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Microbiology (1996), 142,3319-3336

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REVIEW ARTICLE

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Microbial utilization of human signalling molecules Mumtaz Virji Tel: +44 1865 221072. Fax: +44 1865 220479. e-mail: [email protected]

Department of Paediatrics, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK

Keywords : eukaryotic signal transduction, adhesion molecules, cellular invasion

Overview Many pathogenic micro-organisms specifically target host-cell receptors that normally mediate intercellular interactions, attachment to substrata, or receive hormonal, cytokine and other environmental signals. A fundamental question in microbial pathogenesis is how such targeting leads to cellular invasion. In eukaryotic cells, signal transduction involves a complex array of interactive molecules and second messengers that control multiple cellular functions. In this review, several eukaryotic signal transduction systems currently known are presented briefly to set the scene for discussing examples of distinct receptor/signalling systems that are known to be manipulated by pathogenic microbes. The second part of the review will focus on one large subset of receptors, the adhesion molecules, and explore the mechanisms by which microbes engage with them during the course of pathogenesis.

Signalling pathways of eukaryotic cells and some examples of their subversion by pathogens In broad terms, two distinct mechanisms of eukaryotic transmembrane signal transduction are recognized : (a) unimolecular receptor/effector systems that span the plasma membrane with the ligand-binding domain exposed at the cell surface and enzymic effector domain at the cytoplasmic face, and (b) multi-component systems with independent but coupled receptors, transducers and effector molecules. In each case, signals originating at the membrane are passed on to cytosolic molecules in a cascade of activating events and eventually to the nucleus. At the heart of these pathways are phosphorylating enzymes whose compartmentalization is critical in specific signal transduction and in part depends on kinaseanchoring proteins located at various sites in the cell (Mochly-Rosen, 1995). Although distinct pathways may be activated via individual ligand-receptor interactions, many are interrelated (Fig. 1). Moreover, multiple receptor systems can interact and synergize with each other 0002-0896 0 1996 SGM

to regulate cellular events (Rosales e t al., 1995; Clark & Brugge, 1995).

Main categories of eukaryotic signalling pathways Receptor fyrosine kinase pathway (RTK)IRas pathway

RTK pathways have tyrosine kinase activity directly associated with the receptor as a part of the receptor molecule. The membrane-located proto-oncogene product Ras is central to this pathway. Ras is a low-molecularmass GTP-binding protein (small G-protein) whose activation involves conversion from GDP to GTP-bound (activated) form. Binding of ligand (usually growth factors) results in receptor clustering, activation of cytoplasmically located tyrosine kinase domain and autophosphorylation of tyrosine residues at the C terminus. This leads to binding of other proteins such as Grb-2/Sos complex (cytosolic complexes with affinity for phosphorylated receptor as well as Ras) to the receptor and to plasma-membrane-located Ras. Sos activates Ras via exchange of GDP for GTP, leading to activation of other proteins downstream in the cascade which include cytoplasmic serine/threonine kinases (Raf-1, MEK and MAP-kinases). The latter can migrate from the cytosol to the nucleus and deliver signals to the nucleus by phosphorylation of transcription factors. Direct activation of the nuclear factor NF-kB may occur via Raf-1 which can phosphorylate IkB, the inhibitor of NF-kB. Negative modulation of Ras occurs via GAPS, GTPaseactivating proteins, that stimulate intrinsic GTPase activity of Ras (Maruta & Burgess, 1994). Recent evidence suggests that the CAMP and other second messenger signalling pathways are interconnected with the RTK pathway (Rosales e t al., 1995). It appears that phosphorylation of tyrosine plays a key role in Ras activation since other tyrosine kinase pathways (see below) also appear to activate Ras (Maruta & Burgess, 1994). The superfamily of small GTPases include five classes of proteins: Ras (primarily affects cell growth and development), Ran (involved in nuclear protein import), Rab and Arf (monitor and direct vesicular movement) and the 3319

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Receptors:

ECM

Antigen

Pathways:

Integrin / cytoskeletal

NRTK

Growth factors

RTK

;n

I

Focaladhesion complex

I

Hormone

Sev-R/G Prt

n

I - - - - - - -- - - - -

+----

CAMP IP3

activation

gene transcription Fig. 1. Some elements of human cellular receptor-mediated signalling pathways. The pathways and abbreviations are described in the text.

Rho subfamily (involved in regulation of the actin cytoskeleton). Within the Rho subfamily, different proteins participate to direct distinct patterns of actin reorganization. Rho proteins are associated with focal adhesions, Rac with membrane ruffling and NADPH oxidase regulation in some cells, and Cdc42 with actincontaining microspikes or filopodia formation (Aktories & Just, 1995; Vojtek & Cooper, 1995). Non-receptor tyrosine kinase (NRTK) pathways

These pathways are used by receptors often involved in immune recognition. Activation requires receptor aggregation and non-covalent association with other membrane-located proteins such as Src. Src kinases are acylated membrane-associated proteins with Src homology (SH) domains characteristic of many molecules within signalling pathways and which mediate physical association with other components. For example, SH2 domains recognize motifs containing phosphotyrosine, whilst SH3 domains bind to short proline-rich regions (Seed, 1995). Src-like kinases have also been implicated in integrin signalling events (Clark & Brugge, 1995) and in activation of RTK as well as receptors coupled to Gproteins (Erpel & Courtneidge, 1995). Other NRTKs are Syk kinases of haematopoietic cells which are activated by p1 and p3 integrin cross-linking (Clark & Brugge, 1995). 3320

Full activation of Src family kinases requires two opposing enzyme activities : the removal of C-terminal phosphate and phosphorylation of tyrosine proximal to the active site (Seed, 1995). G-proteinpathways

These are utilized by many classes of hormones and involve heptahelical receptors (with seven hydrophobic transmembrane regions, also known as serpentine receptors, Serp-R) (Fig.1) that couple to heterotrimeric G-proteins which are distinct from small monomeric Gproteins. These membrane-associated trimers consist of a, p and y subunits. The Gp and Gy subunits control the function of the G a subunit (Nurnberg e t al., 1995). The G a subunit binds GTP, has GTPase activity and is the effector molecule. Its activation results in activation of other effector enzymes to generate second messengers such as CAMP, 1,2 diacylglycerol (DAG) and inositol triphosphate (IP,) or to regulate ion channels (Nurnberg e t al., 1995). These second messengers are involved in activating protein kinases directly or via Ca2+/calmodulin complexes, leading to activation of cellular events. Specificity and selectivity of signalling via G-proteins is accomplished by coupling of receptors with distinct Gprotein trimers composed of variants of different subunits. Some receptors can activate multiple G-proteins, thus

Microbial utilization of human signalling molecules regulating diverse signal-transduction pathways (Rosales e t al., 1995; Nurnberg e t al., 1995). Pathways used by adhesion molecules Cjttoskeleton-linked path ways (integrin-associated signalling). Integrins are a superfamily of heterodimeric trans-

membrane molecules consisting of one of 16 a chains and one of eight /3 chains giving rise to more than 20 different receptors (Clark & Brugge, 1995). Integrin families and their normal ligands are described in Table 1. Integrins have short cytoplasmic tails without catalytic domains that associate with cytoskeletal proteins such as a-actinin, talin, vinculin, paxillin and tensin in complexes (focal adhesions, Fig. 2) and affect cytoskeletal arrangement via actin microfilaments (Clark & Brugge, 1995). The extracellular domains have Ca2+-binding sites (Fig. 2) implicated to participate in ligand binding. Several integrins interact with matrix proteins and thus have the capacity of forming a physical link between the extracellular matrix (ECM) and cytoskeleton. Two principal mechanisms by which integrins are activated to transduce signals involve conformational change and receptor clustering. Conformation-dependent activation is exemplified by thrombin-mediated activation of gpIIb/IIIa integrin expressed on platelets. Thrombin induces intracellular signalling leading to modulation of the conformation of gpIIb/IIIa, which is then able to bind fibrinogen leading to platelet aggregation. This has been called ‘inside out’ signalling (Richardson & Parsons, 1995). Signal transduction via integrins may reside, at least in part, in cytoskeletal rearrangement which may physically bring together molecular complexes by forming focal adhesions. Inhibition of clustering, which is achieved via cytoskeletal rearrangement, also inhibits tyrosine kinases suggesting kinase activation may be mediated by receptor clustering (Clark & Brugge, 1995 . A tyrosine kinase, focal adhesion kinase (FAK) p ~ 1 2 5 ’ ~ ~is, believed to play an important role in integrin-mediated signal transduction (Rosales e t a l . , 1995; Clark & Brugge, 1995). This kinase localizes to focal adhesion complexes and is stimulated to autophosphorylate at Tyr,,, upon integrin binding to ECM. Studies using the actin-depolymerizing agent cytochalasin D have shown that signal transduction via integrins involves MAP kinase activation in addition to ppl 25FAK phosphorylation and also that signal transduction via integrins requires cytoskeletal reorganization (Rosales e t al., 1995). FAK may interact with Src and recruit Src to focal adhesions causing hyperphosphorylation of focal adhesion structures. There is also evidence for association of other tyrosine kinases with integrins, for example, in monocytes, Syk (Src family) may respond to integrin signalling. Integrin binding to ECM also leads to a rise in intracellular Ca2+ via a 50 kDa integrin-associated protein, IAP shown to bind to some integrins (Rosales e t al., 1995). Stimulation of integrins may be translated into a variety of intracellular signals. For example, monocyte adherence to fibronectin or collagen activates transcription of inflammation mediators such as IL1-/3 and IL8. Integrins may also act in concert with other receptor pathways to

enhance or dampen signals. In particular, activation via growth factor receptors require adherence of cells to ECM via integrins. The small G-protein Rho appears to be critical in integrating signals induced by integrins and growth factor receptors (Clark & Brugge, 1995). Protein tyrosine phosphatases (PTPases) also appear to be involved in regulation of cell adhesion which may involve dephosphorylation of pp125FAK,implicating further the role of the kinase in integrin-mediated adhesion (Rosales e t al., 1995). In addition, PTPases may activate Src kinases by dephosphorylating the negative-regulatory C-terminus phosphotyrosine (Clark & Brugge, 1995). The complexities of integrin/cytoskeletal signalling pathways are discussed in comprehensive reviews by Rosales e t al. (1995) and Clark & Brugge (1995). Pathways used by other adhesion molecules. Mechanisms

and transduction pathways used by the immunoglobulin (Ig) superfamily, cadherins and selectins are much less well understood at present compared with integrin pathways. Structural studies on certain families of transmembrane tyrosine kinases such as the fibroblast growth factor receptor (FGFR) and tyrosine phosphatases have revealed Ig motifs in their extracellular domains. These observations may allude to signal transduction mechanisms of Ig superfamily receptors. Studies on NCAM and N-cadherins appear to suggest that these can act via tyrosine kinases, G-proteins and arachiodonate metabolites. The latter may act as second messengers and activate calcium channels. Cadherins also bind to intracellular proteins called catenins. Tyrosine phosphorylation of catenins or other molecules leads to the detachment of cell-cell contacts mediated by cadherins and results in disruption of epithelial integrity. Additionally, evidence has been provided that adhesion receptors may interact directly with the RTKs such as FGFR (Friesel & Maciag, 1995; Rosales e t al., 1995). The sphingolipid signallingpathway

The agonists that activate this pathway include TNF-a, interferon, IL-1 and vitamin D3. Ligand engaging with the receptor results in activation of a sphingomyelinase that cleaves sphingomyelin to release ceramide and phosphocholine. Ceramide stimulates protein kinases and protein phosphatases leading to activation of transcription factors such as c-jun and NF-KB. It has been implicated in negative regulation of cell growth and in apoptosis (Rosales e t a]., 1995; Divecha & Irvine, 1995). Other pathways

Signal transduction via cytokines involves a family of cytoplasmic tyrosine kinases (Janus kinases, J AKs). Two JAKs are required to activate each other by reciprocal transphosphorylation (Schindler, 1995). These can activate Ras signal pathway (Rosales e t al., 1995) or directly phosphorylate tyrosine in cytoplasmic proteins [termed STATs (signal transducers and activators of transcription)] which migrate to the nucleus and affect transcription of specific genes (Rosales e t al., 1995; Schindler, 1995). 3321

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Table 7. Some known adhesion molecule receptors of pathogenic microbes Familylmember

Natural ligand"

Integrin superfamily fl Integrins [very late activation antigen, VLA (CD29)] VLA-2: u2pl Col, Lm VLA-3: ~ 3 p l Fn, Lm, Col VLA-4: a4p1 Fn, VCAM-1, LFA-1, CR3 VLA-5: ct5/?1 Fn

VLA-6: ~ 6 p l avp1

Microbe/ligand

Echovirus 1 Yersinia invasin Yersinia invasin Yersinia invasin HIV-1 Tat protein (Neisseria meningitidis Opc protein) Shigella Jexneri Ipa proteins Yersinia invasin Yersinia invasin

Bergelson e t al. (1992) Isberg & Tran Van Nhieu (1994) Isberg & Tran Van Nhieu (1994) Isberg & Tran Van Nhieu (1994) Barillari et al. (1993) Virji e t al. (1994a)

ICAM-1/2/3

Histoplasma capsulatum

Wright e t al. (1989)

ICAM-1, C3bi, Fbg, Factor X

Leishmania gp63, LPG, LPS

Russell & Wright (1988)

Histoplasma capsulatum Legionella pnetlmophila Bordetella pertussis FHA Rhodococcus equi Escherichia coli type I fimbriae Histoplasma capsulatum, LPS

Wright e t al. (1989)

Leishmania LPG

Talamas-Rohana e t al. (1990)

Fbg, Fn, vWF, Vn, Tsp

Borrelia burgdorferi

Coburn et al. (1993)

Vn, vWF, Fbg, Tsp

Adenovirus penton base

Wickham e t al. (1993)

HIV-1 Tat protein Coxsackievirus A9 Mycobacteria avium-intracellulare Neisseria meningitidis Opc protein

Barillari e t al. (1993) Roivainen e t al. (1994) Rao e t al. (1993)

Adenovirus penton base

Wickham e t al. (1993)

Lm Fn

$? Integrins (leucams; LFA-1, CD18 group)

LFA-I: aLB;! (CDlla/CD18) CR3, Mac-1: am@

(CDllb/CD18, Mo-1)

CR4, P150,95 :

ICAM-1, Fbg

axB2

(CDllc/CD18, Leu M5)

B3 Integrins (cytoadhesins)

Platelet glycoprotein IIbIIIa : aIIbP3 (CD41ICD61) Vitronectin receptor (VNR) : avP3 (CD51)

p5 Integrins uvp5

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Reference

Vn

Watarai et al. (1996) Isberg & Tran Van Nhieu (1994) Isberg & Tran Van Nhieu (1994)

Bellinger-Kawahara & Horwitz (1990) Relman e t al. (1990) Hondalus e t al. (1993) Gbarah e t al. (1991) Wright e t al. (1989)

Virji e t al. (1994a)

Microbial utilization of human signalling molecules Table 1. (cont.) Familylmember

Natural ligand"

Immunoglobulin superfamily PVR ? CD4 Major histocompatibility complex (MHC) Class I1 ICAM-1 LFA-1, Mac-1

VCAM-1

VLA-4 VLA-4

NCAM (CD56)

Heparan sulphate NCAM

CEA (CD66)

CD66, selectins

Microbe/ligand

Reference

Poliovirus HIV-1,2 ; SIV envelope glycoprotein gp120

Mendelsohn et al. (1989) Harrison (1994)

Human Rhinovirus

Greve e t al. (1989)

(HRV) Coxsackievirus A (CAV) Plasmodium falciparum Plasmodium falciparum Encephalom yocarditis (EMC-D) virus Neisseria meningitidis and Exherichia coli K1 capsulesmolecular mimicry E. coli type 1 fimbriae Neisserial Opa proteins

Crowell & Tomko (1994) Pasloske & Howard (1994) Pasloske & Howard (1994) Huber (1994) Finne e t al. (1983, 1987)

Sauter e t al. (1993) M. Virji and others (unpublished)

Selectin family (LECCAM) ELAM-1 (E-selectin) Sialyl-Lewis", Sialyl-Lewis'

Plasmodium falciiparum

Pasloske & Howard (1994)

Cadherins LCAM LCAM

LCAM LCAM

Shigella ji'exneri Listeria monocytogenes

Sansonetti et al. (1994) Mengaud et al. (1996)

Other CD36 CD44 (HCAM, Hermes antigen)

Col, Tsp Hyaluronate, CD44, Fn, Lm, Col

Plasmodium falciparum Poliovirus

Pasloske & Howard (1994) Shepley & Racaniello (1994)

* Abbreviations : CEA, carcinoembryonic

antigen; Col, collagen; Lm, laminin; Fn, fibronectin; Fbg, fibrinogen ; Vn, vitronectin ; vWF, von Willebrand factor ; Tsp, thrombospondin.

Signalling pathways manipulated by microorganisms

Distinct modes of cellular invasion via induced cytoskeletal events

host proteins and changes in intracellular second messengers. Actin polymerization is a frequent requirement since cytochalasins inhibit invasion. Several enteric pathogens (Salmonella, Shigella) induce pronounced cytoskeletal rearrangements and induce invasion via cellular ' triggering'. Others (Yersinia) induce more localized actin polymerization and this more quiescent invasion is described as 'zippering'. A third class of pathogens, exemplified by Trypanosoma c r q i , do not require actin polymerization and their invasion may be enhanced in the presence of cytochalasin D (Rosenshine & Finlay, 1993; Bliska e t al., 1993; Rodriguez e t al., 1995).

Many enteropathogenic bacteria (some strains of Escherichia coli, Salmonella, Shigella, Yersinia, Listeria) as well as other mucosal pathogens (Haemophiltls inJEzlenXae type b and the pathogenic Neisseriae: N. mening;t;d;s and N . gonorrhoeae) induce endocytosis in host epithelial or endothelial cells (parasite-directed phagocytosis). Signal transduction events in many cases, although utilizing different receptors, are linked to cytoskeletal rearrangement, often involve tyrosine phosphorylation of

Salmonella and Shigella share some similarities in their mechanisms of inducing extensive ruffling of their target cell membrane. Both secrete multiple protein complexes to trigger their uptake (Menard e t al., 1996). Several of the induced signal transduction events in the host cell are, however, distinct. In the case of Salmonella, a marked increase in intracellular calcium occurs and is necessary for invasion (Bliska e t al., 1993). Cellular entry in some cells may involve EGF receptor and tyrosine

Numerous studies have investigated signalling systems utilized by different micro-organisms. Rather than provide an exhaustive list, a small number of recent studies primarily on bacteria are chosen to illustrate a range of signalling pathways used and resulting distinctive outcomes of microbial signalling.

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Fig. 2. Schematic diagram of the representative structures of the major families of adhesion receptors. lntegrins are heterodimers that contain non-covalently associated IX and p subunits. a chains have extracellular Ca2+-bindingdomains important in ligand binding. Cytoplasmic tails of some p integrins interact with focal contact proteins and via these to actin cytoskeleton. Cadherins have five extracellular domains (ECl-EC5) which are heavily glycosylated. The N-terminal domain is involved in homotypic adhesion, the rest of the domains contain Ca*+-bindingproperties. Cadherins interact with the cytoskeleton via catenin proteins. The immunoglobulin (lg) superfamily (SF) is a large family of related molecules which contain various numbers of extracellular lg domains and varying lengths of cytoplasmic domains. They are involved in mediating cell-cell interactions by binding to other lg SF members as well as to integrins (see Table 2). Selectins contain three types of domains. The terminal calcium-dependent (C-type) lectin domain, epidermal growth factor (EGF) domain, and varying numbers of domains found in complement regulatory proteins (CRP). Selectins bind to carbohydrate ligands, particularly sialyl-Lewisxand are involved in endothelial leucocyte interactions.

phosphorylation (Bliska & Falkow, 1993). More than one signal transduction pathways of the host may be used via yet largely undefined Salmonella ligand-host-receptor interactions (Bliska e t al., 1993). Shigella-induced plasma membrane activity is triggered by Ipa antigens. Involvement of the integrin/cytoskeleton pathway including pp125FAK, has been implicated recently in Shigellajexneri interactions with mammalian cells (Watarai e t al., 1996). Endocytosis was mediated by released bacterial proteins (IpaB, IpaC and IpaD) which bound to integrin a5pl in a manner similar to the tissue form of fibronectin. At the site of attachment of bacteria, the integrin a5pl and polymerized actin were co-localized. In addition, protein tyrosine phosphorylation of both ~ ~ 1 and2paxillin 5 ~occurred ~ ~on infection of host cells with Sh. jexneri. Only soluble proteins were found to bind the integrin ; how this leads to bacterial internalization remains to be shown (Watarai e t al., 1996). Dehio et al. (1995) demonstrated that cortactin and pp60c-src (a cortactin tyrosine kinase) are enriched in membrane ruffles

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of host cells that engulf bacteria, They showed tyrosine phosphorylation of cortactin, a cytoskeleton-associated protein. These and other proteins recruited to the sites of Jhigella entry into epithelial cells are components of focal adhesion plaques. In addition, Rho-specific inhibition was shown to impair Shigella entry into host cells and, unlike Salmonella, Ca2+ influx is not required for Shigella entry (Menard e t al., 1996).

Enteropathogenic E. coli (EPEC) are associated with characteristic attaching and effacing (AE) lesions on adhesion to host epithelial cells. A E lesions are characterized by disruption of microvilli at the site of attachment and rearrangement of host cytoskeleton beneath adherent bacteria into a pedestal-like structure containing cytoskeletal elements (actin filaments, a-actinin, ezrin, talin) and tyrosine-phosphorylated proteins. Mutants that do not cause AE lesions are non-invasive (Rosenshine & Finlay, 1993). A membrane-associated protein intimin (product of eaeA) is required for intimate association of bacteria with the host cell and for rearrangement of host-

Microbial utilization of human signalling molecules

Fig. 3. Scanning and transmission electron microscopy showing bacterial uptake by human endothelial cells induced on infection with H. influenzae (a, b) and N. meningitidis (c, d). Cellular processes gradually form around H. influenzae [arrows in (a) and (b)] to envelop bacteria within cellular membrane by a coiling mechanism (b). Relatively greater cytoplasmic membrane activity (arrow, c) can be seen during cellular invasion by N. meningitidis. Arrowheads in (c) show bacteria located under host plasmalemma. (d) Detail of scanning electron micrograph showing the process of phagocytic uptake of a meningococcus (MC) by an endothelial cell. Coiling of membrane protrusions have not been observed in the this case. Both organisms enter host cells within membrane-bound vacuoles. Bars in (a), (b) and (d) 0.5 pm; bar in (c), 1 pm. Electron micrographs were produced with the help of Dr D. J. P. Ferguson.

cell cytoskeleton. However, signal transduction is generated by soluble factors (products of eaeB, cfm and class 5 loci) and the product of eaeB appears to be essential for activation of signalling pathways (Kenny & Finlay, 1995). Signal transduction events shown to occur on EPEC invasion include tyrosine phosphorylation of a 90 kDa protein of the host and elevation of intracellular levels of second messengers (IP, and Ca2+; Kenny & Finlay, 1995; Rosenshine & Finlay, 1993). Studies on the mucosal pathogens H. infEzlenpe and N. meningitidis, which cause bacteraemia, also show differences in their interactions with epithelial and endothelial cells. Endothelial invasion by H. injnenpae type b does not appear to be preceded by extensive plasma membrane activity in the host, and uptake involves processes arising in close vicinity of bacteria. The bacteria are surrounded in a manner that is reminiscent of coiling phagocytosis seen in professional phagocytes (Virji e t al., 1991b ; Fig. 3). Invasion by N. meningitidis, on the other hand, appears to induce relatively greater activity of the host cytoplasmic membrane and numerous processes can be seen by scanning electron microscopy (Fig. 3). The mechanisms of endothelial cell activation by these organisms are clearly distinct and probably utilize different signalling pathways, nevertheless involving the cytoskeleton, as evidenced by inhibition of invasion in the presence of cytochalasin D (Virji e t al., 1991b, 1994a). In N.meningitidis, the outer-membrane protein (OMP) Opc mediates interactions with host-cell integrins by a bridging mechanism utilizing RGD-bearing serum proteins, and leads to cellular invasion (Virji e t al., 1994a). Localized actin polymerization and zippering has been described for the mechanism of entry of Listeria monocytogenes and resembles that for Yersinia. In the latter case, the interactions with host cells occur via Pl integrins (see below). In contrast, L. monoytogenes uses E-cadherin receptors on host cells targeted via its 80 kDa surface

protein, internalin (Mengaud e t al., 1996). Targeting of distinct receptors by the two pathogens probably stimulates different signalling pathways, although, some events, such as tyrosine phosphorylation in host cells, appear to occur in both cases (Mengaud e t al., 1996). Invasion of host cells by the obligate intracellular protozoan parasite, Typanosoma m p i , the cause of Chaga's disease, occurs by an unusual mechanism and appears to be facilitated by disruption of host-cell actin microfilaments. Invasion can be enhanced by cytochalasin D treatment. In addition, pertussis toxin which acts on Gproteins (see below), as well as Ca2+ channel-blockers, inhibit Typ. c r q i entry (Rodriguez e t al., 1995). A recent study has implicated signalling pathways involving the TGF-P receptor for Typ. c r q i invasion of epithelial cells (Ming e t al., 1995). Second messengers

Increases in second messengers such as Ca2+ and IP, in host cells have been shown using peptides derived from Typ. mqi, and resulted in rearrangement of host-cell microfilaments (Rodriguez e t al., 1995). However, increase in inositol phosphate on invasion of cultured epithelial cells with Helicobacterpjlori, was not associated with redistribution of cytoskeletal proteins and was independent of bacterial adherence. This suggested a role for a soluble bacterial factor in stimulating this signalling pathway (Pucciarelli e t al., 1995). Studies on Salmonella, EPEC and verocytotoxin-producing E. coli (VTEC) have also implicated stimulation of second messengers (Kenny & Finlay, 1995 ;Ismaili e t al., 1995 ; Rosenshine & Finlay, 1993). EPEC stimulates host tyrosine kinases and its stimulation of IP, is dependent on host phosphorylation. In contrast, cytosolic IP, and Ca2+elevation that occur on VTEC infection are independent of phosphorylation of host-cell proteins (Ismaili e t a]., 1995).

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G-proteins. The Gram-positive bacterium Streptococctls pnetlmaniae fails to enter resting human cells, but invasion of host cells occur, during inflammatory activation. This involves G-protein-coupled platelet-activating-factor receptor (PAF-R) expressed in activated cells. Bacterial cellwall phosphorylcholine appears to mimic the ligand structure and interacts with PAF-R which is rapidly internalized after interaction with the ligand. However, normal signal transduction (activation of phospholipase C) did not occur on bacterial interaction with PAF-R (Cundell e t al., 1995).

Some bacterial toxins enter eukaryotic cells and act directly on G-proteins. Cholera toxin (CT) and pertussis toxin (PT) ADP-ribosylate heterotrimeric G-proteins (Ga) of the subfamilies G, and Gi. These modifications result in modulation of regulatory functions of the Gproteins and of pathways that include second messengers (Nurnberg e t al., 1995). Several members of the small G-proteins (RhoA, RhoB and RhoC but not Rac or Cdc42) act as substrates for ribosylation by Clostridiztm bottllintlm C3 toxin. Recently, a novel mechanism by which intracellularly acting bacterial toxins act on small G-proteins has been reported. Clostriditlm dzficile toxins A and B, which enter eukaryotic cells by receptor mediated endocytosis, apparently monoglucosylate Rho, Rac and Cdc42. Other members of the Ras superfamily (Ras, Rab and Arf) are not affected. Since Rho proteins are involved in regulation of the actin cytoskeleton and involve second messengers, the effect of these toxins is characterized by destruction of cytoskeleton and morphological changes in the host cell (Aktories & Just, 1995). Protein tyrosine phosphatases

YopH protein of Yersinia is a protein tyrosine phosphatase (PTPase) that appears to break down signal-transduction pathways in many cell types, including those of the immune system, enabling bacteria to avoid phagocytosis. PTPases are also encoded by viruses, for example, the VH1 gene in vaccinia virus encodes a phosphatase that is able to dephosphorylate phosphorylated serine, threonine and tyrosine (Guan & Dixon, 1993). Endotoxin and signal transduction

LPS-mediated signal transduction mechanisms that result in the synthesis and release of inflammation mediators are not clearly defined at present. LPS clearance may involve signal-transduction-independent internalization. Two effector molecules, LBP and CD14, play important roles in LPS interactions with host cells, but LPS internalization may occur independently of these molecules. Diverging pathways of internalization and signal transduction have been examined recently (Gregner e t al., 1995), and it is postulated that additional transducing molecules may activate signal-transduction pathways of the host. Membrane CD14 (mCD14 present on phagocytic cells is involved in surface location of LPS) is a glycosylphosphatidylinositol (GP1)-anchored molecule. In monocytes, cross-linking of mCD14 leads to the activation

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of Src family kinases (Seed, 1995). Epithelial and endothelial cells do not have mCD14. In these cells, LPS from certain Gram-negative bacteria can exert cytopathic effects. In vitro studies show that soluble serum CD14 (sCD14) is apparently required for toxic damage of bovine endothelial cells mediated by H. inftlenxae type b LPS (Patrick e t al., 1992) or of human endothelial cells mediated by N. meningitidis LPS (K. L. R. Dunn & M. Virji, unpublished observations). An interesting aspect of the pathogenesis of these Gram-negative organisms in humans is that LPS-mediated toxicity for vascular endothelial cells is readily observed in the case of N. meningitidis,but not with H. injzmxae type b. For example, purpuric skin lesions that arise as a result of endothelial necrosis and intravascular coagulation, are common in disseminated N.meningitidis infection, but rare during septicaemia caused by H. infzteenxae type b. Similar observations were made during in vitro studies; N. meningitidis but not H. inftlenrae type b cause LPSdependent toxic damage to cultured human umbilical vein endothelial cells (Virji etal., 1991a, b). How N. meningitidis LPSICD14 interactions with human endothelial cells differ from H. inftlenqae LPS/CD14 interactions to deliver signals that are lethal or benign is not known. One explanation lies in differences in the lipid A structures of the LPSs from these bacteria (Dunn e t al., 1995). Lipid A has been shown to mimic ceramide structure and activate ceramide-activated protein kinases (CAPKs) (Joseph e t al., 1994). This signalling pathway normally results in negative-regulation of cell growth and apoptosis (see above). Whether small differences in acylation of lipid A affect interactions with the hypothetical CAPK-activating intermediate remains to be shown. In addition to CD14-mediated mechanisms, members of the integrin family, CR3 (am@) and CR4 (axP2) may also interact with LPS. Signal transduction via CR4 receptors (as assessed by induction of NF-KB translocation) has been reported recently (Ingalls & Golenbock, 1995).

Microbial interactions with adhesion molecules Adhesion molecules comprise a number of different families of molecules with a common overall structure which consists of a surface-exposed ligand-binding domain, a membrane-spanning region and a cytoplasmic tail. Salient structural features and a brief summary of functional aspects of distinct families are shown in Fig. 2. Specific targeting of different adhesion molecules by several micro-organisms is summarized in Table 1.

Opportunism in microbial strategy of host receptor subversion: integrins as common targets Within the repertoire of cell-surface molecules and of adhesion receptors, integrins are a common target for pathogens, perhaps due to their ubiquitous presence on all cell types (Albelda & Buck, 1990). Also, engaging with these receptors provides the best strategy for manipulation of host-cell machinery for entry into cells and

Microbial utilization of human signalling molecules Table 2. Arginine-glycine-aspartic acid (RGD) in integrin targeting of microbes Microbe Adenovirus Picornaviruses : Foot-and-mouth disease virus (FMDV) Echovirus-22 Coxsackievirus-A9 (CAV-9) HIV Bordetella pertassis Borrelia bargdorferi Neisseria meningitidis Streptomyces avidinii Treponema pallidam, Treponema denticola, Tr_ypanosomac r q i Leishmania

Candida albicans

Mechanism RGD sequence in penton base RGD in capsid

RGD sequence in Tat protein RGD sequence in FHA RGD sequence in pertactin RGD-dependent adhesion Utilizes RGD-containing proteins via Opc RGD-like sequence (RYD) in streptavidin Bind RGD of fibronectin

Target receptor avp3, avp5, (a5pl)

Wickham e t al. (1993)

?

Mason e t al. (1994)

CR3 ( a m p ) Epithelial integrins aIIbp3 avp3 (a5pl)

Hyypia et al. (1992) Roivainen e t al. (1994); Chang et al. (1992) Barillari et al. (1993); Brake e t al. (1990) Sandros & Tuomanen (1993) Leininger et al. (1992) Coburn et al. (1993) Virji e t al. (1994a)

a5pl (aIIbB3)

Alon e t al. (1993a, b)

Cell-associated Fn

Thomas e t al. (1985) Dawson & Ellen (1990) Ouaissi et al. (1986) Russell & Wright (1988) ; Soteriadou e t al. (1992) Hostetter (1994)

RGD-like sequence (SRYD) in gp63

CR3 ( a m p )

Binds RGD in C3bi

Cell-associated C3bi

opening up a reservoir of nutrition. In addition, entry into non-phagocytic cells may provide shelter from humoral as well as cellular immune mechanisms. Several distinct opportunistic mechanisms by which microbes subvert integrin receptors and associated signalling pathways may be defined, with molecular mimicry being the most common strategy. Ligand mimicry

Direct interactions of micro-organisms with eukaryotic receptors often occur at the normal host ligand recognition site and may utilize the ligand recognition motif, for example the sequence RGD (Arg-Gly-Asp), a recognition sequence on many ligands of integrins. In addition, as is the case with natural integrin ligands, receptor specificity in microbes may be modified by RGD flanking sequences. Within the RGD motif, aspartic acid is important for recognition by integrins (Ruoslahti & Pierschbacher, 1987) and some integrins that do not recognize RGD sequence nevertheless require an acidic amino acid (Asp or Glu) for binding to ligands (Staunton e t al., 1990; Isberg & Tran Van Nhieu, 1994). In some bacteria, e.g. Yersinia which binds multiple 81 integrins (the majority do not recognize RGD), aspartic acid has been shown to be essential in adhesion (Isberg & Tran Van Nhieu, 1994). Since some pl, /32 as well as /33//35 integrins may bind RGD, expression of this sequence on microbial ligands imparts the potential to interact with multiple in tegrins.

Reference

Microbes that possess RGD or RGD-like sequences. Two of

the virulence-associated proteins of Bordetella pertzlssis (filamentous haemagglutinin and pertactin), contain the RGD sequence which is involved host-cell interactions (see below). Several viruses contain the RGD sequence (Table 2). Other microbes contain RGD-like sequences. For example, streptavidin (a tetrameric analogue of avidin) which is secreted by Streptomyes avidinii, has considerable homology to the RGD-containing integrinbinding domain of fibronectin and may bind to cells via the motif RYDS. It interacts with the specific fibronectin receptor, a5/31. Its specificity for a5pl may be acquired from the RYD flanking sequences (Alon e t al., 1993a, b). Leishmania1 surface-located protease, gp63, is a fibronectin-like molecule and reacts with anti-fibronectin antibodies (Rizvi e t al., 1988). Gp63 does not contain an RGD sequence, but as in streptavidin, a related sequence (SRYD) replaces RGD functionally in recognizing integrins on host macrophages (Soteriadou e t al., 1992). The major receptor for gp63 has been shown to be CR3 (Russell & Wright, 1988). Pseudoligand mimicry, sandwich mechanism:microbes that bind RGDRGD-containing proteins or other adhesion molecules. Microbial adhesion to integrins may also occur

by a bridging or sandwich mechanism involving natural ligands of integrin receptors such as extracellular matrix proteins. In the case of the leucocyte integrin CR3 (amp), the coating of microbes by the ligand, complement component C3bi, renders the microbe resistant to in~

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tracellular killing (see below). Not all sandwich adhesion results in ligand/microbe internalization ; for example, coating of the bacterial surface with fibronectin may result, in some cases, in extracellular location only (Isbcrg, 1991). Several Gram-positive bacteria (staphylococci, streptococci, lactobacilli) interact with fibronectin, collagen, laminin, vitronectin, thrombospondin, elastin, bone sialoprotein and fibrinogen (Patti e t al., 1994). In addition, many Gram-negative bacterial fimbriae adhere to ECM components via pro tein-pro tein or protein-carbo hydrate interactions. Type 1 fimbriae of E. coli and Salmonella have been shown to bind mannoside chains of matrix proteins (Patti e t al., 1994). Type I fimbriae of E. coli also interact with mannosyl residues on CD11/CD18 (ax@) integrins of phagocytes (Gbarah e t al., 1991) and of NCA (CD66) antigens belonging to the Ig superfamily (Sauter e t al., 1993). YadA (Yopl) is a prominent plasmid-encoded OMP and forms multimeric fibrillae on the surface of enteropathogenic Yersiniae. YadA binds to several ECM components and utilizes distinct mechanisms of adhesion to these molecules (Patti e t al., 1994). The spirochaetes Treponema pallidtlm (causing syphilis) and Trep. denticola (an oral commensal) adhere to fibronectin via its RGDS sequence. Such integrin-like interactions with fibronectin may allow them to adhere to host cells via surface-located ligands. It is suggested that treponemes bind to one of the two RGD sequences (in fibronectin dimer) not occupied by host-cell fibronectin receptor (Thomas e t al., 1985; Dawson & Ellen, 1990). Typ. crtlxi may also use a similar mechanism for host-cell adhesion (Ouaissi e t al., 1986). Fibronectin is apparently a common target utilized by many bacteria, as well as by yeasts. Whilst adhesion via receptor ligands may not always result in microbial uptake (Isberg, 1991), affinity of ligand-receptor binding and receptor density are among the factors that determine the fate of the adherent microbe (see below).

Receptor mimicry :microbial mimicry of the integrin structure

interact also with RGD on the Tat protein of human immunodeficiency virus (HIV) (Denis, 1994). It is suggested that the interaction between Tat, present in body fluids of HIV-infected patients, and this opportunistic pathogen, may contribute to the acute susceptibility of AIDS patients to mycobacterial infection.

Candida, a normal commensal yeast of the intestinal tract, becomes pathogenic in immunocompromised patients, neonates and diabetics. The presence of an antigenically, structurally and functionally similar protein to the a subunit of the CD18 Q32) integrins CR3 (am) and CR4 (ax) has been demonstrated in C. albicans (Hostetter, 1994). The yeasts adhere to host epithelial cells via RGDdependent mechanisms and the ligands on the host epithelial cells are reported to be surface-located integrin ligands, C3bi for C. albicans and fibronectin for Candida tropicalis. Two important differences between Candida integrins and mammalian integrins are that the /I2 subunit has not been demonstrated in Candida and binding of the yeast integrin to C3bi is cation-independent. However, Candida may express P,-like integrins and may bind to fibronectin via this molecule (Hostetter, 1994; Klotz e t al., 1992). Complex mimicry Multiple mimicry in Bonletella. The most striking example

of the mimicry of host cellular recognition molecules is seen in Bordetella pertassis filamentous haemagglutinin, FHA (Sandros & Tuomanen, 1993). This protein contains several distinct sites involved in interactions with glycoconjugates, heparin and CR3 (an RGD motif). It also contains sequence similarities to endothelial ligands, to factor X and to C3bi, and represents mimicry of multiple ligands recognized by the leucocyte integrin CR3 ( a m p ) (Sandros & Tuomanen, 1993). In addition to FHA, Bard. pertussis expresses other virulence-related proteins which include pertactin and pertussis toxin. Pertactin also contains an RGD sequence and is implicated in adhesion to epithelial cell integrins. It is suggested that flanking sequences of RGD in FHA and pertactin determine the receptor- and tissue-specificity of interactions of the microbe via R G D (Leininger e t al., 1992). Pertussis toxin subunits S2 and S3 share similarities with selectins. Other mechanisms

Some micro-organisms express proteins that resemble integrins structurally, and antibodies raised against mammalian integrins specifically react with microbial integrinlike structures.

In contrast to the above examples, echovirus does not appear to use mimicry as a mechanism of interacting with the integrin a2pl. Interactions of the virus and the integrin ligands (extracellular matrix proteins) occur by distinct mechanisms, and antibodies that block binding of the virus do not inhibit integrin binding to collagen or laminin. This interaction also results in viral internalization (Bergelson e t al., 1992).

Mycobacteritlm avitlm-intracelltllare is ubiquitous in the environment but is able to act as an intracellular pathogen. Disease due to this organism is rare and usually occurs in immunocompromised hosts, and is responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Integrin pl-reactive antibodies crossreact with bacterial extracts. Also, mycobacteria bind avidly to laminin, collagen I and fibronectin via a plintegrin-like structure and the interactions are cationdependent (Rao e t al., 1992). Mycobacterial integrin can

Adhesion vs integrin-mediated invasion via integrins Yersinia, with its multiple virulence mechanisms, provides an interesting example of complex adhesion/invasion -

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Microbial utilization of human signalling molecules events manipulated via encounter with cellular integrins. Chromosomally coded invasin interacts with several pl integrins and this results in bacterial internalization (Isberg, 1991). The factors that determine uptake of Yersiniae via /?l integrins (which primarily mediate adherence to their ECM ligands) were investigated by Tran Van Nhieu & Isberg (1993). One of the mechanisms leading to invasion may be multiple occupancy on the receptor [as described for Leishmania interactions with CR3 via ligands gp63 and lipophosphoglycan (LPG), which results in internalization ; Talamas-Rohana e t al., 19901. This does not occur in the case of Yersinia invasinlpl interactions since a small region of invasin mediates both binding and invasion (Isberg, 1991). However, unlike the natural ligand, invasin engages with pl integrins via high-affinity interactions. This may allow efficient competition with other ligands and result in ‘zippering’ - a process by which the host cell forms a series of contacts over the surface of the micro-organism, leading to uptake (Tran Van Nhieu & Isberg, 1993; Isberg, 1991). The site of attachment to integrins appears to be unimportant ;high-affinity interactions to any site on a5/?l integrins resulted in internalization. Additionally, receptor clustering achieved by cross-linking of ligands was a requirement for signal leading to uptake (Tran Van Nhieu & Isberg, 1993). It is also suggested that high receptor density may achieve the same end. Thus, upregulation of integrin receptors during many viral and other (e.g. malarial) diseases may help augment widespread invasion. In the absence of the invasin protein, YadA can mediate interactions and invasion also via p1 integrins, and this may occur via a bridging molecule, such as fibronectin, that binds to YadA. Interestingly, internalization by invasion proteins of Yersiniae may be inhibited by other plasmid-encoded proteins, Yops, which appear to act on host cytoskeletal proteins to dephosphorylate. Thus Yersiniae can bind integrins which can lead to cellular invasion, but also have the capacity to modulate subsequent events by interfering with signal transduction via accessory proteins (Bliska e t al., 1993).

The immunoglobulinsuperfamily The Ig superfamily includes a diverse array of receptors defined by the presence of one or more copies of the Ig fold, a compact structure of 60-100 amino acids arranged in facing /?-sheet structures. The receptors are involved in cell-adhesion events, including cell-cell interactions and antigen recognition. Besides being utilized by several bacteria and parasites, the members of the Ig family are targets of several viruses that belong, in the main, to the picornaviridiae (Crowell & Tomko, 1994) (Table 1). Several picornaviruses possess a ‘ canyon’ structure in their capsid which is implicated in adhesion to particular Ig folds of the receptor. Poliovirus receptor (PVR)

Studies on many viral systems particularly implicate cellular receptors in viral tissue tropism. Poliovirus

infection of a limited number of host tissues exemplifies this. The receptor for the virus (PVR) has been shown to be structurally related to members of the Ig superfamily (Mendelsohn e t al., 1989), its function is not known. Although PVR is required for poliovirus infection, tissue tropism of the virus may depend on another adhesion molecule, the lymphocyte homing receptor (CD44, Shepley & Racaniello, 1994). Tissue specific glycosylation of the receptor isoforms may also influence PVR interactions with the virus and determine its tissue tropism (Bernhardt e t al., 1994). NCAM

Molecular mimicry of the adhesion molecule NCAM by certain microbes may involve an additional strategy, distinct from those discussed above. NCAM is a heavily sialylated molecule and neonatal forms of NCAM contain more sialic acid groups than adult NCAM. Bacteria causing meningitis in neonates, such as E. coli K1 and N. meningitidis group B, contain a2,8-linked polysialic acid capsules that resemble structures found in neonates but not in adults. Antibodies to meningococcal capsule have been shown to react specifically with embryonic but not adult NCAM. The success of these microbes in brain tissue colonization may be attributed to their ability to escape host immune detection by mimicking surface glycans on adhesion molecules of the target tissue (Finne e t al., 1983, 1987; Rougon e t al., 1986). CD66 (CEA)

The carcinoembryonic antigen (CEA) family (also known as CD66) includes clinically important tumour markers, such as the CEA, which is up-regulated in epithelial carcinomas. The CEA family also includes cross-reacting antigens, e.g. non-specific cross-reacting antigens (NCAs), biliary glycoproteins (BGPs) and pregnancyspecific glycoproteins (PSGs). The members of the CEA family share antigenic determinants and show high levels of similarity in amino acid sequences (Watt e t al., 1994). The CEA family are targeted by E. coli type 1 fimbriae (Sauter e t al., 1993) as well as by Opa proteins of both N. meningitidis and N . gonorrhoeae (M. Virji and others, unpublished, see below). Multiple receptors of Plasmodium falciparum

Pathology in malaria is associated with cytoadherence of paracytosed erythrocytes. In vitro studies have shown that P. falcz$arum-infected erythrocytes interact with several molecules on endothelial cells, including thrombospondin, CD36, ICAM-1, VCAM-1 and ELAM-1, and also adhere to human monocytes and platelets via CD36 (Berendt eta]., 1989; Pasloske & Howard, 1994). Parasite clones vary in adherence to these receptors, but at least some clones have the potential to interact strongly with multiple receptors (Pasloske & Howard, 1994).

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The selectins E-, P- and L-selectins are involved in inflammatory responses and exhibit lectin-like activity used in functional mimicry by some microbes. The pertussis toxin (PT) produced by Bord. pertzlssis is a hexameric protein with carbohydrate-binding property and recognizes lactosylceramide and gangliosides from epithelial-cell cilia and macrophages. The carbohydrate-recognition region of PT is strikingly similar to the region 15-46 of E- and Pselectins. Moreover, PT subunits S2 and S3 with the carbohydrate-binding domains inhibit neutrophil adhesion to purified selectins, leucocyte adhesion to endothelial cells, and are anti-inflammatory in an animal model for meningitis (Sandros & Tuomanen, 1993). In vitro studies suggest that PT subunits S2 and S3 upregulate CR3 in macrophages, in a manner similar to that observed with selectins. CR3 integrin is also the receptor for Bord. pertzlssis FHA. Thus, P T and FHA function as cooperative adhesins (Sandros & Tuomanen, 1993).

The cadherins The calcium-dependent cadherin adhesion molecules involved in intercellular interactions are utilized by Sb. flexneri and L. monoytogenes. Sansonetti e t al. (1994) have shown that intercellular spread of Sh. flexneri requires the expression of cadherins that maintain the intercellular junctional integrity within intact epithelium. Cadherin expression appears to be required for efficient bacterial anchorage to the internal face of the cytoplasmic membrane of infected cells. In addition, it is required for homotypic interactions between the surface of cell protrusions (containing bacteria) and that of an adjacent cell to facilitate internalization by that cell (Sansonetti e t al., 1994; Menard e t al., 1996). L. monogtogenes surface-located protein, internalin, has been shown to target E-cadherin on epithelial cells for cellular entry (Mengaud e t al., 1996).

Adhesion receptor modulation in pathogenesis Borrelia burgdorferi up-regulates adhesion molecules such as E-selectin, P-selectin, ICAM-1 and VCAM-1 on mouse endothelial cells in vitro, and these may play an important role in the pathogenesis of Borrelia infections (Boggemeyer e t al,, 1994). Such increased expression may conceivably render host cells more susceptible to invasion by other pathogens utilizing these receptors, since receptor density may play a role in microbial location in or out of the host cell (discussed above). Certain microbes appear to induce their own receptors by supplementary mechanisms. Bord. pertzlssis appears to up-regulate CR3 via FHA interactions involving leucocyte signal-transduction complex comprising a /I3 integrin and CD47 (Ishibashi e t al., 1994), as well as via selectin-like function of P T (Sandros & Tuomanen, 1993). In malarial infection, upregulation of receptors has also been observed. Brain endothelium from patients dying of malaria expressed 3330

several of the implicated malarial receptors (CD36, ICAM1, ELAM-1 and VCAM-1; up-regulated or induced), whereas brain tissue from non-infected patients did not show up-regulation of these adhesion molecules (Pasloske & Howard, 1994). The increased expression of some of these receptors may result from increased levels of TNFa observed in malaria patients (Pasloske & Howard, 1994). Respiratory syncytial virus down-regulates adhesion receptors such as LFA-1 and ICAM-1 on mononuclear cells (Salkind et al., 1991). In contrast, parainfluenza virus type 2 up-regulates the expression of ICAM-1 and other receptors on human tracheal epithelial cells (Tosi e t al., 1992), so that these cells are able to bind increased numbers of neutrophils. Modulation of several adhesion molecules has been demonstrated in HIV disease and involves T-cells as well as phagocytic cells (Weeks e t al., 1991). N, meningitidis interactions with human cells an example of the role of multiple adhesive

-

ligands and molecular mimicry in pathogenesis

Interactions via the OMP Opc

N . meningitidis resides in the nasopharynx of its human host and further sequestration occurs from this primary and specific site of colonization. Factors necessary for colonization and for epithelial invasion that lead to serious pathogenic conditions remain to be described fully. Amongst the virulence factors elaborated by the organism are pili (fimbriae), filamentous multimeric protein structures, and OMPs that include the proteins Opa and Opc. Opc, a basic 28 kDa protein, appears to have the capacity to interact with multiple extracellular matrix components and serum proteins (Virji e t al., 1994b), giving the organism the potential to interact with several different integrins by bridging via their respective ligands. This could be an effective strategy for cellular invasion and for adhesion to substrata of damaged mucosa, as well as for penetrating deeper tissues after cellular invasion. Indeed, Opc mediates cellular invasion of cultured epithelial and endothelial cells (Virji e t al., 1992). These interactions can be inhibited by monoclonal antibodies against Opc which appears to be the major requirement on bacteria for this interaction. However, cloned Opc does not confer invasive properties on E. coli, even though the protein is surface-expressed and is immunologically similar to that on N. meningitidis. This suggests that additional bacterial factors may be required in hostcell interactions mediated by Opc. It is also possible that the level of Opc expressed by E. coli is not optimum since efficient interactions of N. meningitidisvia Opc require the protein to be expressed at a high density on the bacterial surface (Virji e t al., 1995). RGD-dependent cellular invasion mediated by Opc

Studies using cultured human endothelial cells have shown that interactions of Opc-expressing N. meningitidis

Microbial utilization of human signalling molecules

Fig. 4. lmmunofluorescence micrograph showing RGD-dependent interactions of N. meningitidis with the apical avp3 integrins of confluent endothelial cells in culture. (a) Control monolayers infected with Opcexpressing meningococci. Bacteria were stained with monoclonal antibody SM82 directed against lipopolysaccharide and rhodamine-conjugated second antibody. (b) RGD-mediated inhibition of interactions of Opc-expressing N. meningitidis (RGD present in serum-supplemented infection medium). RGE-containing peptides were not inhibitory (data not shown). (c) Inhibition of bacterial interactions in the presence of antibody (LM609) against vitronectin receptor (Virji et al., 1994a).

Fig. 5. Mechanism of meningococcal entry into human endothelial cells.

with the apical surface of polarized host cells require serum-derived factors. These factors appear to be RGDcontaining proteins, and RGDS, but not RGES, peptides inhibit bacterial invasion of human endothelial cells. Moreover, antibodies against vitronectin receptor (arvp3, VNR) and fibronectin receptor (a5p1, FNR) inhibit adherence and invasion. VNR appears to be the major receptor involved in serum-dependent apical interactions of N. meningitidis, but FNR may also be involved (Virji e t al., 1994a) (Figs 4, 5). Although pseudoligand mimicry appears to be utilized by Opc-expressing N. meningitidis, it is possible that for signalling further factors are involved in the interactions via the VNR. CR3 has been shown to interact simultaneously with C3bi-coated particles and with microbial glycolipids at distinct sites. Indeed, VNR also exhibits binding sites for ganglioside GD2 (Cheresh e t al., 1987). Gangliosides and LPS share structural similarities in that both are amphipathic with a strongly anionic hydrophilic group and some manner of LPS interaction with vitronectin receptor may be an additional factor required. This is at present a speculation and there is no evidence to support this hypothesis.

Two other lines of evidence support the proposed involvement of integrins, especially of VNR. Capillary endothelial cells derived from human foreskin were shown to be lacking reactivity with monoclonal antibodies against VNR, and indeed are one of the few cells not to express av (Albelda & Buck, 1990). These cells failed to support Opc-mediated adhesion or invasion. Also, treatment of human umbilical vein endothelial cells with certain cytokines that modulate integrin expression, also affects bacterial interactions mediated by Opc (unpublished observations). An interesting feature of the Opc interaction is the requirement for host cytoskeletal function. Attempts to inhibit host-cell invasion by the use of cytochalasin D resulted in inhibition not only of invasion but also of total cell association. This observation is in contrast to cell adhesion mediated by N. meningitidis Opa proteins which increases in the presence of cytochalasin D, although invasion is inhibited (Virji e t al., 1994a). It has been suggested that efficient bacterial internalization requires direct adherence of bacteria to host receptors (Isberg, 1991), and fibronectin-dependent sandwich interactions do not mediate uptake unless receptors are disengaged 3331

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from matrix binding. Affinity of ligand-receptor interactions may also determine attachment/uptake via the same receptor (Isberg, 1991). In the case of N. meningitidiis, serum-protein-dependent Opc interactions results in invasion, suggesting that these are high-affinity interactions with the receptors.

Pilus-dependent interactions and up-regulationof invasion of human endothelial cells mediated by Opc

Meningococcal strains isolated from blood or CNS are invariably capsulate and many, but not all nasopharyngeal isolates also possess capsule. Capsular polysaccharide which coats the outer membrane of the bacteria is protective against host’s immune mechanisms ; for example, it imparts resistance to phagocytosis (McNeil e t al., 1994). Most capsulate meningococci isolated from patients also express pili. These proteins are effective adhesins in capsulate bacteria since unlike other outermembrane adhesins, pili traverse the capsule and are largely unaffected by it (Virji e t al., 1991a, 1995). Pili are used by capsulate organisms to adhere specifically to human epithelial and endothelial cells. In addition, in capsule-deficient bacteria that are often isolated from the nasopharynx, pili may also become accessory proteins to other outer-membrane adhesins and invasins such as Opc described above. In recent studies, a modulatory role of pili in cellular invasion mediated via integrins was demonstrated (Virji e t al., 1995). Pili also augment cytopathic damage of N. meningitidiis (Dunn e t al., 1995). Pili have been shown to be post-translationally modified and in particular contain glycosyl structures of unique composition (Virji e t al., 1993; Stimson e t al., 1995). Whether these structures have significance in host interactions, via lectin-like receptors, remains to be investigated.

Opa proteins and their targeting of CD66

Opa proteins of N. meningitidiis and the related pathogen N. gonorrhoeae are a family of structurally related proteins (Cannon, 1994). Most Opa proteins have been shown to mediate interactions with neutrophils and some also interact with epithelial cells. Recent studies using transfected COS cells expressing several adhesion receptors have shown that most Opa proteins of numerous strains of both N. meningitidis and N . gonorrhoeae interact specificallywith CD66-expressing cells. The adhesion was to the N-terminal domain of the molecule which is largely conserved between different members of CD66. An antibody against the N-terminal domain inhibited Opaexpressing bacterial interactions with human PMN and some epithelial cells known to express CD66. Since the antibody recognizes distinct members of the CD66 family (Watt e t al., 1994), Opa proteins may target distinct CD66 molecules via the largely conserved N-domain and thus interact with diverse cells via the CD66 family of molecules (M. Virji and others, unpublished). 3332

Clinical considerations Clinical conditionsinvolving adhesion molecules, modulation of microbial infection and therapeutic considerations

Leucocyte-adhesion-deficiency syndrome (LAD) is an inherited disorder involving the p2 chain (CD18) of the leucocyte cell adhesion molecules LFA-1, CR3 and CR4, and results in a low number or absence of normal receptors on the cell surface (Arnaout, 1990). The genetic defect can be corrected in vitro by transfection using normal CD18 coding sequences. Patients with LAD develop severe bacterial infections involving oral, respiratory and urogenital mucosa, as well as the skin and the intestine. The infections seem to result from impaired adhesion-dependent functions such as chemotaxis, aggregation and CR3-dependent phagocytosis (Hamacher & Schaberg, 1994). A similar situation has also been observed in the rare condition of impairment in the expression of sialylLewisXon neutrophils, a binding site for endothelial Eselectin (Etzioni e t a/., 1992). These conditions emphasize the importance of CD18 integrins and selectins in host defence against microbes. The potential for intervention in microbial diseases involving adhesion receptors

With the aid of monoclonal antibodies or receptor analogues, it is possible to interfere with specific receptor functions that involve interactions with microbial ligands. The use of such a therapy has obvious associated implications of side-effects since adhesion molecules have central roles in host cellular functions. Receptor blockade may be useful under extreme circumstances, and specific ligands such as RGD-bearing proteins or peptides have also been considered appropriate for use in such intervention. However, RGD peptides may cause endothelium-dependent aortic relaxation (Klotz e t al., 1992) which could lead to exposure of ECM and increased adherence of some microbes. The use of RGD in experimental candidiasis reported decreased yeast load in tissues. A peptide (PepTite-2000) with an amino acid sequence modelled after RGD cell-binding domain of fibronectin was used to modulate hematogenous candidal infections in a rabbit model of infection. The reduction in tissue infiltration is presumed to result from candidal integrins blocked by the fibronectin mimic (Klotz e t al., 1992).

A possible therapeutic potential for anti-adhesion-molecule antibodies has been suggested for management of bacterial meningitis, with intervention targeted at the anti-inflammatory role of antibodies against CD18 cp2) integrins to inhibit the recruitment of white blood cells into the subarachnoid space (Saez-Llorens et al., 1991). The adverse pathophysiological consequences resulting from infiltration were reduced by a combined therapy of dexamethasone to diminish overproduction of cytokines in response to bacterial stimulus and anti-integrin antibodies (Saez-Llorens e t al., 1991). Anti-CD18 (CR3) antibodies have also been effective in increasing survival

Microbial utilization of human signalling molecules of experimental mice after endotoxin challenge, and may be of potential therapeutic value against Gram-negative sepsis and shock (Burch e t a/., 1993).

Concluding remarks Mimicry emerges as a common strategy employed by numerous pathogens in subverting host cellular signalling pathways for invasion. However, invading microbes are constantly subject to host immune surveillance and avoidance of detection by neutralizing antibodies is an additional necessity for survival. T o this end, antigenic variation occurs in many virulence-associated ligands. However, receptor-binding domains need to be conserved, and some microbes have evolved specific structures to protect these sites from neutralizing antibodies. Studies on viruses have provided examples of distinct mechanisms. The canyon structure of picornaviruses provides physical isolation of a conserved binding site. It allows the single Ig domain of the receptor to reach the binding site, but is narrow enough to exclude paired Ig domains of the antibody molecules (Crowell & Tomko, 1994). In contrast, in foot-and-mouth disease virus, the receptor-binding motif RGD is located within a flexible and a highly variable peptide loop protruding from the surface of the virus. The structural flexibility may prevent formation of epitopes recognizable by the host immune system. The ordered structure required for receptor binding may be induced by environmental factors (e.g. pH) at the cell surface or conformational changes induced by the receptor itself (Logan e t a]., 1993; Chapman & Rossmann, 1993). Antigenic variation is common in bacteria such as N. meningitidis, but not all ligands exhibit a high level of variation, for example, Opc structure is largely conserved. However, Opc and several other meningococcal virulence factors are subject to phase variation - which assists immune avoidance. This method may be adopted for ligands with functional roles early in disease prior to antibody formation. Additionally, on/off phase variation has the potential to regenerate phenotypes in microenvironments that may be inaccessible to antibodies. Another widely used mechanism of immune avoidance is host mimicry. Thus, mimicry in different forms is utilized by numerous microbes for dual purposes of avoidance of detection and for subversion of host cellular signalling pathways for invasion. Also, in many cases, factors that determine the natural ligand-specificity also determine tropism of the pathogen. Therefore, studies on microbehost interactions may lead, in the future, also to discoveries of new receptors (as exemplified by the discovery of the novel molecule PVR), receptor functions and mechanisms of eukaryotic signalling pathways.

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