins
(O'Neill et al., 2000).
Cas is ubiquitously expressed, and deletion of this protein is lethal in mouse
embryos (Honda et al., 1998). Fibroblasts from Cas -/- mice exhibit short actin
filaments and display delayed cell spreading as well as reduced migration phenotype
(Honda et al., 1998). Studies have indicated that Cas is involved in cell migration
(Panetti, 2002), cell transformation and the progression of cancer (Behrens et al., 2003;
Burnham et al., 1999; Chodniewicz and Klemke, 2004), and cell survival and
apoptosis (Defilippi et al., 2006; Dolfi et al., 1998; Oktay et al., 1999).
16
FAK
FAK is a 125-kDa protein tyrosine kinase (Hildebrand et al., 1993). It contains an
N terminus with an autophosphorylation site that may interact with the cytoplasmic
domain of β1 integrin (Schaller et al., 1995), a central kinase domain, and also a C
terminus with a focal adhesion targeting sequence (FAT) that interacts with several
proteins, including paxillin (Hildebrand et al., 1995), Cas (Harte et al., 1996; Polte and
Hanks, 1995), and some other proteins that comprise an SH2 domain. The cell-surface
integrin receptor binds ECM, which induces integrin clustering and recruitment of
FAK. FAK is subsequently phosphorylated and becomes associated with Src (Schaller,
1996; Schaller et al., 1994), and this activation of FAK has been implicated in cell
cycle progression, adhesion, and migration (Brown et al., 2005; Cary and Guan, 1999;
Choma et al., 2006; Ilic et al., 1997; Klemke et al., 1998). Like Cas, FAK deficiency is
also lethal in mouse embryos (Ilic et al., 1995).
The Cas-FAK interaction has been shown to increase the tyrosine phosphorylation
of Cas, which in turn causes recruitment of Crk to stimulate cell migration (Cary et al.,
1998; Klemke et al., 1998). The possible downstream effector of Crk is DOCK180 (Gu
et al., 2001), which may act as an activator of Rho GTPase Rac1 (Brugnera et al.,
2002), and the Caenorhabditis elegans counterpart of DOCK180 has been implicated
in the regulation of cell migration and phagocytosis (Wu and Horvitz, 1998). In
addition to cell migration, Cas and FAK have also been found to participate in
host-pathogen interactions, including those involving the bacteria species Yersinia
pseudotuberculosis (Bruce-Staskal et al., 2002; Weidow et al., 2000), Pseudomonas
aeruginosa (Deng et al., 2005), Salmonella typhimurium (Shi and Casanova, 2006),
and Streptococcus pyogene (Humtsoe et al., 2005).
17
Cas SH3 Substrate domain Serine-rich
FAKPTP-1BPTP-PESTPyk2
Pro Pro
YDYVHL
Crk 14-3-3 Src
FAK FATIntergrins
Pro Pro
Cas Paxillin
Y397
SrcKinase
Pro Y925
Fyb
SKAP-HOM Coiled-coil SH3Pro
PHFyb
YEELYAN
Pro YGYI
SKAP-HOMSKAP55
YEDI NLS YDDVFPPPP
YDGIYDDV
SH3-like
NLS
SKAP55Fyn
SLP-76
VASP
SLP-76
Figure 4. Schematic representation showing the YopH targets and the proteins that interact with those
molecules. Pro, proline-rich region; SH3, Src homology 3; FAT, focal adhesion targeting domain; NLS,
nuclear localization sequence; PH, pleckstrin homology domain.
2.2. Professional phagocytes
It is assumed that the first immune cells to encounter infecting Yersinia are the
professional phagocytes and antigen-presenting cells (APCs), which include the
macrophages, neutrophils, and dendritic cells. YopH is essential for the pathogenic
Yersinia, because it impairs the functions of the mentioned immune cells, which is
demonstrated by the finding that yopH mutants are rapidly eliminated upon infection
(Viboud and Bliska, 2005). In mouse macrophage-like J774 cells, besides Cas, Fyb and
SKAP-HOM also serve as substrates for YopH (Black et al., 2000; Hamid et al., 1999).
18
Fyb
Fyb is a haematopoietic cell-specific protein that is also called SLAP-130
(SLP-76-associated protein of 130 kDa) (Musci et al., 1997) and ADAP (adhesion- and
degranulation-promoting adaptor protein) (Geng and Rudd, 2001). Expression of Fyb
occurs in T cells, thymocytes, and myeloid cells, but not in B cells (da Silva et al.,
1997; Geng and Rudd, 2001; Musci et al., 1997). Fyb contains a number of regions
that have the potential to mediate protein-protein interactions; these include several
proline-rich motifs in the N-terminal half of the protein and a C-terminal SH3-like
domain. In addition, Fyb has two putative NLSs and multiple tyrosine-containing
motifs (da Silva et al., 1997). Two YDDV motifs have been shown to be
tyrosine-phosphorylated by Fyn kinase, which is required for binding to the 76-kDa
SH2-domain-containing leukocyte phosphoprotein (SLP-76) (Geng et al., 1999; Raab
et al., 1999). Fyb also harbours a FPPPP motif that has been reported to bind the
Ena/VASP homology 1 (EVH1) domain of enabled/vasodilator-stimulated
phosphoproteins (Ena/VASP), which constitute a family of proteins that bind and
regulate actin (Krause et al., 2000; Renfranz and Beckerle, 2002; Scott et al., 2006).
Two isoforms of Fyb have been identified, Fyb-120 and Fyb-130, which differ with
regard to a 46-amino-acid insert that is found in the C terminus between the two
tyrosine motifs YDGI and YDDV in the 130-kDa form (Veale et al., 1999). Both
Fyb-120 and Fyb-130 bind to the SH2 domains of Fyn and SLP-76 (da Silva et al.,
1997; Musci et al., 1997; Veale et al., 1999). Other dissimilarities between these two
isoforms include more efficient binding of Fyb-120 to SLP-76 and preferential
expression of Fyb-130 in mature T cells. It has been demonstrated that Fyb-130 plays a
role in up-regulation of TCR-induced IL-2 production in mature T cells, and Fyb-120
has an important function in thymic differentiation (Veale et al., 1999).
Fyb-120 is the most well characterized isoform. Studies of Fyb have been focused
on T cells in which Fyb was initially found, and several reports have suggested that
Fyb is included in the TCR signalling pathway (Boerth et al., 2000; da Silva et al.,
1997; Fang et al., 1996; Musci et al., 1997; Raab et al., 1999; Wardenburg et al., 1996;
Wu et al., 1996). Fyb undergoes tyrosine phosphorylation upon TCR activation, and it
forms a complex with Wiskott-Aldrich syndrome protein (WASP), Ena/VASP, Nck,
19
and SLP-76 (Krause et al., 2000). In T cells, Fyb binds SKAP55 (Schraven et al.,
1997), and that association is mediated by the SH3 domain of SKAP55 and the
proline-rich sequence of Fyb (Marie-Cardine et al., 1998a). In macrophages, Fyb forms
a similar complex with SKAP55 homology protein SKAP-HOM (Black et al., 2000;
Bliska et al., 1992; Timms et al., 1999).
Previous studies have provided evidence that Fyb is involved in integrin-regulated
cellular responses. Fyb enhances integrin-mediated adhesion and migration in T cells
and mast cells (Geng et al., 2001; Hunter et al., 2000; Wang et al., 2003), which agrees
with the finding that Fyb -/- T cells failed to enhance integrin-dependent adhesion
(Peterson et al., 2001). This TCR-induced adhesion may be regulated by the
interaction between Fyb and SLP-76 that is mediated by receptor-stimulated
generation of intracellular reactive oxygen species (ROS) (Kwon et al., 2005).
Recently, Kliche et al. (2006) reported that Fyb and SKAP55 participate in
TCR-promoted inside-out signalling through recruitment of the activated small
GTPase Rap1 to the plasma membrane. Fyb has been found to co-localize with F-actin
in membrane ruffles, adhesion plaques/podosomes, and phagocytic cups (Coppolino et
al., 2001; Geng et al., 2001; Koga et al., 2005). The observation that Fyb is a ligand for
the EVH1 domain of the actin-binding Ena/VASP proteins (Krause et al., 2000) might
explain the observation that Fyb co-localizes with cellular structures that exhibit
extensive actin dynamics (Coppolino et al., 2001; Krause et al., 2000). The cited
results indicate that Fyb is involved in actin organization, although it is not necessary
for TCR-induced actin polymerization (Peterson et al., 2001). It is also plausible that
Fyb is required for T cell proliferation (Griffiths et al., 2001; Peterson et al., 2001; Wu
et al., 2006), and it may take part in cell migration and progression to the
multinucleated cell stage in osteoclastogenesis (Koga et al., 2005). Notwithstanding, it
is known that Fyb is dispensable for the development and function of NK cells (Fostel
et al., 2006). The role of Fyb in macrophages is largely unknown, but the finding that
this adaptor protein is rapidly dephosphorylated by the Yersinia antiphagocytic effector
YopH suggests that it may participate in macrophage antibacterial function, perhaps
phagocytosis (Black et al., 2000; Hamid et al., 1999).
20
SKAP-HOM
SKAP-HOM is a cytoplasmic adaptor protein with homology to SKAP55 (Curtis
et al., 2000; Liu et al., 1998), and it is also designated SKAP55R (SKAP55-related
protein) (Marie-Cardine et al., 1998b) and RA70 (retinoic acid-inducible protein)
(Kouroku et al., 1998). Both SKAP-HOM and SKAP55 contain a pleckstrin homology
(PH) and a SH3 domain. However, in contrast to SKAP55, SKAP-HOM includes an
additional N-terminal coiled-coil region, which is assumed to mediate non-covalent
protein-protein interactions by homo- or hetero-oligomeric associations effected via
charged residues (Lupas, 1996; Marie-Cardine et al., 1998b). SKAP55 is expressed
exclusively in T lymphocytes (Marie-Cardine et al., 1997), whereas expression of
SKAP-HOM seems to be essentially ubiquitous (Marie-Cardine et al., 1998b). Both
these proteins can be tyrosine phosphorylated by Fyn kinase, and they have been
implicated in TCR signalling (Marie-Cardine et al., 1997; Marie-Cardine et al., 1998b).
Unlike SKAP55, SKAP-HOM is not tyrosine phosphorylated in resting T cells, but it
is phosphorylated after activation of the cells (Marie-Cardine et al., 1998b; Schraven et
al., 1997).
Timms et al. (1999) detected tyrosine phosphorylation of SKAP-HOM and Fyb in
macrophages adhering to fibronectin, and Black et al. (2000) subsequently confirmed
that SKAP-HOM forms a complex with Fyb and is tyrosine-phosphorylated in
response to macrophage adhesion. Considering those results, along with the knowledge
that both Fyb and SKAP-HOM are targets of YopH in macrophages (Black et al.,
2000), it appears that SKAP-HOM affects actin dynamics under the control of
integrin-mediated signals.
In a study of SKAP-HOM -/- mice (Togni et al., 2005), it was noted that
SKAP-HOM was not required for growth of the animals or development of the
haematopoietic system, nor was it needed for T cell and platelet functions, or for the Fc
or complement receptor-mediated phagocytosis in bone marrow derived macrophages
(BMMs). In contrast, the proliferative responses of anti-IgM- and LPS-stimulated B
cells in knockout mice were significantly decreased. Further experiments demonstrated
that SKAP-HOM is involved in the coupling between B cell receptors (BCRs) and
integrin activation, whereas it is dispensable for BCR-mediated reorganization of the
cytoskeleton.
21
3. Phagocytosis
3.1. Background
Phagocytosis (“cell eating”) entails the engulfment of solid particles (0.5–5μM in
diameter), such as microbial pathogens, apoptotic cells, large immune complexes, and
cell debris. It is a major defence mechanism of the innate immune system of the hosts,
and it is accomplished by professional phagocytes, including macrophages (the
“classic” phagocytes) and neutrophils. However, cells referred to as non-professional
phagocytes are also capable of phagocytosis.
The phagocytic process can be divided into several steps: (i) binding of particles
to the cell surface; (ii) formation of a phagocytic vesicle termed a phagosome; (iii)
maturation of the phagosome to a phagolysosome and digestion of engulfed particles
in the phagolysosome. The initial step of phagocytosis involves recognition of the
particles by special receptors on the plasma membrane of the phagocytes. These
receptors can be either opsonic dependent or opsonic independent. Opsonins in serum
consist of the Fc portion of immunoglobulins (Igs) bound to the particles, and the
complement components C3b and iC3b deposited on the particle surface.
Opsonic-dependent receptors, such as Fc and complement receptors, are recognized by
opsonins supplied by the host. Structural determinants present on the surface of target
particles bind directly to opsonic-independent receptors, which include complement
receptor 3 (CR3), endotoxin receptors, β1 and β3 integrins, mannose receptor, the
β-glucan receptor, the galactose receptor, and scavenger receptors (Tjelle et al., 2000).
The best characterized of these receptors are FcγR (Fcγ receptor for IgG) and CR3
(receptor for complement protein iC3b). In response to Yersinia infection, β1 integrin
expressed on the host cells can bind the bacterial surface protein invasin, and that
mediates uptake of the bacteria in the absence of antiphagocytic Yop effectors (Isberg
and Leong, 1990).
3.1.1 FcγR-mediated phagocytosis
Three classes of Fcγ receptors have been identified, which are designated FcγRI,
FcγRII, and FcγRIII. These receptors differ with regard to their expression patterns,
22
but they share a highly homologous extracellular portion that contains the IgG-binding
domain. Upon receptor clustering induced by binding of IgG-coated particles, the
immunoreceptor tyrosine-based activation motifs (ITAMs) on the intracellular part are
phosphorylated by Src family kinases, which leads to recruitment and activation of the
protein tyrosine kinase Syk. This results in formation of signalling complexes close to
the membrane, in which Syk-mediated phosphorylation of several adaptor proteins
causes activation of downstream pathways that give rise to the phagocytic effect
(Berton et al., 2005). The signalling molecules of those pathways include intracellular
calcium, protein kinase C (PKC), phospholipase D (PLD), phosphatidylinositol
3-kinase (PI3-K), ERK, and GTPases of the Rho family (Garcia-Garcia and Rosales,
2002; May and Machesky, 2001; Swanson and Hoppe, 2004).
3.1.2 CR3-mediated phagocytosis
CR3 is a β2 integrin heterodimer that can bind to several ligands. One of those is
iC3b, which is produced as follows: C3b is produced upon complement activation, and
it binds to microbes, and acts as an opsonin; C3b is subsequently cleaved by plasma
factors to yield iC3b. Ligation of CR3 by iC3b leads to opsonic-dependent
phagocytosis. CR3 can also bind molecules on target particles such as β-glucan to
mediate opsonic-independent phagocytosis (May and Machesky, 2001; Tohyama and
Yamamura, 2006). Unlike FcγR-mediated phagocytosis, it seems that C3-opsonized
particles simply sink into the host cells, with little formation of pseudopodia (Allen
and Aderem, 1996). A plausible explanation for this difference is that CR3-mediated
phagocytosis requires activation of GTPase RhoA, but it does not need the Rac and
Cdc42, which are involved in the extension of pseudopodia that occurs to engulf the
bacteria (Caron and Hall, 1998).
3.1.3 Bacterial uptake mediated by integrin-invasin interaction
Integrins are large heterodimeric transmembrane proteins that are involved in the
spreading, migration, and survival of eukaryotic cells (van der Flier and Sonnenberg,
2001). Integrins can transmit signals to the cytoskeleton when cells adhere to
substrates, and they bind the Yersinia surface protein invasin with high affinity (Isberg
23
and Leong, 1990; Tran Van Nhieu and Isberg, 1993). Integrin-invasin binding is
necessary for uptake of non-opsonized Yersinia (Fallman et al., 1995; Grosdent et al.,
2002; Hudson et al., 2005). In addition to professional phagocytes, non-professional
phagocytes such as epithelial cells and intestinal M cells also can internalize Yersinia
through that binding (Clark et al., 1998; Isberg and Leong, 1990).
The integrin-mediated uptake of Yersinia has not yet been thoroughly elucidated,
although a model has been proposed to explain internalization in non-phagocytic cells
(Fallman et al., 1997). According to that model, Yersinia infection causes invasin on
the bacterium to interact with β1 integrins on the host cell, which provokes clustering
of the integrins and subsequent assembly of focal complex structures. These signalling
complexes transduce signals to the cytoskeleton, resulting in actin reorganization that
allows engulfment of the surface-attached bacterium. It has been shown that the β1
chain of the integrin is critical for downstream signalling during the uptake
(Gustavsson et al., 2002; Van Nhieu et al., 1996). The proteins involved in downstream
signalling include FAK, Cas, Paxillin, c-Src, Syk, WASP, and the small GTPase Rac1
(Alrutz and Isberg, 1998; Alrutz et al., 2001; Andersson et al., 1996; Black and Bliska,
1997; Black et al., 2000; Hudson et al., 2005; McGee et al., 2001; Persson et al., 1997;
Wiedemann et al., 2001). FAK, Cas, and Paxillin are involved in formation of the focal
adhesion complexes that connect integrins with the actin cytoskeleton (Brugge, 1998).
Scibelli et al. (2005) have recently observed that flavoridin (a member of the
disintegrin family) binds β1 integrin with high affinity and thereby prevents the
disruption of focal adhesion complexes and inhibits the uptake of Y. enterocolitica by
HeLa cells.
3.2. Actin dynamics during phagocytosis
Phagocytosis is an actin-dependent process that is mediated by several different
receptors. Internalization of the target particle requires actin polymerization,
pseudopod extension or membrane invagination, membrane recruitment, and closure of
the phagocytic cup forming an intracellular phagosome (Aderem and Underhill, 1999).
24
3.2.1 Actin-based complex
Actin is an ATP-binding protein that exists in a monomeric form (G-actin) and as
polymerized filaments (F-actin). Actin monomers have distinct ends and are assembled
into filaments in a “head-to-tail” manner, which gives the filaments two different ends
that are respectively called barbed (plus) and pointed (minus). Free barbed ends can be
created by uncapping of pre-existing filaments or by de novo nucleation of monomeric
actin, and that serves as a template for actin polymerization. Nucleation of free
monomers is catalyzed by cellular actin nucleation factors, such as the actin-related
protein (Arp) 2/3 complex (Welch et al., 1997). Nucleation of the actin can be
controlled by proteins of the WASP family (Blanchoin et al., 2000; Machesky and
Insall, 1998; Mullins et al., 1998; Zigmond, 2000).
The linkage of the actin cytoskeleton to the signalling pathway is generally
mediated by the interaction between an active Rho family GTPase and a “connector”
on the plasma membrane. Activation of cell surface receptors generates signals that
induce the “connector” to recruit the Arp2/3 complex and thereby initiate growth at the
barbed-end of actin filaments (Higgs and Pollard, 1999). Filaments grow when actin
monomers are added at a fixed angle of 70° from the side of pre-existing actin
filaments, and thus push the plasma membrane forward. Actin filament disassembly
can be executed by cofilin, which is a member of the actin-depolymerizing factor
(ADF)/cofilin family (Carlier et al., 1997; Lappalainen et al., 1998). This ADP-bound
G-actin is exchanged for the ATP-bound form under the influence of profilin and
thereby recycled back to achieve rapid elongation of new barbed ends. Elongation can
be blocked upon depletion of ATP-bound G-actin or by binding of capping proteins to
the barbed end (Pollard et al., 2000).
3.2.2 Regulators of the actin cytoskeleton
Regulation of actin rearrangement during phagocytosis involves various proteins,
including GTPases, WASP, Ena/VASP, and some other actin-binding molecules.
Rho GTPases are small GTP-binding proteins, and most studies have focused on
three members of this family, namely, Rac1, Cdc42, and RhoA. These three Rho
25
GTPases cycle between a GDP-bound (inactive) and a GTP-bound (active) state, and
this process is catalyzed by the following molecules: guanine nucleotide-exchange
factors (GEFs) such as Vav (Garcia-Garcia and Rosales, 2002), which enhance the
switch from bound GDP to GTP; the GTPase-activating proteins (GAPs), which
increase the intrinsic GTP hydrolysis; and the guanine nucleotide dissociation
inhibitors (GDIs), which inhibit both the exchange of GDP for GTP and the hydrolysis
of bound GTP (Van Aelst and D'Souza-Schorey, 1997). In the cytosol, a Rho GTPase
forms a complex with a GDI and is in that way maintained in the GDP-bound inactive
state, which can be released from GDI to be able to convert to the GTP-bound active
form under the action of a GEF. The GTP-bound form interacts with a downstream
effector and is transformed into the GDP-bound state by the action of a GAP. The
GDP-bound molecule then forms a complex with the GDI and returns to the cytosol. In
fibroblasts, Cdc42 generates cell polarity and elicits formation of filopodia; RhoA
activity generates contractile actin bundles (stress fibres) and focal adhesion structures;
and Rac1 activity induces actin polymerization to drive lamellipodial protrusion
(Wittmann and Waterman-Storer, 2001). Studies have indicated that Rho GTPases are
also involved in phagocytosis: Cdc42 controls pseudopod extension during
FcR-mediated phagocytosis (Castellano et al., 2001); RhoA is necessary for
CR3-mediated phagocytosis (Caron and Hall, 1998); and Rac1 controls phagosome
closure during FcR-mediated phagocytosis (Castellano et al., 2001), and it is also
required for β1 integrin-mediated uptake of Y. pseudotuberculosis (Alrutz et al., 2001;
McGee et al., 2001).
The dynamin family of proteins comprises three large GTPases called dynamins
1, 2, and 3. Dynamin 1 is expressed solely in neurons, dynamin 2 is produced
ubiquitously, and dynamin 3 is generated in testis (Urrutia et al., 1997). These proteins
contain an N-terminal GTPase domain, a PH domain that binds phosphatidylinositol
lipids to associate with membranes, and a C-terminal proline-rich region that binds to
SH3 domains of actin-linked proteins such as profilin, mAbp1, and cortactin (Orth and
McNiven, 2003). It has been reported that dynamin 2 is essential for the formation of
clathrin-coated endocytic vesicles (Takei et al., 1995), and it is also involved in
membrane trafficking in the trans-Golgi network (TGN) (Cao et al., 2000; Maier et al.,
26
1996). Moreover, this dynamin variant is required for FcγR-mediated phagocytosis in
macrophages (Gold et al., 1999).
Actin-binding proteins constitute a large family that can be divided into several
groups, only a few of which are listed below. Members of this family include the
WASP proteins, which are located downstream of Rac1 and Cdc42 during cytoskeletal
rearrangement (Blanchoin et al., 2000; Mullins, 2000). The WASP C terminus initiates
the growth of actin filaments by recruiting actin monomers and the Arp2/3 complex.
The Arp2/3 complex contains seven subunits, two of which (Arp2 and Arp3) are
actin-related proteins. During phagocytosis the Arp2/3 complex is co-localized with
F-actin in the phagosome (Alrutz et al., 2001; May et al., 2000). Ena/VASP contains a
central proline-rich domain that binds the G-actin-binding protein profilin and a
C-terminal Ena-VASP-homology domain 2 (EVH2) that binds F-actin (Krause et al.,
2002). It has been shown that VASP, WASP, Fyb, Nck, and SLP-76 form a complex
that represents a link between the actin cytoskeleton and FcγR-mediated phagocytosis
(Coppolino et al., 2001).
3.3. Endocytic fusion and recycling during phagocytosis
Internalization of a particle results a nascent phagosome that fuses with endocytic
compartments to reach maturation (Fig. 5). Finally, the matured phagosome merges
with late endocytic organelles to form a phagolysosome, which is responsible for the
killing and degradation of intracellular pathogens. The phagosome-lysosome fusion is
probably a “kiss-and-run” interaction that involves brief exchange of solutes from the
two compartments without complete intermixing of their fluid membranes (Tjelle et al.,
2000). In addition to transient fusion, phagosome maturation can occur through
complete fusion (Tjelle et al., 2000), but the mechanisms that control
phagosome-endosome merging are still largely unknown. Phagosomes can also fuse
with endoplasmic reticulum (ER) (Desjardins, 2003; Gagnon et al., 2002) and
Golgi-derived vesicles (Fratti et al., 2003).
27
Y
Y
Y
Antig
en
pres
enta
tion
Early endosome
Lateendosome
Lysosome
X
X
X
X
X
X X
XX X
X
XX
X
X
Receptor recycling
XNascent phagosome
phagolysosome
EndocytosisPhagocytosis
X XX
XX
XReceptor recycling
Figure 5. The phagocytic pathway. Microorganisms bind to receptors (X) on the surface of a host cell,
which induces actin polymerization to internalize the microorganisms and form a nascent phagosome. The
phagosome undergoes a series of fusions with endocytic compartments (early endosome, late endosome, and
lysosome) to reach maturation, and microorganisms are completely degraded in the phagolysosome.
Peptides from that process can be presented by major histocompatibility complex (MHC) class I molecules
(active CD8 + T lymphocytes (Lehner and Cresswell, 2004)) or MHC class II molecules (active CD4 + T
lymphocytes (Ramachandra et al., 1999)) to adaptive immune system (Niedergang and Chavrier, 2004).
ADP-ribosylation factor (ARF) and the Rab family members are small
GTPases that are associated with distinct organelle membranes and regulate vesicular
transport (Chavrier and Goud, 1999; Kawasaki et al., 2005). Arf6 has been found to be
activated during FcγR-mediated phagocytosis, and it is necessary for membrane
extension (Niedergang et al., 2003; Zhang et al., 1998). Rab5 and Rab7 are required
for phagosome maturation (Deretic et al., 1997; Via et al., 1997), and, in macrophages,
Rab5 is necessary for recruitment of Rab7 (Vieira et al., 2003). Rab5 is also a key
molecule regulating the earlier events in phagosome-endosome fusion. In a study by
Duclos et al. (2000) it was observed that expression of Rab5 mutants in macrophages
28
caused uncontrolled fusion, which led to the appearance of giant phagosomes that
could begin to mature and acquire lysosome marker LAMP1, but could not kill
intracellular parasites.
Another process in phagolysosome formation occurs through recycling of the
phagosomal membrane to the plasma membrane or other endocytic/phagocytic vesicles.
Rab11 is found in endosomes and nascent phagosomes, and it has been implicated in
this recycling process based on the observation that expression of a
GTP-binding-deficient mutant of Rab11 decreased the rate of transferrin efflux and
impaired FcγR-mediated phagocytosis, whereas expression of a GTPase-deficient
Rab11 mutant had the opposite effect (Cox et al., 2000). Arf6 has also been shown to
target recycling vesicles to the plasma membrane (Radhakrishna and Donaldson, 1997).
Other proteins involved in recycling include PI3-K, soluble
N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the
ATPase NSF (Braun and Niedergang, 2006; Greenberg and Grinstein, 2002).
Macrophages lack a morphologically distinct pericentriolar recycling
compartment and instead have an extensive network made up of transferrin
receptor-positive tubules and vesicles that participate in plasma membrane recycling.
As professional phagocytes, macrophages possess a mechanism for rapid renewal of
the plasma membrane. This is confirmed by the observation that the rate of transferrin
recycling in thioglycollate-elicited murine peritoneal macrophages is about two- to
threefold higher than seen in most other types of cells (Cox et al., 2000).
29
AIM The general aim of the research underlying this thesis was to investigate the
mechanisms of YopH-mediated antiphagocytosis, particularly the roles of the
YopH targets Cas and Fyb. More specific objectives were as follows:
To analyse the effects of Cas during Y. pseudotuberculosis infection.
To explore the functions of Fyb in macrophages and to investigate the
effects of the YopH-induced dephosphorylation of Fyb.
30
RESULTS and DISCUSSION Strain Relevant genotype References
YPIII virulence plasmid cured (Bolin et al., 1982)
MYM(YPIIIpIB29MEKA) yadA::Tn5, yopHMEKypkA (Hakansson et al., 1996)
MYMYopH pyopH (Persson et al., 1997)
MYMYopHC/A pyopHC/A (Persson et al., 1997)
MYMYopHQ 11 N 34 pyopHQ 11 N 34 (Deleuil et al., 2003)
YPIIIpIB102 yadA::Tn5, wild type (Bolin and Wolf-Watz, 1984)
Table I. Y. pseudotuberculosis strains used in the research. MYM is a mutant strain of Y.
pseudotuberculosis that lacks the effectors YopH, YopE, YopK, YopM, and YpkA, but still has intact genes
encoding the proteins that are necessary to regulate, secrete, and translocate Yops (Hakansson et al., 1996).
The MYM strain was used to express YopH and mutants thereof in order to investigate the role of that
effector protein in host cells.
Interaction between YopH and Cas is required for YopH-mediated disruption of
focal adhesion (Paper I)
Previous studies had shown that a catalytically inactive variant of YopH called
YopHC/A forms stable complexes with its substrates in vivo (Guan and Dixon, 1990),
and thus that variant was used to identify the protein targets of YopH in host cells.
YopH targets p130Cas in HeLa cells, fibroblasts, and macrophages (Black and Bliska,
1997; Hamid et al., 1999; Persson et al., 1997), and YopHC/A co-localizes with
p130Cas in adhesion structures of infected cells (Black and Bliska, 1997; Deleuil et al.,
2003; Hamid et al., 1999; Persson et al., 1997). We used p130Cas -/- mouse embryonic
fibroblasts (MEFs) to explore the effects of Cas on host cell responses during Yersinia
infection. The results revealed that YopHC/A does not localize to focal adhesions in
these cells, even though those structures are similar to the ones seen in wild-type
MEFs (Fig. 1A in Paper I). The localization of YopHC/A to focal adhesion structures
31
could be restored by re-expression of full-length Cas in the p130Cas-/- cells (Fig. 1B in
Paper I), which implies that Cas is responsible for the subcellular localization of YopH.
Interestingly, in our study, when Cas lacking the central substrate-binding domain
(∆SDCas) was expressed in p130Cas-/- cells, it was unable to recruit YopHC/A to focal
adhesions, whereas the variant itself was localized to these structures (Fig. 1B in Paper
I). Further immunoprecipitation experiments demonstrated that the central
substrate-binding domain of p130Cas is required to allow association with YopH (Fig.
1C in Paper I). There are 15 tyrosine motifs in the central substrate-binding domain of
Cas that may serve as potential phosphorylation sites for cellular protein tyrosine
kinases (Songyang and Cantley, 1998), and thereby as the target sites of bacterial
phosphatase YopH. Those motifs have been implicated as binding molecules that
transduce signals to control cell motility (Klemke et al., 1998).
It has been observed that host cells infected with Yersinia strains that express
active YopH become round and detached (considered to be the effects of cytotoxicity)
due to the disruption of focal adhesion structures containing p130Cas (Persson et al.,
1997). We found that P130Cas -/- MEFs were less sensitive to this effect, and the
sensitivity could be restored by re-expression of full-length Cas but not by expression
of the substrate-domain-depleted Cas mutant (Fig. 4 and Table 1 in Paper I). These
results suggested that the interaction between YopH and Cas is important for the
cytotoxic effect mediated by YopH. The tyrosine kinase FAK binds to the SH3 domain
of p130Cas (Polte and Hanks, 1995). Our research group has previously shown that
FAK and p130Cas form a complex with YopHC/A in HeLa cells (Persson et al., 1997),
and we obtained similar results when using wild-type fibroblasts (Fig. 2A in Paper I).
However, in macrophages, FAK is not a substrate of YopH but Cas is (Hamid et al.,
1999), and that finding suggested that FAK binds YopH via Cas. In agreement with
that assumption, we noted that FAK did not associate with YopHC/A in p130Cas -/-
MEFs, whereas p130Cas coupled with YopHC/A in FAK -/- MEFs (Fig. 2A in Paper I).
Furthermore, immunofluorescence staining of those cells indicated that Cas, but not
FAK, is required to recruit YopH to focal adhesion structures (Fig. 2B in Paper I).
Both Cas and FAK have been shown to participate in β1 integrin-mediated
internalization of Yersinia in epithelial cells (Alrutz and Isberg, 1998; Bruce-Staskal et
al., 2002; Weidow et al., 2000). Surprisingly, we did not observe any reduced uptake of
32
Yersinia in p130Cas -/- MEFs, as compared to wild-type cells from same littermate (Fig.
3 in Paper I). This suggested that p130Cas is not essential for the uptake of Yersinia,
which was confirmed by the fact that re-expression of full-length Cas in Cas -/- cells
did not have a significant impact on the internalization of the bacteria (Fig. 3 in Paper
I). In the absence of p130Cas, it is possible that other members of the Cas family such
as HEF-1/Cas-L and Sin/Efs can function in uptake, since they are similar to p130Cas
with regard to structure and localization (O'Neill et al., 2000). Presumably, Cas that
lacks the substrate-binding domain acts as a dominant-negative for other members of
the Cas family, which is supported by the observation that expression of ∆SDCas
inhibited uptake of Yersinia by p130Cas -/- MEFs (Fig. 3 in Paper I). FAK has been
implicated to play a role in integrin-mediated uptake (Alrutz and Isberg, 1998), in
consistence with that, internalization of the bacteria was greatly impaired in FAK -/-
MEFs as compared to littermate wild-type cells (Fig. 3 in Paper I). Except for that
FAK-dependent uptake of Yersinia, Bruce et al. (2002) have reported a
FAK-independent pathway that requires Pyk2, as well as p130Cas, Crk, and Src. Pyk2
is a FAK-related proline-rich protein tyrosine kinase (Avraham et al., 1995) that has
been found to associate with Cas in FAK -/- MEFs (Ueki et al., 1998). Expression of
Pyk2 is elevated in both FAK -/- MEFs (Sieg et al., 1998) and normal macrophages
(Bruce-Staskal et al., 2002). Recently, Owen et al. (2006) have suggested that FAK is
required for invasin-integrin mediated uptake of Y. pseudotuberculosis by macrophages,
while Pyk2 is essential for YadA/ECM-integrin mediated uptake. The mechanism of
Cas/Pyk2-mediated uptake is still unclear, since Pyk2 does not contain the sequences
in FAK that have been shown to bind to integrins (Klingbeil et al., 2001; Schaller et al.,
1995). It is possible that Cas/Pyk2 functions the same as Cas/FAK to bring Cas to Src
kinase and thereafter to induce phosphorylation of Cas. That phosphorylation results in
the recruitment of Crk and activation of Rho GTPase Rac1, which causes the
cytoskeletal rearrangement (Ridley, 2000; Weidow et al., 2000) that is a prerequisite of
bacteria internalization. YopH dephosphorylates Cas in infected cells and thereby
disrupts the Cas-mediated signalling process, which in turn leads to impaired uptake of
Yersinia.
33
YopH N terminus binds Fyb and it is important for YopH-mediated effects in
macrophages (Paper II)
Macrophages are professional phagocytes that are believed to be targets for
pathogenic Yersinia to inject virulence effectors, Yops, via the type III secretion system
(Marketon et al., 2005). YopH is one of the Yops that is delivered into macrophages
and blocks their capacity for phagocytosis (Fallman et al., 2002), and it also appears to
be highly critical for the ability of pathogenic Yersinia to remain in the lymphoid
system and establish an infection (Logsdon and Mecsas, 2003; Trulzsch et al., 2004;
Viboud and Bliska, 2005). In the mouse macrophage-like J774 cells, YopH selectively
dephosphorylates Cas, Fyb, and SKAP-HOM (Black et al., 2000; Hamid et al., 1999).
Of all the substrates of YopH, Fyb is the only one that is a haematopoietic-specific
protein. We initiated our studies of Fyb by investigating the way it interacts with YopH
(Paper II). GST-YopHC/A was used to pull down lysates of J774 cells that were
infected with MYM or with MYM expressing active YopH or catalytically inactive
YopHC/A. The results showed that YopH interacted with tyrosine-phosphorylated Fyb
(Fig. 1 in Paper II). In addition, proteins associated with YopHC/A exhibited enhanced
phosphorylation, which agrees with earlier findings and probably indicates that
YopHC/A protects Fyb from endogenous phosphatases (Hamid et al., 1999). To
ascertain which region in YopH is involved in the interaction with Fyb, we performed
in vitro binding studies using different variants of His-Fyb and GST-YopH protein
fragments (Fig. 2, a and b in Paper II). This showed that the 1–130 amino acids in the
N-terminal region of YopH were linked to tyrosine phosphorylated Fyb (Fig. 2d in
Paper II), which is similar to what has been reported regarding the interaction of YopH
with Cas (Black et al., 1998).
Research has revealed that the C-terminal PTPase domain of YopH harbours an
additional substrate-binding domain, which interacts with Cas in a
phosphotyrosine-dependent manner. It has been suggested that this domain co-operates
with the N-terminal substrate-binding domain of YopH to promote efficient recognition
of Cas by YopH in epithelial cells (Montagna et al., 2001). However, the
substrate-binding ability of the YopH N terminus is about 12-fold higher than that of
34
the phosphatase domain (Montagna et al., 2001). The three-dimensional structure of
the YopH N-terminal region has been determined (Evdokimov et al., 2001), and it is
known that residues Q 11 , V 31 , A 33 , and N 34 are involved in substrate binding (Deleuil
et al., 2003; Montagna et al., 2001). Our immunoprecipitation analyses using
YopHQ 11 N 34 confirmed that these residues are also necessary for the interaction of
YopH with Fyb (Fig. 2e in Paper II).
Our next intention was to reveal the biological importance of the YopH
N-terminal substrate-binding region with regard to the effects that this protein has on
macrophages. An important task of YopH is to block phagocytosis during Yersinia
infection (Fallman et al., 2002), and the present immunoprecipitation experiments
showed that YopHQ 11 N 34 has a reduced capacity to dephosphorylate Fyb in
macrophages (Fig. 3a in Paper II). That finding is in analogy with a previous study
conducted by our research team, showing that MYMYopHQ11N 34 was less efficient
than MYMYopH at blocking bacterial uptake by HeLa cells (Deleuil et al., 2003).
Similar results were obtained using the more phagocytosis-efficient J774 cells (Fig. 3b
in Paper II). YopH also has toxic effects on cultured cells, and we found that J774 cells
infected with MYMYopHQ 11N 34 were less sensitive to the YopH-mediated
cytotoxicity compared to cells exposed to MYM expressing wild-type YopH (Fig. 3c in
Paper II). Those observations suggested that the N-terminal substrate-binding domain
of YopH is necessary for both a full antiphagocytic effect and cytotoxicity towards
macrophages. In addition, the substract-binding ability of YopH N terminus is also
important for the virulence of Y. pseudotuberculosis (Deleuil et al., 2003).
The role of Fyb in macrophages is not yet fully understood. Nonetheless, the
fact that YopH targets Fyb implies that this immune-cell-specific adaptor protein plays
an important role in the antimicrobial function of macrophages.
mAbp1 is a novel interactor of Fyb (Paper III and unpublished data)
Previous studies have suggested that Fyb is involved in integrin-mediated cell
adhesion (Geng et al., 2001), which indicates that this protein is associated with
signalling to the actin cytoskeleton. Furthermore, Krause et al. (2000) have reported
35
that Fyb is localized to actin-rich structures in cells, potentially via interaction between
the FPPPP motif of Fyb and the EVH1 domain of Ena/VASP proteins.
To study the function of Fyb in macrophages, and especially in phagocytosis, we
first investigated the effects of the Fyb-Ena/VASP interaction on localization of Fyb to
actin-rich cellular structures. Surprisingly, our results showed that the FPPPP motif is
not required for the association of Fyb with such structures in J774 cells (Fig.1 in
Paper III). Also, a pull-down assay using GST-Fyb truncated variants and J774 cell
lysates revealed that only a minor portion of VASP binds Fyb (supplementary data Fig.
1 in Paper III). Taken together, these findings indicated that an additional part of Fyb
directly or indirectly mediated the linking between Fyb and the actin cytoskeleton.
α-helical
PXXP YXXP
mAbp1 SH3ADF-H/cofilin
F-actinF-actin DynaminFgd1HPK-1Piccolo
SrcSyk
To identify new Fyb-interacting proteins in immune cells, we performed a yeast
two-hybrid screen using a mouse lymphoma cDNA library. This approach detected a
novel interactor of Fyb called mammalian actin-binding protein 1 (mAbp1), which is
also known as SH3P7 (Sparks et al., 1996) and HIP-55 (haematopoietic progenitor
kinase 1 (HKP1) interacting protein of 55kDa) (Ensenat et al., 1999). The protein
mAbp1 is ubiquitously expressed, and it contains a single actin-depolymerizing factor
homology (ADF-H) domain in its N terminus, which is followed by an α-helical
structure region, a stretch of PXXP and YXXP motifs, and a C-terminal SH3 domain
(Fig. 6) (Ensenat et al., 1999; Kessels et al., 2001; Larbolette et al., 1999).
Figure 6. Schematic representation of mAbp1 and the proteins that interact with mAbp1.
Our in vitro interaction studies showed that the N-terminal part (residues 1–339) of
Fyb, which contains several proline-rich regions, binds to the mAbp1 SH3 domain (Fig.
4 E, F, and G in Paper III). We also used immunofluorescence staining of J774 cells to
determine the subcellular localization of mAbp1. These experiments revealed mAbp1
in the perinuclear region, as well as in the leading edge of cells where it co-localized
with F-actin (Fig. 3B in Paper III). Those locations correspond to what has previously
36
Fyb 1-339 Fyb 1-547 Fyb 548-783
Figure7. Overexpression of GFP-Fyb
truncated variants in HeLa cells.
been shown for Fyb in other types of
cells (Geng et al., 2001). In accordance
with that, co-staining of Fyb and mAbp1
demonstrated that these proteins
co-localized in areas displaying high actin
dynamics, namely, in cell edges and
ruffles (Fig. 3C in Paper III). The
co-localization of Fyb and mAbp1 was
observed solely at the cell edges and
neither of these proteins was found in punctuated structures resembling focal
adhesions. The latter observation agrees with published data indicating that Fyb is not
localized to adhesion structures (Krause et al., 2000). Overexpression of the Fyb N
terminus in HeLa cells led to distinct accumulation of the protein in the edges of the
cells (Fig. 7), which further supports the notion that the Fyb-mAbp1 interaction is
responsible for the localization of Fyb in actin-rich structures.
Additional infection experiments using Yersinia and J774 cells showed that both
Fyb and mAbp1 were tyrosine dephosphorylated by YopH, but this did not affect the
Fyb-mAbp1 interaction (Fig. 5 in Paper III). In accordance with that observation,
co-staining of Fyb and mAbp1 revealed both these proteins at the cell edges even in
the presence of active YopH (supplementary data Fig. 2 in Paper III). Hence, the
Fyb-mAbp1 interaction is not interrupted by YopH, which is consistent with studies in
vitro in which it was found that the association between Fyb and mAbp1 did not
depend on the tyrosine phosphorylation of those two proteins. It has previously been
reported that mAbp1 is localized to areas that harbour newly formed actin structures,
and such assignment of the protein does not require tyrosine phosphorylation (Kessels
et al., 2000). Moreover, in our experiments, the tyrosine phosphorylation of Fyb, but
not mAbp1, was stimulated by the β1 integrin engagement caused by the Yersinia
infection (Fig. 5, A and B in Paper III). This supports a model in which tyrosine
phosphorylation of Fyb is involved in the receptor-mediated clustering of β1 integrin,
whereas such phosphorylation of mAbp1 is less important.
37
Fyb has been reported to form a complex with Nck, VASP, and WASP that is
translocated to phagocytic cups during FcγR-mediated phagocytosis (Coppolino et al.,
2001). However, Fyb does not bind directly to Nck or WASP. Fyb is probably tyrosine
phosphorylated upon stimulation and can therefore operate via SLP-76 to associate
with Nck and Vav. The latter protein is a GEF of Rho GTPases and it has WASP as a
downstream effector. Thus WASP activates the Arp 2/3 complex, which results in local
actin rearrangements at the contact site. However, our observation that
tyrosine-dephosphorylated Fyb co-localized with mAbp1 in the cell edges implicated
an additional way for Fyb to connect actin cytoskeleton. Indeed, the yeast orthologue
of mAbp1 has been found to be an activator of the Arp 2/3 complex, and it is critical
for regulating the actin cytoskeleton (Goode et al., 2001; Qualmann and Kessels,
2002). mAbp1 is also co-localized with Arp 2/3 in the leading edges of newly formed
lamellipodia, though it lacks the acid regions that are required for the activating
capacity of Arp 2/3 (Kessels et al., 2000). Accordingly, our finding of mAbp1 as an
interactor of Fyb suggests a novel linkage occurs between the Fyb-containing complex
and the F-actin network.
mAbp1 influences spreading of macrophages and antiphagocytosis mediated by
pathogenic Yersinia (Paper IV and unpublished data)
Considering mAbp1, the ability to bind F-actin and the observed localization to
F-actin-rich structures suggested that this protein participates in the regulation of actin
dynamics. However, we found that neither RNA interference (RNAi) of mAbp1 nor
overexpression of the different variants of mAbp1 (Fig. 1A in Paper IV) in HeLa cells
affected β1 integrin-mediated cell spreading (Fig. 4A in Paper IV and unpublished
data), which also concurs with findings in human embryonic kidney cells (Mise-Omata
et al., 2003). This does not rule out the possibility that mAbp1 is involved in regulating
F-actin dynamics, probably not for forces leading to that kind of membrane extensions
in these cell types. In contrast to what has been observed regarding mAbp1 in HeLa
cells, ectopic expression of full-length mAbp1 in J774 cells increased both the
38
spreading areas and the number of spreading cells, whereas expression of the
mAbp1 W/A mutant and the two SH3 variants resulted in impaired spreading (Fig. 4 B
and C in Paper IV). These results indicated that both the actin-binding region and the
SH3 domain of mAbp1 are required to induce an increase in spreading. The
actin-binding region is likely important for localization to the cell periphery where the
dynamic rearrangement and growth of the F-actin network occurs, whereas the SH3
domain likely contributes by binding proteins important for this event, such as Fgd1,
which is the Cdc42 GEF that stimulates F-actin reorganization via the Rho GTPase
Cdc42 (Hou et al., 2003; Olson et al., 1996), or other proteins binding the mAbp1 C
terminus. The finding that mAbp1 greatly promoted cell spreading specifically in
macrophages also suggested that mAbp1 might have different functions in
haematopoietic and non-haematopoietic cells. The mAbp1 protein interacts with at
least two haematopoietic-specific proteins, Fyb (Paper III) and HPK1 (Ensenat et al.,
1999), the latter of which is a protein serine/threonine kinase and a member of the
MAPK kinase kinase kinases family (Han et al., 2003). In T cells, mAbp1 is mainly
associated with HPK1 (Ensenat et al., 1999) and that complex has been found to
regulate TCR signalling, such as T cell proliferation, cytokine production, and immune
responses (Le Bras et al., 2004; Chen and Tan, 2000; Han et al., 2005). HPK1 can
activate JNK, which has been implicated in cell migration (Huang et al., 2004).
However, it is still unclear whether mAbp1 and HKP1 form a complex in
macrophages, and the role of HPK1 in that cell type is largely unknown. Interestingly,
Fyb, the other haematopoietic-specific protein that can interact with mAbp1, has been
implicated in integrin signalling in T cells (Peterson et al., 2001). In macrophages, Fyb
is a target of YopH (Black et al., 2000; Hamid et al., 1999), which is responsible for
the impairment of β1 integrin-mediated cytoskeletal rearrangments (Fallman et al.,
2002). Hence, mAbp1 might influence the β1 integrin-mediated spreading via the
association with Fyb.
The mAbp1 protein has also been implicated in the coordination of endocytic and
cytoskeletal activities with the evidence that this protein binds directly to the following
molecules: dynamin, which is the GTPase that controls fusion of endocytic vesicles
(Kessels et al., 2001); and piccolo, which is the presynaptic zinc finger protein that can
39
interact with both profilin and actin to participate in synaptic vesicle endo- and
exocytosis (Fenster et al., 2003). Thus, via the mentioned proteins, mAbp1 might
influence F-actin-dependent events that are essential for membrane invagination and
vesicle trafficking. Inasmuch as that mAbp1 is involved in endocytosis (Connert et al.,
2006; Kessels et al., 2000; Kessels et al., 2001; Mise-Omata et al., 2003; Sauvonnet et
al., 2005 and Fig. 2B in Paper IV), we conducted experiments to determine whether
this protein also participates in the phagocytic process, which in many ways resemble
endocytosis. Expression of the different mAbp1 variants in HeLa and J774 cells
indicated that mAbp1 is not required for internalization of the non-virulent Y.
pseudotuberculosis strain YPIII (Fig. 3, A and B in Paper IV). In contrast, HeLa cells
overexpressing mAbp1 showed a slight increase in uptake of the wild-type Y.
pseudotuberculosis strain YPIIIpIB102 (Fig. 3A in Paper IV). Furthermore, enforced
expression of mAbp1 W/A (in which residue 411 is mutated to Ala, a change that blocks
interaction with the proline-rich region of associated proteins such as Fyb (Paper III))
in both HeLa and J774 cells led to impaired uptake of YPIIIpIB102 (Fig. 3, A and B in
Paper IV). Hence, mAbp1FL W/A might acts as a dominant-negative protein by
occupying sites for endogenous mAbp1 and thereby reducing the phagocytic capacity
of the cells even further. These results suggested that mAbp1 can participate in, but is
not essential for, β1 integrin-mediated uptake of Yersinia, and support for that
assumption was provided by assays of the uptake of such bacteria by HeLa cells with
mAbp1 RNAi (Fig. 3D in Paper IV). The exact role of mAbp1 in this process awaits
further analysis.
Role of the Fyb/SKAP-HOM complex in macrophages (unpublished data)
To investigate the role of Fyb in macrophages, we used both overexpression of
Fyb and RNAi to knock down Fyb in J774 cells. However, we failed to maintain
overexpression of Fyb, whereas Fyb RNAi reduced the expression with about 70%
(Fig. 8). In addition, the level of SKAP-HOM, but not mAbp1, was also impaired in
Fyb RNAi cells (about 60% reduction, Fig. 8). SKAP-HOM is also a substrate of
YopH in macrophages, and it binds Fyb via its SH3 domain (Liu et al., 1998). These
40
results suggested that the level of SKAP-HOM protein depends on the presence of Fyb.
Similar findings regarding Fyb and SKAP55 have been obtained in T cells (Huang et
al., 2005), in which Fyb binds SKAP55 to protect against proteolysis of SKAP55. In
addition, deficiency of SKAP-HOM does not alter expression level of Fyb in T cells
(Togni et al., 2005). Hence, a possible explanation for the unsuccessful overexpression
of Fyb in our experiments is that the enforced expression of Fyb may have increased
the level of SKAP-HOM, which has been reported to inhibit the growth of myeloid
cells (Curtis et al., 2000).
0
0,2
0,4
0,6
0,8
1
1,2
Fyb SKAP-HOM mAbp1
Rel
ativ
e ex
pres
sion
J774FybRNAi
Figure 8. Relative expression of proteins in J774 and Fyb RNAi cells. Results are the mean ± SD of three
separate experiments.
Fyb and SKAP-HOM have been identified in complex with SHPS-1 in
macrophages (Timms et al., 1999). SHPS-1 is also known as SIRPα (signal regulatory
protein α) (Kharitonenkov et al., 1997), BIT (brain Ig-like molecule with
tyrosine-based activation motifs) (Sano et al., 1997), or p84 (Chuang and Lagenaur,
1990), which is an Ig-like transmembrane receptor that contains cytoplasmic
immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Fujioka et al., 1996).
Tyrosine phosphorylation of those motifs mediates association between SHPS-1 and
tyrosine phosphatase SHP-1, and in that way SHPS-1 recruits SHP-1 to the
macrophage cell membrane. SHP-1 is generally viewed as a negative regulator of cell
signalling. Moreover, SHPS-1 is extremely abundant in macrophages (Veillette et al.,
1998), and hence it has been suggested that protein participates in the negative control
of macrophage functions (Oshima et al., 2002; Yamao et al., 2002). Recruitment of Fyb
to SHPS-1 occurs through SKAP-HOM (Timms et al., 1999). Both Fyb and
SKAP-HOM are the specific targets of the antiphagocytic effector YopH in
41
macrophages, thus they might be involved in the transmission and/or regulation of
signals from integrins. In addition, SHPS-1 also has been implicated in
integrin-mediated signalling (Inagaki et al., 2000; Maile et al., 2003; Tsuda et al.,
1998). To ascertain whether Fyb/SKAP-HOM acts via SHPS-1 to influence uptake of
Yersinia, we treated J774 cells with SHPS-1 antibodies for 30 min and then subjected
them to a Yersinia uptake assay. Those antibodies bind to the extracellular Ig-like part
of SHPS-1 and thereby inhibit the activation through this region. There was no obvious
difference between antibody-treated and untreated cells with regard to their ability to
internalize YPIII or YPIIIpIB102 (Fig. 9, A and B). This is an unexpected finding,
since it has been shown that SHPS-1 negatively regulates macrophage phagocytosis of
antibody-coated red blood cells (Ikeda et al., 2006; Oshima et al., 2002). Perhaps the
amounts of SHPS-1 antibodies we used were insufficient to result in a significant
difference between the treated and untreated cells. However, it is also possible that
different signalling pathways or molecules participate in the phagocytosis of different
types of particles. In other words, previous studies have used antibody-coated red
blood cells in uptake assays, which represent FcγR-mediated phagocytosis, whereas
we focused on β1 integrin-mediated internalization of pathogens.
A B
0
20
40
60
80
100
Untreated 1μg/ml 10μg/ml
YPIII
pIB
102
upta
ke (%
)
0
20
40
60
80
100
Untreated 1μg/ml 10μg/ml
YPIII
upt
ake
(%)
Figure 9. Uptake of YPIII (A) and wild-type YPIIIpIB102 (B) by J774 cells. The J774 cells were left
untreated or were treated with 1 or 10 μg/ml anti-SHPS-1 antibodies before performing the uptake assays.
Results are the mean ± SD of three separate experiments.
It is still not clear whether formation of a Fyb/SKAP-HOM/SHPS-1 complex is
induced by Yersinia infection or if it is mediated by tyrosine phosphorylation. To
address that issue, we used J774 cells infected with MYM, MYM expressing wild-type
YopH, or catalytically inactive YopHC/A; uninfected cells were used as controls. Cell
42
lysates were immunoprecipitated with SKAP-HOM antibodies, and immunocomplexes
were detected with Fyb, SHPS-1, or SKAP-HOM antibodies. The results showed that a
major portion of cellular Fyb was bound to SKAP-HOM (Fig. 10, A and B), and, even
after an infection time of one hour, this interaction was not abolished in the presence of
active YopH, and that Yop induces tyrosine dephosphorylation of Fyb and
SKAP-HOM (Fig. 10B). This observation further confirmed the previous finding that
Fyb binds SKAP-HOM in a phosphotyrosine-independent manner (Black et al., 2000).
In contrast, an interaction between SHPS-1 and SKAP-HOM was detected only in the
lysates of cells infected with MYMYopHC/A (Fig. 10A). The role of SHPS-1 during
Yersinia infection has not been fully elucidated, although it is apparently not required
for internalization of the bacteria. It has been suggested that MyD-1, which is the
bovine homologue of SHPS-1, can promote the attachment of APCs to CD4 + T cells
(Brooke et al., 1998), and hence Fyb/SKAP-HOM may act through SHPS-1 to activate
the adaptive immune system.
Figure 10. Immunoprecipitation of J774 cell lysates with anti-SKAP-HOM antibodies. A: J774 cells
were left uninfected or infected with MYM, or MYMYopH, or MYMYopHC/A for 45 min. B: J774 cells
were left uninfected or infected with MYMYopH for various times.
Another transmembrane receptor, PIR-B (paired immunoglobulin-like receptor B),
has been reported to form a complex with Fyb/SKAP-HOM in macrophages during
Yersinia infection (Black et al., 2000). PIR-B is a negative regulator of β2
integrin-mediated spreading of BMMs (Pereira et al., 2004), but its role in Yersinia
43
uptake has not yet been explained. Similar to SHPS-1, PIR-B also contains
cytoplasmic ITIM motifs that can be tyrosine phosphorylated and thereby mediate
association of PIR-B with SHP-1 (Pereira and Lowell, 2003). It is not known whether
the extracellular domains of these receptors bind directly to invasin, or if they must
associate with an integrin receptor to induce activation of the Fyb/SKAP-HOM
signalling complex. However, we cannot rule out the possibility that Fyb/SKAP-HOM
is primarily cytosolic or that it interacts with some other type of transmembrane
receptor in J774 cells.
The functions of Fyb in macrophages have been obscure, although the discovery
that this adaptor is a substrate for the bacterial antiphagocytic factor YopH suggested
that it is involved in phagocytosis. However, in our experiments, Fyb RNAi did not
change J774 cells with regard to the internalization of Yersinia (data not shown). It has
been shown that the retinoic-acid-induced protein designated PML-RARalpha target
gene encoding an adaptor molecule-1 (PRAM-1) shares structural homologies with
Fyb (two tyrosine motifs and an SH3-like domain), and it also binds the adaptors
SLP-76 and SKAP-HOM (Moog-Lutz et al., 2001). Recently, it was also noted that,
like Fyb, PRAM-1 can bind mAbp1 (Denis et al., 2005). Experiments on PRAM-1 -/-
mice have demonstrated that PRAM-1 is necessary for integrin-mediated
adhesion-dependent production of reactive oxygen intermediates and cellular
degranulation, but not for other integrin-dependent responses, such as cell spreading
and activation of several signalling pathways in neutrophils (Clemens et al., 2004).
PRAM-1 is expressed and regulated during normal human myelopoiesis (in
haematopoietic tissue, such as bone marrow and peripheral blood leukocytes), whereas
there is only negligible expression of this protein in BMMs and mast cells, even
though they contain PRAM-1 mRNA (Clemens et al., 2004). It is still unclear whether
knock down of Fyb can affect the expression of PRAM-1 or if expression of PRAM-1
in Fyb -/- cells can restore the function of Fyb.
Macrophages constitute the first line of defence in lymphoid tissues, and they are
considered to be the major targets for the antiphagocytic effectors of Y.
pseudotuberculosis. Besides phagocytosing pathogens, macrophages perform the
44
important functions of killing the invaders and presenting antigens to the adaptive
immune system. It is plausible that Fyb is involved in the killing of bacteria or other
immune responses.
45
CONCLUSIONS
During infection, pathogenic Y. pseudotuberculosis can inject virulence effectors
(Yops) into host cells to interfere with the antipathogenic machinery. YopH is a PTPase,
and as such it blocks phagocytosis by targeting proteins that mediate the signalling
associated with cytoskeletal rearrangements.
The present research has shown the following:
1. In non-professional phagocytes, YopH binds the substrate domain in Cas,
and this interaction is required for YopH-mediated disruption of focal
adhesions and cytotoxicity, which are associated with antiphagocytosis.
2. In macrophages, the N terminus of YopH binds tyrosine-phosphorylated Fyb,
and this domain is important for YopH-mediated antiphagocytosis.
3. The protein mAbp1 is a novel interactor of Fyb in macrophages. The
binding between these two proteins occurs via the SH3 domain in mAbp1
and the N terminus in Fyb. mAbp1 is implicated to influence β1
integrin-mediated spreading and the antiphagocytic effects of pathogenic
strains of Y. pseudotuberculosis.
4. Fyb binds another YopH target, SKAP-HOM, in a tyrosine-independent
manner, and the endogenous level of SKAP-HOM is regulated by Fyb in
macrophages.
The functions of Fyb and mAbp1 in macrophages are the subjects of our ongoing
research.
46
ACKNOWLEDGEMENTS
First, I would like to express my gratitude to my supervisor, Maria Fällman, for
giving me the chance to be her graduate student. I had almost no background in
molecular biology experiments, could you believe it? Thank you, Maria, for your
great guidance and strong support throughout my studies.
I want to thank the following former members of “Maria’s girls”: Nivia Hamid,
Karen McGee, Fabienne Deleuil, Anna Gustavsson, Lena Mogemark, Ritu
Andersson, and Nina Ericsson for their assistance in the lab. Karen, Anna, and
Fabienne were very supportive at the beginning of my studies. Thank you Lena for
your excellent collaboration, and Ritu for your help with my son’s daycare. Also
thanks to all for the tasty contributions to Friday’s group meeting. Karen and Lena, I
miss your soft chocolate cakes; Anna, you made such a beautiful birthday cake, I
could not believe you made it yourself! I also want to thank the present members of
our group, Anna Fahlgren, Sara Carlsson, and Anders Fagerström, for sharing
good times in the lab. Special thanks to Sara for help with the manuscript. I am also
grateful to the former and present subjects and summer students for helping with my
experiments.
Thanks to the all my teaching colleagues. It’s almost Christmas time again, and I
still remember when I got a card and chocolate from students during my first year of
teaching. Edmund, I will never forget that funny lab report you shared with us:
“Bacteria look like the needles that fall off the Christmas tree.”
I am also grateful to all the past and present members of the “Journal Club” and
the “Lab meeting” for your constructive discussions and suggestions. Thank you
everybody at the department for help with the equipment and other things…..!
Special thanks to Patricia Ödman for correcting the English in this thesis.
Thanks to my Chinese friends: Jufang Wu, Bo Huang, Guangwei Ou,
Changchun Chen, Professor Jin and his wife Gu. Shall we go fishing somewhere
again next summer? Suyan Wang and her family, we had a good time together in
Greece during that EMBO course!
I am very grateful to my parents, Yongqiang Ji and Suyun Yuan. I miss you very
much! Mama, you always understand me. Dad and mama, I think you are the best
47
感谢父母多年来的支持!parents! I love you! Thanks to my brother Tao Ji and
his wife Min Zhang for their support and understanding during these years. Min, good
luck with your own thesis!
Last, but not at all least, I want to thank my husband Jian. You helped me
enormously at the beginning of my studies, you are my “second supervisor” of
molecular biology, and without you I would never have got this far! My little boy,
Yuanyang, you bring me so much joy everyday! I love you!
This work was supported by the Swedish Medical Research Council, the Swedish
Cancer Foundation, the King Gustaf Vth 80 Year Foundation, the Medical Research
Foundation at Umeå University, and the J.C. Kempes Memorial Foundation.
48
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