Bacterial pathogenesis: exploiting cellular adherence

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Bacterial pathogenesis: exploiting cellular adherence Erin C Boyle
and B Brett Finlayy Cell adhesion molecules, such as integrins, cadherins, the immunoglobulin superfamily of cell adhesion molecules and selectins, play important structural roles and are involved in various signal transduction processes. As an initial step in the infectious process, many bacterial pathogens adhere to cell adhesion molecules as a means of exploiting the underlying signaling pathways, entering into host cells or establishing extracellular persistence. Often, bacteria are able to bind to cell adhesion molecules by mimicking or acting in place of host cell receptors or their ligands. Recent studies have contributed to our understanding of bacterial adherence mechanisms and the consequences of receptor engagement; they have also highlighted alternative functions of cell adhesion molecules. Addresses Department of Microbiology and Immunology, Biotechnology Laboratory, Wesbrook Building, Room 237, 6174 University Boulevard, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
e-mail: [email protected] y e-mail: [email protected]

Current Opinion in Cell Biology 2003, 15:633–639 This review comes from a themed issue on Cell-to-cell contact and extracellular matrix Edited by Eric Brown and Elisabetta Dejana 0955-0674/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S0955-0674(03)00099-1

Abbreviations CAM cell adhesion molecule CAS Crk-associated substrate CEACAM carcinoembryonic antigen-related CAM EC1 extracellular domain 1 ECM extracellular matrix FAK focal adhesion kinase FcR Fc receptor IgCAM immunoglobulin superfamily of CAMs ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine-based inhibitory motif Leb Lewis B Lex Lewis X LRR leucine-rich repeat Opa opacity-associated

Introduction Adherence to host cell surfaces is often the first step in the establishment of bacterial disease. For extracellular pathogens, adherence allows bacteria to withstand the mechanical clearing mechanisms of the host; for intracellular pathogens, adherence is often a prerequisite for uptake (invasion). www.current-opinion.com

Adhesins are bacterial components that mediate interaction between the bacterium and the host cell surface. Bacterial engagement of host cell receptors can be a means of targeting a pathogen to a particular niche, coopting underlying signaling pathways, establishing persistent infections and inducing invasion [1]. Invasion affords bacteria protection from immune detection and facilitates access to deeper tissues. Many bacterial pathogens have evolved the capacity to adhere to CAMs (cell adhesion molecules). CAMs are cell-surface receptors that mediate cell–cell and cell– extracellular-matrix (ECM) interactions. Generally, they can be classified into four main groups: integrins, cadherins, members of the immunoglobulin superfamily of CAMs (IgCAMs), and selectins [2]. Bacteria are able to bind to CAMs by mimicking or acting in place of host cell receptors or their ligands. In this review, we will concentrate on a few recent examples of the interaction between bacteria and CAMs during infection.

Integrins and Yersinia Perhaps the best-characterized bacterial adhesin is invasin, an outer membrane protein of the Gram-negative diarrhea-causing pathogens Yersinia enterolitica and Y. pseudotuberculosis [3]. During the initial stages of the infectious process, Yersinia translocates rapidly from the intestinal lumen to the lymph nodes via invasion into M cells. M cells are specialized cells that sample antigens from the lumen and present them to underlying immune cells [4]. Several intestinal pathogens exploit this property of M cells and use them as portals for crossing the intestinal epithelium and establishing infection [5]. Invasin mediates high-affinity binding to a subset of b1 integrins (a3b1, a4b1, a5b1, a6b1 and avb1) presented at the apical surface of M cells [6,7]. Invasin binds a5b1 integrin with 100-fold higher affinity than its natural substrate, fibronectin [8]. Data from crystal structures, competitive inhibition studies, mutagenesis and monoclonal antibody inhibition studies reveal that invasin binds b1 integrins at an overlapping or identical site to fibronectin [3,9,10]. Although they lack sequence similarity, there is considerable receptor recognition similarity between invasin and fibronectin [11]. This illustrates how Yersinia is able to efficiently mimic and compete with host cell molecules to establish an infection. High-affinity binding by invasin and invasin self-association mediate b1 integrin recruitment and multimerization, Current Opinion in Cell Biology 2003, 15:633–639

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Figure 1

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Model for the signaling pathways leading to Yersinia invasion. Yersinia infection increases autophosphorylation of the protein tyrosine kinase Pyk2. Phosphorylated Pyk2 probably activates Src-family kinases, which phosphorylate CAS. The adaptor protein Crk is recruited to phosphorylated CAS, which results in a downstream signal that activates Rac1. In a parallel pathway, engagement of b1 integrins (red) by invasin (blue) leads to FAK autophosphorylation. Both FAK and Src are required for Yersinia invasion. Src is probably recruited via FAK, and, through an indirect mechanism, FAK–Src interaction can lead to Rac1 activation. Both pathways require Rac1. Rac1 might activate WAVE, which can then bind the Arp2/3 complex, leading to actin polymerization and bacterial engulfment. P, phosphate; WAVE, Wiskott–Aldrich syndrome protein family verprolin-homologous protein.

which appear to be critical for bacterial uptake [8,12]. High-affinity binding facilitates circumferential binding of integrins about the bacterial surface, which culminates in a zipper-like mode of entry. Multimerization of b1 integrins seems to be important for recruitment of cytoskeletal and signaling proteins required for transmission of downstream signals. Specific residues within the cytoplasmic domain of the b1 chain are necessary for invasinmediated internalization of Yersinia [13,14]. Both the actin cytoskeleton and microtubules [15] are involved in uptake, as well as the tyrosine kinases Pyk2 and Src, Rac, the Arp2/3 complex, focal adhesion kinase (FAK), Crk-associated substrate (CAS) and Crk [3,16,17,18] (Figure 1). YadA, another Yersinia adhesin, interacts with b1 integrins indirectly by binding to ECM proteins such as fibronectin and collagen [19]. Although invasin has generally been considered the primary Yersinia adhesin, recent evidence suggests YadA-mediated invasion constitutes a second efficient uptake pathway that functions after invasinmediated translocation although the intestinal barrier [20]. Because of differences in gene regulation, YadA could be important under environmental conditions where invasin expression is repressed.

Cadherins and Listeria monocytogenes Listeria monocytogenes is the etiological agent of listeriosis, a severe food-borne disease that can lead to gastroenterCurrent Opinion in Cell Biology 2003, 15:633–639

itis, meningo-encephalitis in immunocompromised individuals, and spontaneous abortions in pregnant women [21]. This organism has the remarkable ability to cross the intestinal, blood–brain and fetoplacental barriers. L. monocytogenes is a facultative intracellular pathogen and can induce its own uptake into cells that are normally non-phagocytic using a zipper-like mechanism similar to that used by Yersinia. Internalin (also called InlA) is a surface-exposed bacterial adhesin that mediates entry into epithelial cells (e.g. the human enterocyte-like epithelial cell line Caco-2) by binding the epithelial cell receptor E-cadherin. Internalin contains an amino-terminal leucine-rich repeat (LRR) region, which is necessary and sufficient to promote internalization into cells expressing E-cadherin [22]. The binding site of internalin on E-cadherin was mapped to the extracellular amino-terminal domain (known as extracellular domain 1 [EC1]), which is responsible for the adhesive specificity of homotypic interactions between neighboring cells. Oral infection of mice with L. monocytogenes is an inefficient means of studying disease because bacterial translocation across the intestinal epithelium is very low. Moreover, colonization of the brain or fetoplacental unit has never been observed upon oral or intravenous inoculation of mice. The conundrum facing researchers was www.current-opinion.com

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that despite the importance of internalin in invasion of human epithelial cells in vitro, a role for internalin after intravenous or oral infection of mice was never observed. Recently, it was found that mouse E-cadherin could not mediate entry of L. monocytogenes [23]. Ultimately, the molecular basis for this species specificity came down to a single amino acid difference between mouse and human E-cadherin. Recently, the crystal structure of the LRR region of internalin and the LRR region complexed to the EC1 domain of human E-cadherin was reported [24]. The crystal structure provides a structural explanation for the stringent species specificity of internalin [24]. The subsequent development of a transgenic mouse model in which mice express human E-cadherin exclusively in enterocytes of the small intestine has demonstrated that internalin mediates invasion of enterocytes in vivo and facilitates crossing of the intestinal barrier [25,26]. These studies reveal that internalin is a bona fide virulence factor and illustrates the importance of using an appropriate model system. Future studies await the development of transgenic mice that express human E-cadherin in all tissues, which might facilitate the study of brain and fetoplacental tropism. Several important questions remain: How does Listeria access E-cadherin in vivo if its expression is limited to adherens junctions and basolateral surfaces? How does Listeria hijack E-cadherin and exploit it as a means of uptake? E-cadherin is indeed intimately associated with both signaling molecules and the actin cytoskeleton. A recent study revealed that the interaction between the cytoplasmic tail of E-cadherin and a- and b-catenin is Figure 2

Listeria

α β

involved in actin-dependent uptake of Listeria [27] (Figure 2). More studies are needed to determine the signaling events involved in internalin-mediated invasion.

IgCAMs and Neisseria gonorrhoeae Neisseria gonorrhoeae is a human-specific pathogen that infects mucosal tissues of the genitourinary tract. Initial attachment to mucosal surfaces is mediated by neisserial type-IV pili [28]. Subsequently, intimate adherence is conferred by colony opacity-associated (Opa) proteins, a family of phase-variable outer-membrane proteins that act as adhesins and facilitate invasion [29,30]. A single gonococcal strain can possess up to 11 different Opa variants. A few Opa variants bind heparan sulfate proteoglycans on epithelial cells or use vitronectin and fibrinogen to bind indirectly to integrins. Most Opa proteins target carcinoembryonic antigen-related CAM (CEACAM) receptors, which are members of the IgCAMs and are widely distributed throughout human host tissues [31]. The function of CEACAM receptors is not completely understood; however, it is known that they mediate cell–cell adhesion and play a role in cell cycle control and cell differentiation [31]. N. gonorrhoeae specifically targets four CEACAM family members: CEACAM1, -3, -5 and -6. In human epithelial cells, engagement of CEACAM receptors by Opa proteins results in bacterial engulfment [32–34]. In the polarized epithelial cell line T84, Opa-mediated binding to CEACAM1, -5 or -6 induces invasion, transcytosis and release of bacteria at the basolateral surface [35]. In neutrophils, Opa proteins mediate adherence and opsoninindependent phagocytosis when targeted to CEACAM1, -3 and -6 receptors [32,34,36]. In certain cases, N. gonorrhoeae is capable of triggering expression of its own cellular receptor. CEACAM1 expression is upregulated in endothelial cells in an NF-kB-dependent manner in response to gonococcal infection or purified lipopolysaccharride [37]. Consequently, this probably reinforces Opa-mediated binding and invasion. Figure 3

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Signaling involved in internalin–E-cadherin-mediated uptake of L. monocytogenes. The bacterial adhesin internalin (pink) binds the epithelial CAM E-cadherin (blue). a- and b-catenin act as adapters between the cytoplasmic tail of E-cadherin and the actin cytoskeleton. This link to the actin cytoskeleton is required for invasion of L. monocytogenes. www.current-opinion.com

Differences in cell-surface modifications during N. gonorrhoeae invasion depends on the CEACAM receptor used. (a) CEACAM3mediated invasion induces impressive, extended surface projections at the site of adherence. (b) CEACAM1 and -6 (shown) invasion results in the tight envelopment of bacteria within pseudopods. Reproduced from [38], by permission of Oxford University Press. Current Opinion in Cell Biology 2003, 15:633–639

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Interestingly, recent studies have shown that the engagement of different CEACAM receptors by N. gonorrhoeae results in distinct signaling consequences and mechanisms of entry (Figure 3) [38,39]. Adherence and invasion of gonococci using CEACAM1 or -6 results in actin- and Rho-GTPase-independent pseudopod formation [38]. Conversely, invasion via CEACAM3 induces actin-driven membrane projections involving Rac1 and Cdc42. The neutrophil-restricted CEACAM3 receptor has an immunoreceptor tyrosine-based activation motif (ITAM) encoded in its cytoplasmic domain. ITAM motifs are associated with signaling activation of T-cell, B-cell and Fc receptors (FcRs). In fact, engagement of CEACAM3 is quite similar to engagement of FcR (Figure 4).

Both FcR-mediated phagocytosis and Opa-mediated invasion via CEACAM3 involve Cdc42, Rac1, actin polymerization, Syk kinase, phospholipase C, phosphoinositide 3-kinase, and also requires tyrosine phosphorylation of specific residues within the ITAM motif [38,39]. Therefore, Billker et al. [38] suggest that CEACAM3 might act as a phagocytic receptor in vivo and could consequently be an important component of the host innate immune response. CEACAM1, conversely, possesses an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplamic tail. ITIM motifs are known to recruit phosphatases, such as SHP-1, which dampen signaling events. A role for the

Figure 4

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Neisseria Arp 2/3

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Comparison of the signaling pathways involved in (a) CEACAM3-mediated invasion by N. gonorrhoeae and (b) FcR-mediated phagocytosis. Src-family kinases (Hck and Fgr in [a] and Lyn and Hck in [b]) phosphorylate tyrosine residues within the receptor’s ITAM motif. Syk tyrosine kinase is recruited to the phosphorylated ITAM motif, where it is activated. Syk then transmits a downstream signal that leads to the activation of Rac and Cdc42, which might activate N-WASP, leading to actin polymerization via the Arp2/3 complex. IgG, immunoglobulin G; N-WASP, neuronal Wiskott–Aldrich syndrome protein; P, phosphate. Current Opinion in Cell Biology 2003, 15:633–639

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CEACAM1 ITIM motif was recently identified in T lymphocytes [40]. Opa-mediated ligation of CEACAM1 receptors on CD4þ T cells downregulates activation and proliferation of infected cells. This bacterial strategy to subvert the normal immune response is dependent on the ITIM motif. The authors speculate that Opa– CEACAM1-mediated immunosuppression could account for the fact that gonococcal infection does not result in protective immunity, despite causing a significant inflammatory response; it could also account for why gonococcal infection seems to increase an individual’s susceptibility to other infections [41,42]. An interesting question that follows from this research is whether different invasion mechanisms result in different consequences for intracellular bacteria. For example, both complement-receptor-3-mediated uptake of Bordetella pertussis and CD48-mediated uptake of Escherichia coli into mast cells results in intracellular survival of bacteria through avoidance of the oxidative burst — an innate immune response consisting of a rapid and transient synthesis of antibacterial reactive oxygen and nitrogen intermediates [43,44]. Generation of transgenic mice expressing specific human CEACAM receptors will allow assessment of the contribution of Opa–CEACAM interactions in vivo.

Selectin mimicry and Helicobacter pylori Helicobacter pylori infects more than half the world’s population. Although asymptomatic in most cases, it can lead to gastritis, peptic ulcers and gastric cancer [45]. H. pylori is primarily thought to be an extracellular pathogen. It has the remarkable ability to colonize the gastric mucosa and persist within a host for decades. If the mechanical clearing mechanisms present within the stomach are considered, it seems likely that adhesion is a crucial aspect of the ability of H. pylori to persist and cause disease. The best-characterized adhesin of H. pylori is BabA, which binds Lewis B (Leb), a fucosylated histo-blood group antigen present on red blood cells and in the gastrointestinal mucosa [46]. Recently, a second receptor–adhesin interaction was identified [47]. When gastric biopsy tissue from H. pylori-infected individuals was infected with a babA mutant, adherence was still observed. Moreover, binding of the babA mutant was not blocked by pretreatment of bacteria with soluble Leb antigen. This suggests there is another adhesin, possibly specific to inflamed (i.e. H. pylori-infected) tissue, that binds a receptor distinct from that of BabA. Using thin layer chromatography, mass spectrometry and nuclear magnetic resonance, sialyl-dimeric-Lewis x (Lex) glysophospholipid was identified as a second receptor. This was confirmed using competitive inhibition and monoclonal antibody inhibition studies. Using a retagging method [46], this sialyl-dimeric-Lex-binding bacterial www.current-opinion.com

adhesin was identified as the bacterial outer membrane protein SabA. Sialylated glycoconjugates are low in healthy gastric mucosa but are expressed during gastritis. Upon inflammation, sialyl-Lex serves as a receptor for selectins and mediates the initial steps of leukocyte migration through the endothelium [48]. Recent studies demonstrate that H. pylori infection stimulates expression of sialyl-Lex at the surface of the gastric mucosa [47]. H. pylori is therefore another example of a bacterial pathogen capable of inducing expression of its own receptor, which can then be exploited for further adherence. Using selectin mimicry, H. pylori can take advantage of the host’s own mechanism by which it initiates an immune response to infection. By specifically targeting sites of inflammation, H. pylori might benefit nutritionally from exudates released upon cell damage and could take advantage of the exposed ECM components for further colonization. Adherence to sialylated Lex expressed during chronic inflammation might therefore contribute to the exceptional ability of H. pylori to establish persistent infections.

Conclusions Ubiquitously expressed and intimately associated with downstream signaling pathways, CAMs make ideal anchors for pathogen adherence and effective media for communication with host cells. In this review, we summarize only a few examples of bacterial exploitation of CAMs. A wide range of pathogens have evolved to bind integrins [43,49]. Uropathogenic E. coli [50] and Haemophilis influenzae [51] are other pathogens that target CEACAM receptors, while Anaplasma phagocytophila (human granulocytic ehrlichiosis agent) employs selectin mimicry by specifically binding fucosylated P-selectin glycoprotein ligand-1 (PSGL-1) [52]. Pathogens can be useful models in the study of receptor engagement and function. In the future, it will be important to establish further how pathogens hijack cellular receptors and exploit them as a means of uptake. Elucidation of bacterial adherence and invasion mechanisms will contribute to our understanding of the pathogenesis of these organisms. Cooperation between microbiologists and cell biologists will be crucial in this pursuit.

Update Recently, CagA, a H. pylori protein delivered into host cells using a type IV secretion system [53], was shown to induce the recruitment of ZO-1 and other tight junction components to the site of bacterial attachment on polarized epithelial cells [54]. Tight junction structure and function was altered as a result of CagA and this is likely to contribute to disruption of gastric epithelial integrity during H. pylori infection. The interaction between CagA and tight junction components might target or retain H. pylori at cell–cell junctions. Alternatively, recruitment Current Opinion in Cell Biology 2003, 15:633–639

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of tight junction components to the site of bacterial adherence could assemble a powerful signaling scaffold through which H. pylori could communicate with the host cells.

Acknowledgements We would like to thank AH Bouton for helpful discussion, as well as O Billker and TF Meyer for allowing reproduction of their photographs. EC Boyle is supported by the Natural Sciences and Engineering Research Council of Canada (NSERC). BB Finlay is a Howard Hughes Medical Institute (HHMI) International Research Scholar, a Canadian Institute for Health Research (CIHR) Distinguished Investigator, and the University of British Columbia Peter Wall Distinguished Professor. Operating grants from HHMI, CIHR, and the Canadian Bacterial Disease Network support work in BB Finlay’s laboratory.

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Current Opinion in Cell Biology 2003, 15:633–639