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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

Enteropathogenic and enterohaemorrhagic Escherichia coli use a novel infection strategy to colonize the gut epithelium, involving translocation of their own receptor, Tir, via a type III secretion system and subsequent formation of attaching and effecting (A/E) lesions. Following integration into the host cell plasma membrane of cultured cells, and clustering by the outer membrane adhesin intimin, Tir triggers multiple actin polymerization pathways involving host and bacterial adaptor proteins that converge on the host Arp2/3 actin nucleator. Although initially thought to be involved in A/E lesion formation, recent data have shown that the known Tir-induced actin polymerization pathways are dispensable for this activity, but can play other major roles in colonization efficiency, in vivo fitness and systemic disease. In this review we summarize the roadmap leading from the discovery of Tir, through the different actin polymerization pathways it triggers, to our current understanding of their physiological functions.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

Commensal Escherichia coli, which was discovered in 1885, is the predominant facultative anaerobe of the human gut microbiota. It colonizes the gastrointestinal tract within hours after birth and establishes a mutual beneficial relationship with the host throughout life. However, through acquisition of pathogenicity islands (PAI), prophages and plasmids, several highly adapted E. coli clones have evolved the ability to efficiently colonize specific anatomical sites and cause disease. Pathogenic E. coli can cause a variety of diarrhoeal diseases (Clements et al., 2012), and some (i.e. extra-intestinal E. coli – ExPEC) are capable of infecting extra-intestinal sites such as the urinary tract (uropathogenic E. coli – UPEC) or the bloodstream and/or the central nervous system, particularly in the neonate (Manges and Johnson, 2012).

Traditionally, diarrhoeagenic E. coli have been divided into six pathotypes (Clements et al., 2012): (i) enterotoxigenic E. coli (ETEC), which expresses the heat stable (ST) and/or heat labile (LT) enterotoxins, (ii) enteroinvasive E. coli (EIEC), which harbours a 220 kb virulence plasmid encoding the ipa operon that mediates invasion of gut epithelial cells, (iii) diffuse adherent E. coli (DAEC), a heterogenous group of E. coli with variable virulence characteristics, (iv) enteroaggregative E. coli (EAEC), which encodes the enteroaggregative heat-stable toxin (EAST1), as well as the aggregative adherence fimbriae (AAF) that promote the formation of bacterial aggregates on the mucosal surface, (v) Shiga toxin- (Stx-) producing E. coli (STEC; aka verocytotoxigenic E. coli or VTEC), a group of pathogens that includes the enterohaemorrhagic E. coli (EHEC), an STEC subset that encodes the locus of an enterocyte effacement (LEE) pathogenicity island (PAI) (Nataro and Kaper, 1998), and (vi) enteropathogenic E. coli (EPEC), which carry the LEE but does not produce Stx and are subdivided into typical or atypical EPEC based on the presence or absence, respectively, of the EPEC Adherence Factor (EAF) (Girón et al., 1991) plasmid that encodes the bundle forming pili required for localized adherence on epithelial cells (Trabulsi et al., 2002). New pathotypes are also emerging, such as the E. coli O104:H4 strain, an Stx-producing EAEC strain that caused a lethal outbreak in Germany in 2011 (Frank et al., 2011; Rohde et al., 2011).

Of the E. coli pathotypes, EPEC and EHEC are unique in colonizing the intestinal mucosa via ‘attaching and effacing’ (A/E) lesions, characterized by effacement of the intestinal brush border, intimate bacterial attachment to the plasma membrane of enterocytes, and accumulation of electron dense material, consisting of mostly actin filaments, under the adherent bacteria (Wong et al., 2011; Clements et al., 2012). This hallmark of EHEC and EPEC has been the subject of considerable study for the last two and a half decades. Although these related pathogens utilize highly homologous virulence factors to promote A/E lesions, which are morphologically indistinguishable, EPEC and EHEC utilize distinct signalling pathways to generate filamentous actin. Thus, these studies have provided fascinating examples of how microbial pathogens can hijack related mammalian actin assembly signalling cascades in multiple ways. The diversity and redundancy of signalling suggest that A/E lesion formation plays an important role in the pathogenesis of these infectious agents, a supposition that has recently been tested in animal models.

Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

While E. coli was first linked with severe diarrhoeal illnesses in the early 1900s (Robins-Browne, 1987), the first EPEC strain was isolated in 1945 during an investigation of an infantile diarrhoeal outbreak in Hillingdon Hospital in Middlesex, England (Bray, 1945). EHEC O157:H7 was first identified as a new diarrhoeagenic pathogen in 1982 during an investigation of an outbreak of gastrointestinal illness, which was traced to consumption of contaminated hamburgers (Riley et al., 1983).

In 1987, the visualization of electron-dense structures, termed ‘pedestals’, beneath bacteria attached to host cells offered the first clue towards understanding the EPEC virulence mechanism. Later, it was realized that this dense matter consisted of filamentous actin, formed following attachment of EPEC to cultured epithelial cells (Knutton et al., 1989). This unusual phenotype had not been observed previously for other human pathogens and prompted genetic screens that led to the identification of the first essential virulence factor for intimate EPEC attachment to cultured cells: an outer membrane adhesin, intimin, encoded by the eae gene (Jerse and Kaper, 1991; Donnenberg et al., 1993a). Intimin consists of a conserved 700 aa N-terminal region that contains a beta barrel autotransporter domain that anchors it to the outer membrane and an allelic variable 280 aa C-terminal region that is exposed on the bacterial outer surface (Frankel et al., 1995). This latter domain is comprised of two Ig-like and one C-type lectin-like domains and is homologous to the Yersinia pseudotuberculosis invasin protein that binds tightly to beta-1 integrins, to promote bacterial entry into mammalian cells (Isberg and Falkow, 1985; Isberg and Leong, 1990; Hamburger et al., 1999). EPEC intimin is critical for binding to cultured mammalian cells, establishing colonization in animal models and causing disease in human volunteers (Donnenberg et al., 1993b; 1993a). In 1992, an EHEC O157:H7 eae gene was identified, which shared 83% sequence homology with EPEC eae (Yu and Kaper, 1992).

A second observed phenotype that led to key discoveries of EPEC and EHEC virulence factors was the invasion of cultured cells by typical EPEC. Although EPEC is not known as an invasive pathogen during human infection, this in vitro phenotype provided an important readout in an additional genetic screen to identify a battery of mutants incapable of cellular entry. Many of these mutants were also deficient in formation of actin pedestals and intimate attachment (Donnenberg et al., 1990). Today we know that these mutants are deficient in the biogenesis of a LEE-encoded type III secretion system (T3SS) that is capable of translocating bacterial effectors into host cells (Jarvis et al., 1995; Goosney et al., 2000; Clements et al., 2012).

Since its discovery in 1995, LEE-encoded genes not devoted to the biogenesis of the T3SS were better defined and found to include eae, gene regulators, chaperones and T3SS translocated effector proteins (McDaniel et al., 1995; Goosney et al., 2000). EPEC and EHEC also share a large number of effectors (known as non-LEE encoded effectors or Nle's) that are encoded by genetic elements other than the LEE (Tobe et al., 2006; Iguchi et al., 2009). The total number of T3SS effectors expressed by individual EPEC and EHEC isolates is not fixed and ranges from 22 to 50 (Tobe et al., 2006; Iguchi et al., 2009).

The next breakthrough in our understanding of the virulence associated with the A/E phenotype was the observation that some of the EPEC LEE PAI genes were required to promote tyrosine phosphorylation of a protein, termed Hp90 (Human protein 90 kDa), that localized at the tips of actin pedestals beneath sites of bacterial attachment (Rosenshine et al., 1992). Post-translational modification of Hp90 was linked to both invasiveness and formation of actin pedestals. Moreover, it was demonstrated that Hp90 bound to the bacteria in an intimin-dependent manner (Rosenshine et al., 1992; 1996). Hp90 was believed to be a host trans-membrane receptor for intimin, analogous to the integrin receptors of invasin (Isberg and Leong, 1990). However, in 1997, Kenny and co-workers discovered that Hp90 was actually a T3SS effector that localizes in the host plasma membrane and binds to intimin, and thus renamed Hp90 the translocated intimin receptor, or Tir (Kenny et al., 1997). This discovery was independently confirmed by Deibel and colleagues, who renamed Hp90 EspE (Deibel et al., 1998), although the name Tir is more widely used. The delivery of a self-receptor into the host cell by a bacterium to effect tight pathogen-host binding was a surprising and previously unknown pathogenic strategy.

Our current molecular understanding of actin pedestal formation

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

Tir, a unique paradigm for host cell attachment and signalling by a microbial pathogen

Upon translocation into host cells, Tir inserts into the apical membrane such that the N- and C-terminal portions of Tir are cytosolic, with helical transmembrane domains traversing the host apical membrane. The central (∼ 100-residue) portion, known as the Intimin-Binding Domain (IBD), forms two helices separated by a hairpin loop on the external surface of the host cell (Kenny, 1999; Luo et al., 2000). This loop binds the C-terminus of intimin with high affinity (Kd of ∼ 10 nM for EPEC and less than 1 nM for EHEC) thus facilitating extremely tight attachment of these pathogenic E. coli to the host cell (DeVinney et al., 1999; Ross and Miller, 2007). Based on sequence and antigenic variation within the 280 aa variable C-terminal region, a classification scheme for intimin was developed, becoming an important epidemiological marker alongside the O:H serotypes. The most common intimin types are α, expressed by EPEC belonging to the EPEC1 evolutionary branch; β, expressed by EPEC2 and some non-O157 EHEC (e.g. O26) and γ, which is mainly found in EPEC O55:H7 and EHEC O157:H7 (McGraw et al., 1999). Although intimin types are strain-specific, they are interchangeable between the strains and can interact with any Tir IBD (DeVinney et al., 1999).

In addition to its function as a bacterial receptor, Tir serves as a signalling effector that elegantly initiates the recruitment of host adaptors and actin nucleators from inside the host cytosol, a strategy commonly used by intracellular pathogens to exert pathophysiological effects. Although EPEC and EHEC Tirs are homologous in structure and share ∼ 60% identity (Kenny, 1999) they use divergent mechanisms of signalling that then converge on the activation of common actin nucleators to produce pedestals that are indistinguishable when examined by electron microscopy or fluorescence imaging of actin (Fig. 1).

figure

Figure 1. Actin pedestals as visualized by transmission electron microscopy appear as electron dense protrusions beneath intimately attached bacteria (A). By immunofluorescence microscopy, actin pedestals can be visualized with phalloidin-conjugated fluorophores. EHEC (blue; DAPI stained) induces actin pedestals on infected HeLa cells (B) and HCT8 intestinal cells (C) stained with Alexa-488-conjugated phalloidin (green). White arrowheads point to typical actin pedestals for these cell types. Tir stained with Alexa-568 (red) colocalizes with actin at the tips of actin pedestals, forming yellow foci beneath intimately adhered bacteria.

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Both intimin and Tir appear to be adapted for multimerization. The beta barrel region of intimin, which anchors intimin to the bacterial outer membrane, and the IBD of Tir each encode dimerization domains, giving rise to a model in which the two proteins might efficiently form higher-order ‘lattices’ essential for downstream signalling (Luo et al., 2000; Touzé et al., 2003).

Within an infected host cell, Tir interacts with a number of host proteins through its N- and C-terminal cytosolic domains. The N-terminal tail is functionally interchangeable between EHEC and EPEC Tirs and binds various focal adhesion proteins, thereby potentially linking Tir to the cytoskeleton (Goosney et al., 2000; Kenny, 2001). However, such activity is insufficient for actin pedestal formation (Kenny, 2001). Both EPEC and EHEC require the C-terminal tail, the portion of Tir that is most divergent between these two highly related pathogens (DeVinney et al., 1999). Notably, intimin binding enhances post-translocation modification of the C-terminus of Tir inside the host cell. These modifications, when fully characterized, were pivotal in revealing the mechanism of actin pedestal formation (Kenny et al., 1997; Campellone et al., 2004a; Campellone and Leong, 2005).

EPEC pedestal formation pathways

Host modification of the C-terminus of EPEC Tir is critical for its activity. This domain contains multiple tyrosine, threonine and serine residues that are available for potential host phosphorylation. Serine/threonine phosphorylation, which changes the apparent molecular weight of Tir on SDS-PAGE where it appears as a 90 kDa protein (similar to the MW of Hp90), has been suggested to play a role in Tir insertion into the apical membrane, but otherwise does not contribute to actin pedestal formation. Tyrosine phosphorylation of EPEC Tir, on the other had, is absolutely required for efficient actin polymerization (Kenny et al., 1997). Multiple protein kinases, including members of the Abl and Src kinase families, have been localized to EPEC pedestal tips and thus implicated in pedestal formation; broad inhibition of both these classes of kinases by non-specific pharmacological inhibitors can inhibit EPEC pedestals (Swimm et al., 2004; Hayward et al., 2009). However, EPEC can still induce pedestal formation on a variety of cultured cells lacking individual kinases from the Abl or Src families, suggesting that EPEC Tir is capable of utilizing multiple, redundant, tyrosine kinases for Tir phosphorylation in vitro (Swimm et al., 2004).

Of the Tir C-terminal tyrosine residues, phosphorylation of residue Y474 (in EPEC1 strain E2348/69) is the most critical for focal actin assembly and triggers the major pathway by which EPEC1 strains initiate pedestal formation in vitro (Fig. 2, pathway 1) (Kenny et al., 1997; DeVinney et al., 2001). In clinical isolates, this critical tyrosine residue may be in other close positions due to small insertions or deletions. This pedestal formation pathway utilizes the host adaptor Nck, which contains an SH2 domain capable of binding a 12-residue region of EPEC Tir that includes the critical phosphorylated Y474 (Gruenheid et al., 2001; Campellone et al., 2002). Nck, found at the tips of EPEC-induced actin pedestals, also contains three SH3 domains that may directly or indirectly recruit the host actin nucleation promoting factor N-WASP (Gruenheid et al., 2001; Buday et al., 2002). N-WASP, in turn, recruits and activates the actin nucleating Arp2/3 complex (Snapper and Rosen, 1999; Miki and Takenawa, 2003).

figure

Figure 2. EPEC1 and EHEC use different mechanisms of Tir activated signalling cascades that converge on common host actin nucleation factors, N-WASP and Arp2/3 to form actin pedestals. The major EPEC1 actin pedestal pathway requires phosphorylation of the critical Tir residue Y474, which recruits Nck (pathway 1). Nck can activate N-WASP directly or through WIP. The major EHEC actin pedestal pathway requires Tir residues NPY458 and the EHEC effector EspFU/TccP (pathway 2). EspFu/TccP is linked to Tir via host adaptors IRTKS and IRSp53 and activates N-WASP. Minor EPEC actin assembly pathways utilize Tir residues Y474 or Y454 to initiate Nck independent activation of N-WASP (pathways a and b). A minor actin assembly pathway for EHEC utilizes EspFu, but does not require N-WASP for Arp2/3 activation (pathway c).

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Due to similarities in the use of Nck and N-WASP by vaccinia virus for ‘actin comet tail’ formation, a similar mechanism of actin assembly has been proposed for EPEC (Campellone and Leong, 2005). Vaccinia produces A36R, a Nck-binding protein, that, like EPEC Tir, is tyrosine phosphorylated by the host. An eight-residue sequence in the A36R Nck-binding region is nearly homologous to the Y474 region of EPEC Tir (Campellone and Leong, 2005). Recruitment of Nck by A36R leads to indirect activation of N-WASP through the intermediary adaptor, WASP Interacting Protein (WIP) (Moreau et al., 2000). WIP contains N-terminal proline-rich domains that are capable of binding Nck's SH3 domains; its C-terminal WASP-binding domain (WBD) activates N-WASP by binding to the N-WASP N-terminal WH1 domain (Moreau et al., 2000). Ectopic overexpression of the WBD decreases vaccinia actin comet tail formation, presumably by a dominant-negative effect, and various mutations in the WH1 domain of N-WASP can abrogate its recruitment and actin comet tail formation by vaccinia (Moreau et al., 2000).

A role for WIP in pathogen-mediated actin assembly appears to depend on the experimental system. Garber and colleagues showed that both EPEC and vaccinia are still able to induce actin assembly in a cell line lacking WIP (Garber et al., 2012). N-WASP also contains a proline-rich domain that is capable of binding Nck directly, and thereby leading to its activation (Antón et al., 1998). As is the case for the kinases(s) responsible for EPEC Tir phosphorylation, seemingly disparate observations concerning the mechanism of N-WASP activation by Nck and the role of WIP may indicate a degree of redundancy in the N-WASP activation pathway.

Although the major pathway for EPEC pedestal formation requires Nck, Nck-independent mechanisms of actin pedestal formation have also been uncovered, reflecting redundant pedestal formation pathways (Fig. 2, pathways a and b). EPEC infection of Nck-deficient murine embryonic fibroblasts still generate roughly one-fifth the number of the actin pedestals generated upon infection of wild-type fibroblasts. Most of this activity, also requires Y474, reaffirming its role as a key residue for triggering actin assembly by EPEC Tir. However, even in the absence of Y474, about 2–5% of bound EPEC can generate actin pedestals, a rate more than 10-fold higher than for a Tir-deficient EPEC strain. An additional tyrosine residue at position 454 of EPEC Tir is critical for this minor Nck-independent actin assembly activity (Campellone and Leong, 2005). Initially considered of marginal importance, this pathway was later shown to be highly related to the major pathway of actin pedestal formation by EHEC.

EHEC pedestal formation pathways

A critical first indication that EHEC and EPEC utilized distinct pedestal formation pathways came from studies showing that tyrosine phosphorylated proteins were absent from EHEC actin pedestals (DeVinney et al., 2001). In fact, EHEC Tir lacks the equivalent of EPEC Tir Y474, and EHEC does not require Nck for actin pedestal formation (Kenny, 1999; DeVinney et al., 2001). Consistent with distinct pathways of actin assembly, EHEC tir cannot complement EPEC tir mutants for actin pedestal formation unless modified to encode the critical Y474-containing region of EPEC Tir (Kenny, 2001; Campellone et al., 2002). Co-infection of cultured tir and eae (encoding intimin) deletion mutants of EPEC and EHEC revealed that the latter requires an additional EHEC-specific effector for pedestal formation (DeVinney et al., 2001; Kenny, 2001). These observations pointed to different mechanisms of actin pedestal formation for EPEC and EHEC (Fig. 2).

Experiments using chimeric Tir proteins suggested that the key to differences in EPEC and EHEC Tir lie in the C-terminus. While the N-terminus and IBD of EHEC and EPEC Tir are highly similar and functionally interchangeable, their cytosolic C-terminal domains are only ∼ 41% identical (DeVinney et al., 1999). Further studies of this region revealed that an NPY sequence at residues 456–458 of EHEC Tir is critical for actin pedestal formation (Brady et al., 2007). EHEC Tir residue Y458 corresponds to EPEC Tir Y454, the residue previously shown to be essential for triggering one of the minor Nck-independent actin assembly pathways in EPEC pedestal formation, described above.

Instead of utilizing tyrosine phosphorylation and Nck recruitment, the EHEC Tir NPY-mediated pathway acts through a different category of host adaptors (Fig. 1, pathway 2). The NPY458 sequence of EHEC Tir, and likely the equivalent sequence of EPEC Tir as well, binds to IRTKS and IRSp53, members of the I-Bar subfamily of membrane deforming and remodelling proteins. These proteins link to Tir via their N-terminal IMD domains, which also form ‘zeppelin'-shaped homodimers associated with convex membrane protrusions (Vingadassalom et al., 2009; Weiss et al., 2009; de Groot et al., 2011; Zhao et al., 2011). The C-terminal regions of IRTKS and IRSp53 contain additional motifs, such as an SH3 domain, that link their membrane deforming activity to signalling proteins and actin regulators (Vingadassalom et al., 2009; Weiss et al., 2009).

A paradox in this Tir NPY-mediated pedestal pathway is that it promotes pedestal formation with low efficiency for EPEC, yet with high efficiency for EHEC (Campellone and Leong, 2005; Campellone et al., 2006). This riddle was solved by the identification of an additional non-LEE-encoded bacterial effector, termed EspFU (a homologue of the effector EspF encoded on prophage U) or TccP (Tir cytoskeleton coupling protein) (Garmendia et al., 2004; Campellone et al., 2004b). Found in EHEC O157:H7, EspFU/TccP also greatly enhances NPY458 mediated pedestal formation if expressed in EPEC1 (Campellone et al., 2006).

Discovered before the Tir NPY458 residues and IRTKS/IRSp53, EspFU/TccP provided structural insights that helped elucidate both the identity of its linker to EHEC Tir and the mechanism of N-WASP activation. EspFU/TccP consists of an N-terminal secretion signal, followed by (in nearly all isolates) between three and eight repeating elements (Garmendia et al., 2005; 2006). Each functional repeating unit contains two structurally and functionally distinct domains, an N-terminal helical section that binds and activates N-WASP, and a C-terminal proline-rich domain with multiple canonical SH3 binding PxxP motifs that are recognized by the SH3 domain in the C-terminus of IRTKS and IRSp53 (Garmendia et al., 2006; Cheng et al., 2008; Aitio et al., 2010).

EspFU/TccP acts through structural mimicry and, remarkably, has evolved to outcompete some endogenous host ligands, making it a most efficient hijacker of mammalian signalling pathways. SH3 domains are an abundant and common motif for affecting cellular interactions. The proline-rich domains of EspFU/TccP recognize the SH3 domain of IRTKS with high affinity (Aitio et al., 2010), potentially allowing it to outcompete Eps8, a host actin modulator that appears to be a major binding partner of IRSp53 (Funato et al., 2004; Disanza et al., 2006). Like EspFU/TccP, Eps8 and has been shown to bind the SH3 domains of IRTKS and IRSp53 via multiple PxxP motifs (Aitio et al., 2010). Although the IRTKS/IRSp53-binding sequences of Eps8 and EspFU/TccP are nearly identical, the latter binds to the IRTKS SH3 domain with higher affinity due to the presence of a tryptophan, rather than an alanine, between tandem PxxP motifs. Thus, EspFU/TccP's tryptophan ‘switch’ may allow EHEC to usurp control of IRTKS/IRSp53 from natural host ligands (Aitio et al., 2012).

Similarly, the N-WASP-activating helical domain of EspFU/TccP acts by high-affinity mimetic displacement. N-WASP's C-terminal VCA domain, which functions to activate Arp2/3, is normally maintained in an autoinhibited state, due to an intramolecular interaction between an amphipathic helix in this domain and a hydrophobic groove located the GBD domain in the central portion of N-WASP (Snapper and Rosen, 1999; Miki and Takenawa, 2003; Cheng et al., 2008). Endogenous N-WASP activators such as Cdc42 disrupt this autoinhibition by binding to the GBD and indirectly disrupting this interaction by altering the GBD structure (Snapper and Rosen, 1999; Miki and Takenawa, 2003). In contrast, EspFu/TccP's helical domain is structurally similar to the helix in the N-WASP VCA domain and can directly displace it from the GBD, thereby activating N-WASP (Cheng et al., 2008).

A notable finding is that more than 99% of EspFU/TccP alleles possess at least three repeats of the functional (i.e. helical- and proline-rich-containing) domains, suggesting that multiple repeats confer a selective advantage to EHEC (Garmendia et al., 2005). Multiple EspFU/TccP repeats synergistically enhance activation of Arp2/3 in vitro (Campellone et al., 2008; Sallee et al., 2008). In addition, we speculated above that the dimerization domains of intimin and Tir might promote a higher-order structure conducive to efficient actin signalling. In fact, the presence of dimerization activity in IRTKS/IRSp53 indicates that three of the four components in this signalling cascade, i.e. intimin, Tir, IRTKS/IRSp53 each have the capacity to multimerize and the fourth, TccP/EspFU, encodes multiple repeat elements. A compelling hypothesis is that EHEC utilizes a series of both bacterial and mammalian components to promote the formation of a large multimeric complex at the host cell membrane, one that efficiently generates filamentous actin and enhances EHEC's ability to attach to colonic epithelium.

Finally, just as EPEC encodes multiple actin assembly pathways, EHEC triggers yet another alternative pathway for pedestal formation in a cell culture model (Fig. 2, pathway c). Low levels of actin assembly can be observed when embryonic fibroblasts from N-WASP knockout mice are infected with an intimin-expressing E. coli strain that delivers EHEC Tir and EspFU/TccP. This N-WASP-independent pathway depends on multiple repeats of EspFU/TccP, again suggesting a functional role for multiple repeats (Vingadassalom et al., 2010) but the critical elements of this pathway are yet unidentified.

TccP2/EspFM-induced actin polymerization in EPEC2 and non-O157 EHEC

Typical EHEC O157:H7 also possesses a pseudogene of EspFU/TccP encoded on prophage CP-933 M/Sp4, and thus called TccP2/EspFM, which lacks an intact type III secretion signal (Ogura et al., 2007). Beta-glucuronidase-positive and sorbitol-fermenting EHEC O157 strains harbour an intact tccP2/espFM gene as well as an espFU/tccP gene (Ogura et al., 2007). Although they carry divergent N-terminal secretion signals, EspFU/TccP and TccP2/EspFM share the same repeat structure, and EspFU/TccP and TccP2/EspFM are functionally interchangeable in EHEC O157 strains (Ooka et al., 2007).

Unexpectedly, EspFU/TccP was also found in EPEC strains of the serotype O119:H6, which express Tir that contains both NPY458 and Y474 equivalents (Ooka et al., 2007). Infections of cultured cells have shown that EPEC O119:H6 can simultaneously utilize the Tir:Nck and Tir:IRTKS/IRSp53:EspFU/TccP pathways (Fig. 3) (Whale et al., 2007). Later, this infection strategy was found to be common among EPEC strains belonging to the EPEC2 evolutionally lineage and non-O157:H7 strains, as they encode both a functional TccP2/EspFM and a Tir protein that can be tyrosine phosphorylated (Whale et al., 2006; Ogura et al., 2007). These results show the existence of yet another level of redundancy and suggest that Tir-induced actin polymerization is under strong selective pressure.

figure

Figure 3. EPEC2 and non-O157 EHEC can use the major pathways of both typical EPEC1 and EHEC O157 to generate actin pedestals. Tir in these strains contain both a Y474 equivalent, which initiates actin assembly through Nck (pathway 1), and an NPY458 equivalent, which initiates actin assembly through IRTKS/IRSp53 and an EspFU/TccP homologue, TccP2/EspFM (pathway 2).

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Map and EspH impact on Tir-induced actin polymerization

Map and EspH have both been implicated in filopodia formation and modulation of Tir-induced actin polymerization in undifferentiated epithelial cells. While Map is a Cdc42 guanine nucleotide exchange factor (GEF) (Huang et al., 2009), EspH is a RhoGEF inhibitor that acts by competitively binding to the tandem Dbl-homology and pleckstrin-homology (DH-PH) domains of Dbl-family mammalian RhoGEFs (Dong et al., 2010). Ectopic expression of EspH leads to cell detachment, while infections with strains either missing or overexpressing EspH result in short or elongated pedestals respectively. The cytotoxicity of EspH could be offset by expression of the bacterial RhoGEFs EspM2 and EspT (Wong et al., 2012a). EPEC infection leads to accumulation of WIP at the tip of the Tir-induced actin pedestals (Garber et al., 2012; Wong et al., 2012b) in a process involving EspH, which itself taggers actin polymerization in a Tir-dependent mechanism (Wong et al., 2012b).

Activation of Cdc42 by Map leads to transient filopodia formation during early stages of infection; interaction of Map with NHERF1 via its PDZ-biding motif is needed for filopodia stabilization (Berger et al., 2009). Recovery from the filopodial signals requires phosphorylation of Tir tyrosine Y474 and activation of the actin polymerization pathway as either infection of Nck-proficient cells with EPEC expressing TirY474 or infection of Nck-deficient cells with wild-type EPEC results in persistence of filopodia (Berger et al., 2009). The relevance of these pathways in EPEC-induced microvilli effacement was recently reported following infection of polarized Caco-2 cells (Dean et al., 2013). Taken together, these data show the existence of an elaborated cross-talk between Tir and other effectors that subvert actin dynamics.

In vivo and ex vivo infection models

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

Rabbit

In 1983 and 1985 Moon et al. and Tzipori et al. used rabbit ligated ileal loops and gnotobiotic piglets to describe the A/E lesion (Moon et al., 1983; Tzipori et al., 1985). Subsequently, Robins-Browne et al. showed that rabbit EPEC (REPEC) strains were unable to colonize the intestinal tract of guinea pigs and were detected in distal intestine of conventional mice but without evidence of A/E lesions (Robins-Browne et al., 1994).

Using the rabbit model Marchès et al. have shown that intimin and Tir are essential for A/E lesion formation in vivo (Marchès et al., 2000). In infant rabbits infected with EHEC O157:H7, colonization efficiency of the espFU/tccP mutant was similar to the parental wild-type strain in the ileum but was reduced in the large bowel at 7 days post infection (Ritchie et al., 2008). Infection of rabbits with EHEC Δmap mutant resulted in reduced colonization specifically in the small intestine, while infection with EHEC ΔespH resulted in reduced colonization throughout the intestine. However, neither Map nor EspH were needed for A/E lesion formation in the rabbit model (Ritchie and Waldor, 2005).

Bovine and porcine

As EHEC can colonize large farm animals, they could potentially be employed as infection models, although for obvious reasons they cannot be used for routine investigation. EHEC and EPEC strains have been shown to cause A/E lesions on the bovine gut mucosa using experimental bovine and porcine challenges (Girard et al., 2005; Naylor et al., 2005) and intestinal in vitro organ cultures (IVOC) (Girard et al., 2005; 2009), as well as following inoculation of bovine ligated intestinal loops (Vlisidou et al., 2006). The bovine ileal loop and IVOC, as well as the gnotobiotic piglet (Ritchie et al., 2008), models confirmed a role for intimin and Tir, but revealed that EspFU/TccP is not required for A/E lesion formation on these mucosal surfaces, although expression of TccP/EspFU was associated with larger aggregates of mucosally adherent bacteria in the latter model (Ritchie et al., 2008). Moreover, neither Tir Y474 nor TccP2/EspFM were needed for A/E lesion formation following infection of bovine IVOC with EPEC strains E2348/69 (EPEC1) and B171 (EPEC2) (Girard et al., 2009).

Human intestinal organ cultures (hIVOC) and xenographs

Knutton et al. have utilized human IVOC (hIVOC) to show that EPEC can efficiently form A/E lesions on the human gut mucosa (Knutton et al., 1987). Later, Phillips et al. found that EHEC specifically causes A/E lesions on human Peyer's patches (PP) (Phillips et al., 2000). Moreover, the human IVOC model revealed that infection with EPEC E2348/69, but not EHEC O157, resulted in the accumulation of tyrosine phosphorylated proteins and Nck under attached bacteria, while infection with either strains resulted in the localization of N-WASP. Recruitment of N-WASP and A/E lesion formation following EPEC infection were independent of Tir residue Y474. In contrast, while EspFU/TccP was not needed for A/E lesion formation, recruitment of N-WASP was not detected following infection with the EHEC tccP/espFU mutant. Infection of human IVOC with a clinical atypical EPEC O125:H6 isolate, which naturally lacks both EspFU/TccP and the equivalent of Y474, also resulted in the formation of typical A/E lesions (Bai et al., 2008). These results suggest that EPEC can recruit N-WASP to infection sites by a Tir Y474-independent mechanism, but the relationship, if any, of this pathway to the minor pathways described above is not yet known.

Infection of hIVOC with EPEC Δmap and ΔespH mutants revealed that while both induced brush border remodelling and produced typical A/E lesions, there were some differences from the wild-type strain; elongation of non-effaced microvilli, particularly in the espH mutant, appeared to be attenuated and some bacteria that had clearly formed pedestals had come away, leaving a ‘pedestal footprint’ (Shaw et al., 2005). The fact that this kind of pedestal footprints were not seen following infection with parental wild-type strain suggests that Map and EspH might modulate Tir interaction with the cytoskeleton, which in turn impacts Tir–intimin interaction.

Human intestinal xenografts in severe combined immunodeficiency (SCID) mice allow for experimentation with the live human tissue with minimal ethical conflicts. Recent work has shown that the model faithfully recapitulates the tissue tropism of EHEC to the colon, where it forms A/E lesions in a T3SS-dependent manner (Golan et al., 2011). This model has also been used to show that flagellin of EHEC O157:H7 is sufficient to eliciting colonic inflammation (Miyamoto et al., 2006). In contrast to the IVOC model, the use of xenografts allows for the analysis of innate immune responses to infection. However, the use of SCID animals precludes employing this model to study the adaptive response. An additional major difference from the natural infection setting is the lack of microbiota in the xenografts, which are for the most part sterile.

Mouse

Infections of mice with EPEC and EHEC isolates, with or without pre-treatment with antibiotics to deplete the endogenous gut flora, have been reported, but in the absence of A/E lesions and close association of the bacteria with the mucosa, the usefulness of this model is somewhat questionable. For this reason, Citrobacter rodentium, which shares a characteristic mechanism of colonization (i.e. A/E lesion formation) and T3SS effectors with EPEC and EHEC, is arguably one of the best murine models of bacterial infection available, as it reflects a natural and physiological host–pathogen interaction in immunocompetent animals (reviewed in Mundy et al., 2005). Importantly, not only are virulence factors generally interchangeable between C. rodentium and the human pathogens (Frankel et al., 1995), but from the available volunteer and intestinal explant studies, it is clear that C. rodentium has predictive value in assessing the role of E. coli virulence factors in humans.

Deng et al. were the first to report that the Tir Y474 pathway was not needed for A/E lesion formation following infection of mice with C. rodentium, which carries the equivalent Y474 residue at position Y471 (Deng et al., 2003). Later, Girard et al. reported that expression of EspFU/TccP in C. rodentium (which is naturally espFU/tccP negative) did not enhance virulence (Girard et al., 2009). In 2010 Crepin et al. reported that both the Tir-dependent Nck and IRTKS/IRSp53 pathways are dispensable for colonization and A/E lesion formation (Crepin et al., 2010). Similar to EPEC on human IVOC, recruitment of Nck, but not N-WASP, in infected mice was Y471-dependent. In mixed infections, the wild-type C. rodentium out-competed the tir tyrosine mutants. These results show that although not needed for A/E lesion formation, the ability to stimulate Tir-induced actin polymerization pathways provide a competitive colonization advantage.

Citrobacter rodentium does not produce Stx, the phage-encoded toxin required for the most serious manifestations of EHEC disease in humans, such as haemorrhagic colitis or haemolytic uraemic syndrome, the triad of anaemia, thrombocytopenia and renal failure. To generate a murine infection model that permits the analysis of Stx-mediated disease in the context of infection of conventional mice, C. rodentium was lysogenized with λstx2dact, a naturally occurring Stx-encoding lambdoid phage. Infection with C. rodentium (λstx2dact) mimics several key features of EHEC disease, including the induction of pro-inflammatory cytokines and intestinal inflammation (Mallick et al., 2012). More importantly, Stx induced renal inflammation and dysfunction, similar to HUS in human EHEC disease. A/E lesions were apparent in this model, and not dependent on Stx. Furthermore, C. rodentium (λstx2dact) expressing the Tir mutant Y471F, which promotes cell attachment but is incapable of stimulating Nck-dependent actin assembly, colonized colonic epithelia less efficiently than wild type, a defect that was associated with an absence of intestinal or renal inflammation. Notably, expression of the Tir Y471F mutant was specifically associated with a 16-fold lower risk of lethality. These findings suggest that this model recapitulates both the unique mode of epithelial colonization and Stx-mediated pathology.

What are the physiological roles of Tir-induced pedestal formation?

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References

While Tir and intimin are essential virulence factors needed for colonization and disease, the physiological role of Tir-mediated actin polymerization is not well defined. However, a number of infection models have shown that while the Tir:Nck and Tir:IRSTKS/IRSp53:TccP/EspFU pathways are not needed for A/E lesion formation, they promote efficient colonization and in vivo fitness of the pathogen. First, EPEC and EHEC have been observed to undergo two-dimensional actin-based motility on the cell surface, a process that could promote the spread of daughter cells and the formation on the epithelium surface of large aggregates, which have been associated with pedestal formation in infected piglets. Second, in conjunction with other effectors, Tir impacts the integrity of tight junctions and focal adhesions, thus affecting cell exfoliation. Third, Tir-induced disruption of tight junctions might contribute to diarrhoea and/or translocation of Stx across the gut epithelium, consistent with the association of pedestal formation with toxin-mediated renal disease in the C. rodentium (Stx2dact) model. Finally, although EPEC is considered an extracellular pathogen, a minority of EPEC strains express EspT, which activates Rac1 and promotes bacterial entry into epithelial cells via the ‘trigger’ mechanism (Bulgin et al., 2009). Following invasion, these strains reside within E. coli-containing vacuoles (ECV); localization of Tir to the ECV membrane was shown to induce actin polymerization around it that was needed for intracellular bacterial replication. Therefore Tir-induced actin polymerization is essential for infection by the small clade of invasive EPEC.

An additional possible function of the actin pedestal is the formation of a localized signalling hub that co-ordinates bacterial modulation of host cell functions. Indeed, multiple host proteins (e.g. cortactin, talin ezrin, annexin) as well as T3SS effectors (e.g. Map, EspH, EspF) are recruited to the pedestal structure and it is possible that within the pedestals these components are organized in a specific architecture that allows a special and temporal control of effector functions (Munera et al., 2012).

Taken together, the emerging data on Tir-induced actin polymerization suggest that while actin pedestal formation may not be required for generating A/E lesions, these pathways have major roles in virulence and pathogenesis, which likely is responsible for their widespread preservation among diverse strains and the existence of redundant mechanisms in clinical isolates.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Key milestones for intimate attachment: the LEE PAI, effector proteins and intimin
  5. Our current molecular understanding of actin pedestal formation
  6. In vivo and ex vivo infection models
  7. What are the physiological roles of Tir-induced pedestal formation?
  8. Acknowledgements
  9. References
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