Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42-independent, actin-rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir-independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia-like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule - a protein that targets mitochondria and induces their dysfunction. We show that Map-induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP-CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map-induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase-activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map-induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co-ordinately regulated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.     

The enteropathogenic E. coli effector EspH promotes actin pedestal formation and elongation via WASP-interacting protein (WIP)

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are diarrheagenic pathogens that colonize the gut mucosa via attaching-and-effacing lesion formation. EPEC and EHEC utilize a type III secretion system (T3SS) to translocate effector proteins that subvert host cell signalling to sustain colonization and multiplication. EspH, a T3SS effector that modulates actin dynamics, was implicated in the elongation of the EHEC actin pedestals. In this study we found that EspH is necessary for both efficient pedestal formation and pedestal elongation during EPEC infection. We report that EspH induces actin polymerization at the bacterial attachment sites independently of the Tir tyrosine residues Y474 and Y454, which are implicated in binding Nck and IRSp53/ITRKS respectively. Moreover, EspH promotes recruitment of neural Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex to the bacterial attachment site, in a mechanism involving the C-terminus of Tir and the WH1 domain of N-WASP. Dominant negative of WASP-interacting protein (WIP), which binds the N-WASP WH1 domain, diminished EspH-mediated actin polymerization. This study implicates WIP in EPEC-mediated actin polymerization and pedestal elongation and represents the first instance whereby N-WASP is efficiently recruited to the EPEC attachment sites independently of the Tir:Nck and Tir:IRTKS/IRSp53 pathways. Our study reveals the intricacies of Tir and EspH-mediated actin signalling pathways that comprise of distinct, convergent and synergistic signalling cascades.     

Enteropathogenic E. coli translocated intimin receptor, Tir, interacts directly with alpha-actinin

Enteropathogenic Escherichia coli (EPEC) triggers a dramatic rearrangement of the host epithelial cell actin cytoskeleton to form an attaching and effacing lesion, or pedestal. The pathogen remains attached extracellularly to the host cell through the pedestal for the duration of the infection. At the tip of the pedestal is a bacterial protein, Tir, which is secreted from the bacterium into the host cell plasma membrane, where it functions as the receptor for an EPEC outer membrane protein, intimin [1]. Delivery of Tir to the host cell results in its tyrosine phosphorylation, followed by Tir-intimin binding. Tir is believed to anchor EPEC firmly to the host cell, although its direct linkage to the cytoskeleton is unknown. Here, we show that Tir directly binds the cytoskeletal protein alpha-actinin. alpha-Actinin is recruited to the pedestal in a Tir-dependent manner and colocalizes with Tir in infected host cells. Binding is mediated through the amino terminus of Tir. Recruitment of alpha-actinin occurs independently of Tir tyrosine phosphorylation. Recruitment of actin, VASP, and N-WASP, however, is abolished in the absence of this tyrosine phosphorylation. These results suggest that Tir plays at least three roles in the host cell during infection: binding intimin on EPEC; mediating a stable anchor with alpha-actinin through its amino terminus in a phosphotyrosine-independent manner; and recruiting additional cytoskeletal proteins at the carboxyl terminus in a phosphotyrosine-dependent manner. These findings demonstrate the first known direct linkage between extracellular EPEC, through the transmembrane protein Tir, to the host cell actin cytoskeleton via alpha-actinin.     

Talin, a host cell protein, interacts directly with the translocated intimin receptor, Tir, of enteropathogenic Escherichia coli, and is essential for pedestal formation

Enteropathogenic Escherichia coli (EPEC) is able to inject its own receptor, a transmembrane protein called translocated intimin receptor, Tir, into the host epithelial cell. The bacterium then uses an outer membrane protein, intimin, to bind to Tir and remains firmly attached to the host cell surface for the duration of the infection. The bacterium is also able to trigger the rearrangement of several host cell proteins, culminating with the formation of an actin-rich, pedestal-like structure beneath the EPEC adherence site. Although several cytoskeletal proteins are rearranged following EPEC infection, the exact role played by these proteins during pedestal formation remains unknown. We report here that talin, an integrin-binding protein, is recruited by EPEC and associates directly with Tir. By surface plasmon resonance (SPR), the predicted value for the dissociation constant ( K _D) for Tir–talin binding was 1.86 × 10⁻⁷ M. We also demonstrate that microinjection of anti-talin antibodies into HeLa cells resulted in the complete inability to focus actin filaments beneath the attached bacterium. These findings demonstrate that talin is essential for EPEC-induced pedestal formation in infected cells.     

Crk Adaptors Negatively Regulate Actin Polymerization in Pedestals Formed by Enteropathogenic Escherichia coli (EPEC) by Binding to Tir Effector

Infections by enteropathogenic Escherichia coli (EPEC) cause diarrhea linked to high infant mortality in developing countries. EPEC adheres to epithelial cells and induces the formation of actin pedestals. Actin polymerization is driven fundamentally through signaling mediated by Tir bacterial effector protein, which inserts in the plasma membrane of the infected cell. Tir binds Nck adaptor proteins, which in turn recruit and activate N-WASP, a ubiquitous member of the Wiskott-Aldrich syndrome family of proteins. N-WASP activates the Arp2/3 complex to promote actin polymerization. Other proteins aside from components of the Tir-Nck-N-WASP pathway are recruited to the pedestals but their functions are unknown. Here we investigate the function of two alternatively spliced isoforms of Crk adaptors (CrkI/II) and the paralog protein CrkL during pedestal formation by EPEC. We found that the Crk isoforms act as redundant inhibitors of pedestal formation. The SH2 domain of CrkII and CrkL binds to phosphorylated tyrosine 474 of Tir and competes with Nck to bind Tir, preventing its recruitment to pedestals and thereby inhibiting actin polymerization. EPEC infection induces phosphorylation of the major regulatory tyrosine in CrkII and CrkL, possibly preventing the SH2 domain of these proteins from interacting with Tir. Phosphorylated CrkII and CrkL proteins localize specifically to the plasma membrane in contact with EPEC. Our study uncovers a novel role for Crk adaptors at pedestals, opening a new perspective in how these oncoproteins regulate actin polymerization.     

Tir Is Essential for the Recruitment of Tks5 to Enteropathogenic Escherichia coli Pedestals

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that infects the epithelial lining of the small intestine and causes diarrhea. Upon attachment to the intestinal epithelium, EPEC uses a Type III Secretion System to inject its own high affinity receptor Translocated intimin receptor (Tir) into the host cell. Tir facilitates tight adhesion and recruitment of actin-regulating proteins leading to formation of an actin pedestal beneath the infecting bacterium. The pedestal has several similarities with podosomes, which are basolateral actin-rich extensions found in some migrating animal cells. Formation of podosomes is dependent upon the early podosome-specific scavenger protein Tks5, which is involved in actin recruitment. Although Tks5 is expressed in epithelial cells, and podosomes and EPEC pedestals share many components in their structure and mechanism of formation, the potential role of Tks5 in EPEC infections has not been studied. The aim of this study was to determine the subcellular localization of Tks5 in epithelial cells and to investigate if Tks5 is recruited to the EPEC pedestal. In an epithelial MDCK cell line stably expressing Tks5-EGFP, Tks5 localized to actin bundles. Upon infection, EPEC recruited Tks5-EGFP. Tir, but not Tir phosphorylation was essential for the recruitment. Time-lapse microscopy revealed that Tks5-EGFP was recruited instantly upon EPEC attachment to host cells, simultaneously with actin and N-WASp. EPEC infection of cells expressing a ΔPX-Tks5 deletion version of Tks5 showed that EPEC was able to both infect and form pedestals when the PX domain was deleted from Tks5. Future investigations will clarify the role of Tks5 in EPEC infection and pedestal formation.     

Enterohaemorrhagic and enteropathogenic Escherichia coli Tir proteins trigger a common Nck-independent actin assembly pathway

The Tir proteins of enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are each translocated into the host plasma membrane where they promote F-actin pedestals in epithelial cells beneath adherent bacteria, but the two proteins act by different means. The canonical EPEC Tir becomes phosphorylated on tyrosine residue 474 (Y474) to recruit the host adaptor protein Nck, and also stimulates an inefficient, Nck-independent pathway utilizing tyrosine residue 454 (Y454). In contrast, the canonical EHEC Tir lacks Y474 and instead utilizes residues 452-463 to recruit EspF(U), an EHEC-specific effector that stimulates robust Nck-independent actin assembly. EHEC Tir Y458 and EPEC Tir Y454 are both part of an asparagine-proline-tyrosine (NPY) sequence. We report that each of the EHEC Tir NPY residues is required for EspF(U) recruitment and pedestal formation, and each of the EPEC Tir NPY residues is critical for inefficient, Nck-independent pedestal formation. Introduction of EspF(U) into EPEC dramatically enhanced Nck-independent actin assembly by EPEC Tir in a manner dependent on NPY(454). These results suggest that EPEC and EHEC Tir trigger a common Nck-independent actin assembly pathway and are both derived from an ancestral Tir molecule that utilized NPY to stimulate low-level pedestal formation.     

Involvement of the intermediate filament protein cytokeratin-18 in actin pedestal formation during EPEC infection

While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474. Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein. Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization. This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection.     

EspFU is a translocated EHEC effector that interacts with Tir and N-WASP and promotes Nck-independent actin assembly

Several microbial pathogens including enteropathogenic E. coli (EPEC) exploit mammalian tyrosine-kinase signaling cascades to recruit Nck adaptor proteins and activate N-WASP-Arp2/3-mediated actin assembly. To promote localized actin "pedestal formation," EPEC translocates the bacterial effector protein Tir into the plasma membrane, where it is tyrosine-phosphorylated and binds Nck. Enterohemorrhagic E. coli (EHEC) also generates Tir-dependent pedestals, but in the absence of phosphotyrosines and Nck recruitment. To identify additional EHEC effectors that stimulate phosphotyrosine-independent actin assembly, we systematically generated EHEC mutants containing specific deletions in putative pathogenicity-islands. Among 0.33 Mb of deleted sequences, only one ORF was critical for pedestal formation. It lies within prophage-U, and encodes a protein similar to the known effector EspF. This proline-rich protein, EspFU, is the only EHEC effector of actin assembly absent from EPEC. Whereas EHEC Tir cannot efficiently recruit N-WASP or trigger actin polymerization, EspFU associates with Tir, binds N-WASP, and potently stimulates Nck-independent actin assembly.     

A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC)-like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category.     

Esp-independent functional integration of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC) into host cell membranes

The pathogenesis of enteropathogenic Escherichia coli (EPEC) is characterized by the type III secretion system-dependent exploitation of target cells that results in attaching and effacing (A/E) lesions, actin rearrangements and pedestal formation. This pathology is mediated by effector proteins which are translocated by the type III secretion system into the host cell such as the translocated intimin receptor (Tir) and several E. coli secreted proteins (Esp). Secretion of virulence proteins of EPEC is tightly regulated. In response to Ca(2+), Esp secretion is drastically reduced, whereas secretion of Tir is increased. Membrane insertion of Tir, secreted under low Ca(2+) conditions, is therefore independent of Esp. Furthermore, espB and espD mutant strains of EPEC, unable to form the translocation pore, still translocate Tir into host cells membranes. This autointegrated Tir is functional, as it is able to complement a tir mutant strain in recruiting actin to bacterial contact sites. The uptake of Tir into the host cell appears to depend on the C-terminal part of the protein, as deletion of this part of Tir prevents autointegration. Together, our results demonstrate that under conditions of limited Ca(2+) an alternative mechanism for Tir integration can trigger the induction of A/E lesions.     

Clustering of Nck by a 12-residue Tir phosphopeptide is sufficient to trigger localized actin assembly

Enteropathogenic Escherichia coli (EPEC) translocates effector proteins into mammalian cells to promote reorganization of the cytoskeleton into filamentous actin pedestals. One effector, Tir, is a transmembrane receptor for the bacterial surface adhesin intimin, and intimin binding by the extracellular domain of Tir is required for actin assembly. The cytoplasmic NH2 terminus of Tir interacts with focal adhesion proteins, and its tyrosine-phosphorylated COOH terminus binds Nck, a host adaptor protein critical for pedestal formation. To define the minimal requirements for EPEC-mediated actin assembly, Tir derivatives were expressed in mammalian cells in the absence of all other EPEC components. Replacement of the NH2 terminus of Tir with a viral membrane-targeting sequence promoted efficient surface expression of a COOH-terminal Tir fragment. Artificial clustering of this fusion protein revealed that the COOH terminus of Tir, by itself, is sufficient to initiate a complete signaling cascade leading to pedestal formation. Consistent with this finding, clustering of Nck by a 12-residue Tir phosphopeptide triggered actin tail formation in Xenopus egg extracts.     

Synergistic roles for the Map and Tir effector molecules in mediating uptake of enteropathogenic Escherichia coli (EPEC) into non-phagocytic cells

Enteropathogenic Escherichia coli (EPEC) are a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. Enteropathogenic Escherichia coli encode a type III secretion system (TTSS) to transfer effector proteins into host cells, a process which is essential for virulence. In addition to generation of A/E lesions, the TTSS is also implicated in the ability of EPEC to invade cultured cells but the effector proteins responsible for promoting invasion have not been identified. In this paper we confirm the requirement of TTSS in EPEC invasion and demonstrate important roles for the Map and Tir effector molecules. Whereas in trans expression of Tir in the tir mutant restored invasion to wild-type levels, similar complementation of the map mutation by in trans expression of Map results in a hyperinvasive phenotype. The Map effector protein has two distinct functions within host cells, mediating Cdc42-dependent filopodia formation and targeting mitochondria to elicit dysfunction. The former function appears to be related to Map's ability to promote invasion as this was inhibited by interference with Cdc42 signalling. Conversely, Map targeting to mitochondria is not necessary for invasion. Promotion of EPEC invasion by Tir appears to involve interaction with intimin but is independent of pedestal formation, and intimin-Tir interaction is neither necessary nor sufficient for invasion. Comparison of the invasiveness of strains lacking Tir and/or Map with wild-type or mutant strains expressing the effectors in trans provides evidence that Map and Tir stimulate invasion by synergistic mechanisms. This synergism, which is in stark contrast to the antagonistic actions of Map and Tir in regulating filopodia and pedestal formation, further illustrates the complex interplay between EPEC effectors.     

Enteropathogenic Escherichia coli Tir translocation and pedestal formation requires membrane cholesterol in the absence of bundle-forming pili

Enteropathogenic Escherichia coli (EPEC) is a significant cause of paediatric diarrhoea worldwide. Virulence requires adherence to intestinal epithelial cells, mediated in part through type IV bundle-forming pili (BFP), and the EPEC protein Tir. Tir is inserted into the enterocyte plasma membrane (PM), resulting in the formation of actin-rich pedestals. Tir is translocated by the type III secretion system (TTSS), through a pore comprised of EPEC proteins inserted into the PM. Here, we demonstrate that in the absence of BFP, EPEC adherence, effector translocation and pedestal formation are dependent on lipid rafts. Lipid raft disruption using methyl-beta-cyclodextrin (MbetaCD) decreased adherence by an EPEC BFP-deficient strain from 85% to 1%. Translocation of the effectors Tir and EspF was blocked by MbetaCD treatment, although the TTSS pore still formed. MbetaCD treatment after Tir delivery decreased pedestal formation by EPEC from 40% to 5%, but not by the related pathogen E. coli O157:H7 which uses a different Tir-based mechanism. In contrast, EPEC expressing the BFP can circumvent the requirement for membrane cholesterol. This suggests that lipid rafts play a role in virulence of this medically important pathogen.     

A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12-residue sequence of EPEC Tir containing Y474 that confers actin-signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation-dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.     

Enterohaemorrhagic Escherichia coli Tir requires a C-terminal 12-residue peptide to initiate EspF-mediated actin assembly and harbours N-terminal sequences that influence pedestal length

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) both utilize type III secretion systems that translocate the effector protein Tir into the plasma membrane of mammalian cells in order to stimulate localized actin assembly into 'pedestals'. The Tir molecule that EPEC delivers is phosphorylated within its C-terminus on tyrosine-474, and a clustered 12-residue phosphopeptide encompassing this residue initiates an efficient signalling cascade that triggers actin polymerization. In addition to Y474, tyrosine-454 of EPEC Tir is phosphorylated, although inefficiently, and promotes actin polymerization at low levels. In contrast to EPEC Tir, EHEC Tir lacks Y474 and triggers pedestal formation in a phosphotyrosine-independent manner by interacting with an additional effector protein, EspF(U). To identify EHEC Tir sequences that regulate localized actin assembly, we circumvented the strict requirements for type III translocation and directly expressed Tir derivatives in mammalian cells by transfection. Infection of Tir-expressing cells with a Tir-deficient EHEC strain demonstrated that ectopically expressed Tir localizes to the plasma membrane, is modified by mammalian serine-threonine kinases and is fully functional for actin pedestal formation. Removal of portions of the cytoplasmic N-terminus of Tir resulted in the generation of abnormally long pedestals, indicating that this region of EHEC Tir influences pedestal length. In the presence of the entire N-terminal domain, a 12-residue peptide from the C-terminus of EHEC Tir is both necessary and sufficient to recruit EspF(U) and initiate actin pedestal formation. This peptide encompasses the portion of EHEC Tir analogous to the EPEC Tir-Y454 region and is present within the Tir molecules of all pedestal-forming bacteria, suggesting that this sequence harbours a conserved signalling function.     

The bacterial effectors EspG and EspG2 induce a destructive calpain activity that is kept in check by the co‐delivered Tir effector

Bacterial pathogens deliver multiple effector proteins into eukaryotic cells to subvert host cellular processes and an emerging theme is the cooperation between different effectors. Here, we reveal that a fine balance exists between effectors that are delivered by enteropathogenic E. coli (EPEC) which, if perturbed can have marked consequences on the outcome of the infection. We show that absence of the EPEC effector Tir confers onto the bacterium a potent ability to destroy polarized intestinal epithelia through extensive host cell detachment. This process was dependent on the EPEC effectors EspG and EspG2 through their activation of the host cysteine protease calpain. EspG and EspG2 are shown to activate calpain during EPEC infection, which increases significantly in the absence of Tir – leading to rapid host cell loss and necrosis. These findings reveal a new function for EspG and EspG2 and show that Tir, independent of its bacterial ligand Intimin, is essential for maintaining the integrity of the epithelium during EPEC infection by keeping the destructive activity of EspG and EspG2 in check.     

Dynamin is required for F-actin assembly and pedestal formation by enteropathogenic Escherichia coli (EPEC)

After attaching to human intestinal epithelial cells, enteropathogenic Escherichia coli (EPEC) induces the formation of an actin-rich pedestal-like structure. The signalling pathway leading to pedestal formation is initiated by the bacterial protein Tir, which is inserted into the host cell plasma membrane. The domain exposed on the cell surface binds to another bacterial protein, intimin, while one of the cytoplasmic domains binds the adaptor protein Nck. This leads to recruitment of other cytoskeletal proteins including neural Wiskott-Aldrich syndrome protein (N-WASP) and Arp2/3, resulting in focused actin polymerization at the site of bacterial attachment. In this study we investigated the role of the large GTPase dynamin 2 (Dyn2) in pedestal formation. We found that in HeLa cells, both endogenous and overexpressed Dyn2 were recruited to sites of EPEC attachment. Recruitment of endogenous Dyn2 required the presence of Tir, Nck and N-WASP but was independent of cortactin and Arp2/3. Knock-down of Dyn2 expression by RNA interference reduced actin polymerization and pedestal formation. Overexpression of dominant-negative mutants of Dyn2 also reduced pedestal formation and prevented recruitment of N-WASP, Arp3 and cortactin, but not Nck. Together, our results indicate that Dyn2 is an integral component of the signalling cascade leading to actin polymerization in EPEC pedestals.     

Enterohaemorrhagic and enteropathogenic Escherichia coli use a different Tir-based mechanism for pedestal formation

Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild-type EPEC Tir and the non-tyrosine-phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.     

CesT is a bivalent enteropathogenic Escherichia coli chaperone required for translocation of both Tir and Map

Map is an enteropathogenic Escherichia coli (EPEC) protein that is translocated into eukaryotic cells by a type III secretion system. Although not required for the induction of attaching and effacing (A/E) lesion formation characteristic of EPEC infection, translocated Map is suggested to disrupt mitochondrial membrane potential, which may impact upon subsequent functions of the organelle such as control of cell death. Before secretion, many effector proteins are maintained in the bacterial cytosol by association with a specific chaperone. In EPEC, chaperones have been identified for the effector proteins translocated intimin receptor (Tir) and EspF, and for the translocator proteins EspB and EspD. In this study, we present evidence that the Tir-specific chaperone, CesT, also performs a chaperone function for Map. Using a combination of biochemical approaches, we demonstrate specific interaction between CesT and Map. Similar to other chaperone-effector pairings, binding is apparent at the amino-terminus of Map and is indicated to proceed by a similar mechanism to CesT:Tir interaction. Map secretion from a cesT mutant strain (SE884) is shown to be reduced and, importantly, its translocation from this strain after infection of HEp-2 cells is almost totally abrogated. Although other chaperones are reported to have a bivalent binding specificity, CesT is the first member of its family that chaperones more than one protein for translocation.