The bacterial virulence factor NleA is required for the disruption of intestinal tight junctions by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) is a diarrhoeal pathogen that adheres to epithelial cells of the small intestine and uses a type III secretion system to inject effector proteins into host cells. EPEC infection leads to disruption of host intestinal tight junctions that are important for maintaining intestinal barrier function. This disruption is dependent on the bacterial type III secretion system, as well as the translocated effectors EspF and Map. Here we show that a third type III translocated bacterial effector protein, NleA, is also involved in tight junction disruption during EPEC infection. Using the drug Brefeldin A, we demonstrate that the effect of NleA on tight junction integrity is related to its inhibition of host cell protein trafficking through COPII-dependent pathways. These results suggest that NleA's striking effect on virulence is mediated, at least in part, via its role in disruption of intestinal barrier function.     

Cytokeratin 18 interacts with the enteropathogenic Escherichia coli secreted protein F (EspF) and is redistributed after infection

Enteropathogenic Escherichia coli (EPEC) pathogenesis requires the delivery of effector proteins into host cytosol by a type III secretion system. The effector protein EspF, while critical for disruption of epithelial barrier function through alteration of tight junctions, is not required for bacterial viability or attachment. Yeast two-hybrid analyses revealed host intermediate filament (IF) protein cytokeratin 18 (CK18) as an interacting partner of EspF. This was confirmed by co-immunoprecipitation of extracts from EPEC-infected epithelial cells. EPEC infection increased detergent-soluble CK18 amounts without significantly altering CK18 expression. The adaptor protein 14-3-3 binds to CK18 and modulates its solubility. EPEC infection promoted CK18/14-3-3 interactions, corresponding to the increase of CK18 in the soluble fractions. 14-3-3 also co-immunoprecipitated with EspF, suggesting that CK18, 14-3-3 and EspF may form a complex in infected cells. The 14-3-3zeta isoform was co-immunoprecipitated with CK18, suggesting the involvement of specific signalling pathways. Immunofluorescence studies revealed a dramatic alteration in the architecture of the IF network in EPEC-infected epithelial cells. IF fragmentation, evident at 2 h post infection, progressed to a collapse of this network at later time points. The secretion mutant (DeltaescN) failed to alter CK18 solubility and IF morphology, while deletion of espF partially impaired the ability of EPEC to induce CK18 modifications. These results suggest that modifications in 14-3-3 interactions and IF network, modulated by type III secreted proteins, may be an important step in EPEC pathogenesis.     

Attaching and effacing pathogen-induced tight junction disruption in vivo

Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption.     

A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway

Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway. These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE). In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus. Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells. Although these results do not indicate an obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.     

Role of EspF in host cell death induced by enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in children in developing countries. Many EPEC genes involved in virulence are contained within the locus of enterocyte effacement (LEE), a large pathogenicity island. One of the genes at the far righthand end of the LEE encodes EspF, an EPEC secreted protein of unknown function. EspF, like the other Esps, is a substrate for secretion by the type III secretory system. Previous studies found that an espF mutant behaved as wild type in assays of adherence, invasion, actin condensation and tyrosine phosphorylation. As EPEC can kill host cells, we tested esp gene mutants for host cell killing ability. The espF mutant was deficient in host cell killing despite having normal adherence. The addition of purified EspF to tissue culture medium did not cause any damage to host cells, but expression of espF in COS or HeLa cells caused cell death. The mode of cell death in cells transfected with espF appeared to be pure apoptosis. EspF appears to be an effector of host cell death in epithelial cells; its proline-rich structure suggests that it may act by binding to SH3 domains or EVH1 domains of host cell signalling proteins.     

Enteropathogenic Escherichia coli EspF is targeted to mitochondria and is required to initiate the mitochondrial death pathway

Enteropathogenic Escherichia coli (EPEC) is a causative agent of infant diarrhoea in developing countries. The EspF protein is the product of the espF gene found on the locus of enterocyte effacement, the key pathogenicity island carried by EPEC and enterohemorrhagic E. coli. EspF is injected from adherent EPEC into host cells via a type III secretion system and was previously shown to induce apoptotic cell death and to be required for disruption of host intestinal barrier function. In this work, we show by immunofluorescence and fractionation studies that EspF is targeted to host mitochondria. The N-terminal region of EspF serves as a mitochondrial import signal and, when expressed within cells, can target hybrid green fluorescent protein to mitochondria. Assessment of mitochondrial membrane potential in infected epithelial cells indicated that EspF plays a role in the mitochondrial membrane permeabilization induced by EPEC infection. Furthermore, EspF was associated with the release of cytochrome c from mitochondria into the cytoplasm and with caspase-9 and caspase-3 cleavage. These findings indicate a role for EspF in initiating the mitochondrial death pathway.     

Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein

The human intestinal pathogen, enteropathogenic Escherichia coli (EPEC), causes diarrhoeal disease by a mechanism that is dependent on the injection of effector proteins into the host cell. One effector, EspF, is reported to be required for EPEC to disrupt tight junction integrity of intestinal cells and increase the paracellular movement of molecules, which is likely to contribute to diarrhoea. Here, we show that not one but three EPEC-encoded factors play important roles in this process. Thus, the Map (Mitochondria-associated protein) effector is shown to: (i) be as essential as EspF for disrupting intestinal barrier function, (ii) be able to function independently of EspF, (iii) alter tight junction structure and (iv) mediate these effects in the absence of mitochondrial targeting. Additionally, the outer membrane protein Intimin is shown to be crucial for EspF and Map to disrupt the intestinal barrier function. This function of Intimin is completely independent of its interaction with its known receptor Tir, revealing a physiologically relevant requirement for Intimin interaction with alternative receptor(s). This work demonstrates that EPEC uses multiple multifunctional proteins to elicit specific responses in intestinal cells and that EPEC can control the activity of its injected effector molecules from its extracellular location.     

Enteropathogenic Escherichia coli effector EspF interacts with host protein Abcf2

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of infant diarrhoea in developing countries. The EspF effector protein is injected from EPEC into host cells via a type III secretion system and is involved in the disruption of host intestinal barrier function. In addition, EspF is sorted to mitochondria and has a role in initiating the mitochondrial death pathway. To clarify the manner in which EspF affects host cells, we sought to identify eukaryotic EspF-binding proteins using affinity purification. Abcf2, a protein of unknown function and member of the ABC-transporter family, bound EspF in this assay. An interaction between EspF and Abcf2 was confirmed in a yeast two-hybrid system, by colocalization and by co-immunoprecipitation from EPEC-infected cells. Levels of Abcf2 were decreased in cells infected with EPEC in an EspF dose-dependent manner. Knock-down of Abcf2 expression by RNA interference increased EspF-induced caspase 9 and caspase 3 cleavage. In addition, Abcf2-knocked down cells showed increased caspase 3 cleavage upon treatment with the apoptosis inducing agent staurosporine. These results indicate that EspF induces or facilitates host cell death by targeting and interfering with the putative protective function of Abcf2.     

EPEC effector EspF promotes Crumbs3 endocytosis and disrupts epithelial cell polarity

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inject effector proteins into host intestinal epithelial cells causing diarrhoea. EPEC infection redistributes basolateral proteins β1‐integrin and Na⁺/K⁺ ATPase to the apical membrane of host cells. The Crumbs (Crb) polarity complex (Crb3/Pals1/Patj) is essential for epithelial cell polarisation and tight junction (TJ) assembly. Here, we demonstrate that EPEC displaces Crb3 and Pals1 from the apical membrane to the cytoplasm of cultured intestinal epithelial cells and colonocytes of infected mice. In vitro studies show that EspF, but not Map, alters Crb3, whereas both effectors modulate Pals1. EspF perturbs polarity formation in cyst morphogenesis assays and induces endocytosis and apical redistribution of Na⁺/K⁺ ATPase. EspF binds to sorting nexin 9 (SNX9) causing membrane remodelling in host cells. Infection with ΔespF/pespFD3, a mutant strain that ablates EspF binding to SNX9, or inhibition of dynamin, attenuates Crb3 endocytosis caused by EPEC. In addition, infection with ΔespF/pespFD3 has no impact on Na⁺/K⁺ ATPase endocytosis. These data support the hypothesis that EPEC perturbs apical–basal polarity in an EspF‐dependent manner, which would contribute to EPEC‐associated diarrhoea by disruption of TJ and altering the crucial positioning of membrane transporters involved in the absorption of ions and solutes.     

Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for bacterial pathogenesis: critical role of the 16th leucine residue in EspF

The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features. EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors. In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), because EspF activity for importing into the mitochondria was abrogated by MTS deletion mutants. Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished EspF activity. Infection of HeLa cells with wild type but not the espF mutant (DeltaespF) decreased mitochondrial membrane potential (DeltaPsi(m)), leading to cell death. The DeltaPsi(m) decrease and cell death were restored in cells infected with DeltaespF/pEspF but not DeltaespF/pEspF(L16E), suggesting that the 16th leucine in the MTS is a critical amino acid for EspF function. To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with DeltaespF(CR), DeltaespF(CR)/pEspF(CR), or DeltaespF(CR)/pEspF(L16E)(CR). These results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.     

Identification of the secretion and translocation domain of the enteropathogenic and enterohemorrhagic Escherichia coli effector Cif, using TEM-1 beta-lactamase as a new fluorescence-based reporter

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) strains are human and animal pathogens that inject effector proteins into host cells via a type III secretion system (TTSS). Cif is an effector protein which induces host cell cycle arrest and reorganization of the actin cytoskeleton. Cif is encoded by a lambdoid prophage present in most of the EPEC and EHEC strains. In this study, we analyzed the domain that targets Cif to the TTSS by using a new reporter system based on a translational fusion of the effector proteins with mature TEM-1 beta-lactamase. Translocation was detected directly in living host cells by using the fluorescent beta-lactamase substrate CCF2/AM. We show that the first 16 amino acids (aa) of Cif were necessary and sufficient to mediate translocation into the host cells. Similarly, the first 20 aa of the effector proteins Map, EspF, and Tir, which are encoded in the same region as the TTSS, mediated secretion and translocation in a type III-dependent but chaperone-independent manner. A truncated form of Cif lacking its first 20 aa was no longer secreted and translocated, but fusion with the first 20 aa of Tir, Map, or EspF restored both secretion and translocation. In addition, the chimeric proteins were fully able to trigger host cell cycle arrest and stress fiber formation. In conclusion, our results demonstrate that Cif is composed of a C-terminal effector domain and an exchangeable N-terminal translocation signal and that the TEM-1 reporter system is a convenient tool for the study of the translocation of toxins or effector proteins into host cells.     

E. coli secreted protein F promotes EPEC invasion of intestinal epithelial cells via an SNX9‐dependent mechanism

Enteropathogenic Escherichia coli (EPEC) infection requires the injection of effector proteins into intestinal epithelial cells (IECs) via type 3 secretion. Type 3‐secreted effectors can interfere with IEC signalling pathways via specific protein–protein interactions. For example, E. coli secreted protein F (EspF) binds sorting nexin 9 (SNX9), an endocytic regulator, resulting in tubulation of the plasma membrane. Our aim was to determine the mechanism of EspF/SNX9‐induced membrane tubulation. Point mutation of the SNX9 lipid binding domains or truncation of the EspF SNX9 binding domains significantly inhibited tubulation, as did inhibition of clathrin coated pit (CCP) assembly. Although characterized as non‐invasive, EPEC are known to invade IECs in vitro and in vivo. Indeed, we found significant invasion of Caco‐2 cells by EPEC, which, like tubulation, was blocked by pharmacological inhibition of CCPs. Interestingly, however, inhibition of dynamin activity did not prevent tubulation or EPEC invasion, which is in contrast to Salmonella invasion, which requires dynamin activity. Our data also indicate that EPEC invasion is dependent on EspF and its interaction with SNX9. Together, these findings suggest that EspF promotes EPEC invasion of IECs by harnessing the membrane‐deforming activity of SNX9.     

EspJ effector in enterohemorrhagic E. coli translocates into host mitochondria via an atypical mitochondrial targeting signal

EHEC is a bacterial pathogen causing diarrhea and hemorrhagic colitis in humans. To exert virulence, EHEC exploits a subset of effectors that are translocated into host cells via the type III secretion system. EspJ, which was recently identified as a type III secreted effector, is conserved in related pathogens such as EPEC and Citrobacter rodentium. However, the exact function of EspJ remains unclear. In the present study, we found that EspJ was unstable in host cells, which might be attributable to the N‐terminal part beginning from amino acid number 59. Using stable forms of EspJ derivatives, we demonstrated for the first time that EspJ has the ability to translocate into mitochondria via an atypical mitochondrial targeting signal at the N terminus (1–36 a.a.) of EspJ. It has been reported that a mitochondrial targeting effector, EspF, disrupts the mitochondrial membrane potential, resulting in an induction of host cell death. To further investigate EspJ function in mitochondria, HeLa cells were infected with wild‐type EPEC, an isogenic EspJ‐mutant or an EspJ‐overexpressing strain. The result of LDH release assay using an EspJ‐mutant showed that the EspJ effector appears not to be involved in cytotoxicity.     

Enteropathogenic Escherichia coli infection leads to appearance of aberrant tight junctions strands in the lateral membrane of intestinal epithelial cells

Infection of intestinal epithelial cells with enteropathogenic Escherichia coli (EPEC) disrupts tight junction (TJ) architecture and barrier function. The aim of this study was to determine the impact of EPEC on TJ protein interactions and localization. Human intestinal epithelial cells (T84) were infected for 1, 3 or 6 h with EPEC. To probe the TJ protein-protein interactions, co-immunoprecipitations were performed. The associations between ZO-1, occludin and claudin-1 progressively decreased after infection. Corresponding morphological changes were analysed by immunofluorescence confocal microscopy. Tight junction proteins progressively lost their apically restricted localization. Freeze-fracture electron microscopy revealed the appearance of aberrant strands throughout the lateral membrane that contained claudin-1 and occludin as determined by immunogold labelling. These structural alterations were accompanied by a loss of barrier function. Mutation of the gene encoding EspF, important in the disruption of TJs by EPEC, prevented the disruption of TJs. Tight junction structure normalized following eradication of EPEC with gentamicin and overnight recovery. This is the first demonstration that a microbial pathogen can cause aberrant TJ strands in the lateral membrane of host cells. We speculate that the disruption of integral and cytoplasmic TJ protein interactions following EPEC infection allows TJ strands to form or diffuse into the lateral plasma membrane.     

Translocation of enteropathogenic Escherichia coli across an in vitro M cell model is regulated by its type III secretion system

Enteropathogenic Escherichia coli (EPEC) is an extracellular pathogen that utilizes a type III secretion system (TTSS) to modulate diverse host cell processes including cytoskeletal dynamics, tight junction permeability and macrophage phagocytosis. Some EPEC strains exhibit selective tropism for the specialized follicle-associated epithelium (FAE) overlying lymphoid follicles in the gut, which is a major site of uptake of inert particulates and pathogens, but do not translocate from the intestinal lumen in significant numbers. We have investigated the interaction of EPEC with FAE using an established in vitro model of the specialized FAE in which polarized enterocyte-like Caco-2 cells cocultured with the Raji B cell line undergo a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells found within FAE. Having confirmed that coculture with Raji B cells induces brush border reorganization and enhances particle transport across Caco-2 cells, we investigated translocation of bacteria across the M cell model. While Salmonella translocation was markedly upregulated by Raji coculture, transport of wild-type EPEC occurred at similarly low levels across both native Caco-2 and Caco-2/Raji-cocultured layers. Translocation rates were markedly higher for EPEC strains lacking either functional TTSS or the effector protein EspF. These observations resemble previously reported data on the inhibition of macrophage phagocytosis by EPEC, which has also been reported to be dependent on TTSS and EspF. Furthermore, as with macrophage phagocytosis, enhanced translocation of a TTSS mutant was blocked by wortmannin, implicating inhibition of phosphatidyl inositol 3-kinase-mediated signalling in the regulation of M cell translocation by EPEC.     

SepL, a protein required for enteropathogenic Escherichia coli type III translocation, interacts with secretion component SepD

Enteropathogenic Escherichia coli (EPEC), an important cause of infantile diarrhoea in the developing world, disrupts host cell microvilli, causes actin rearrangements and attaches intimately to the host cell surface. This characteristic phenotype, referred to as the attaching and effacing (A/E) effect, is encoded on a 36 kb pathogenicity island called the locus of enterocyte effacement (LEE). The LEE includes genes involved in type III secretion and translocation, the eae gene encoding an outer membrane adhesin known as intimin, the tir gene for the translocated intimin receptor, a regulator and various genes of unknown function. Among this last group is sepL. To determine the role of SepL in EPEC pathogenesis, we constructed and tested a non-polar sepL mutant. We found that this sepL mutant is deficient for A/E and that it secretes markedly reduced quantities of those proteins involved in translocation (EspA, EspB and EspD), but normal levels of those proteins presumed to be effectors (Tir, EspF and EspG). Despite normal levels of secretion, the mutant strain was unable to translocate EspF and Tir into host cells and formed no EspA filaments. Fractionation studies revealed that SepL is a soluble cytoplasmic protein. Yeast two-hybrid and affinity purification studies indicated that SepL interacts with the LEE-encoded protein SepD. In contrast to SepL, we found that SepD is required for type III secretion of both translocation and effector proteins. Together, these results demonstrate that SepL has a unique role in type III secretion as a functional component of the translocation system that interacts with an essential element of the secretion machinery.     

Enteropathogenic E. coli effectors EspG1/G2 disrupt microtubules,contribute to tight junction perturbation and inhibit restoration

Enteropathogenic Escherichia coli (EPEC) uses a type 3 secretion system to transfer effector proteins into the host intestinal epithelial cell. Several effector molecules contribute to tight junction disruption including EspG1 and its homologue EspG2 via a mechanism thought to involve microtubule destruction. The aim of this study was to investigate the contribution of EspG‐mediated microtubule disruption to TJ perturbation. We demonstrate that wild type EPEC infection disassembles microtubules and induces the progressive movement of occludin away from the membrane and into the cytosol. Deletion of espG1/G2 attenuates both of these phenotypes. In addition, EPEC infection impedes barrier recovery from calcium switch, suggesting that inhibition of TJ restoration, not merely disruption, prolongs barrier loss. TJs recover more rapidly following infection with ΔespG1/G2 than with wild type EPEC, demonstrating that EspG1/G2 perpetuate barrier loss. Although EspG regulates ADP‐ribosylation factor (ARF) and p21‐activated kinase (PAK), these activities are not necessary for microtubule destruction or perturbation of TJ structure and function. These data strongly support a role for EspG1/G2 and its associated effects on microtubules in delaying the recovery of damaged tight junctions caused by EPEC infection.     

The enteropathogenic Escherichia coli effector protein EspF decreases sodium hydrogen exchanger 3 activity

Enteropathogenic Escherichia coli (EPEC) have been previously shown to alter sodium hydrogen exchanger 3 (NHE3) activity in human intestinal epithelial cells. To further characterize these observations, PS120 fibroblasts transfected with NHE3 were studied. EPEC E2348/69 infection decreased NHE3 activity in PS120 fibroblasts. The effect on NHE3 was enhanced when PS120 cells were co-transfected with the scaffolding/regulatory proteins NHERF1 or NHERF2 or EBP50 and E3KARP respectively. The decrease in NHE3 activity was dependent on an intact type III secretion system, although intimate attachment mediated by translocated intimin receptor was not required. Despite its ability to bind to NHERF proteins, the EPEC effector Map had no impact on the regulation of NHE activity. Instead, EspF was found to be responsible for decreased NHE3 activity. However, neither EspF-induced apoptosis nor the interaction of EspF with sorting nexin-9, an endocytic protein, were involved.     

The type III effector EspF coordinates membrane trafficking by the spatiotemporal activation of two eukaryotic signaling pathways

Bacterial toxins and effector proteins hijack eukaryotic enzymes that are spatially localized and display rapid signaling kinetics. However, the molecular mechanisms by which virulence factors engage highly dynamic substrates in the host cell environment are poorly understood. Here, we demonstrate that the enteropathogenic Escherichia coli (EPEC) type III effector protein EspF nucleates a multiprotein signaling complex composed of eukaryotic sorting nexin 9 (SNX9) and neuronal Wiskott-Aldrich syndrome protein (N-WASP). We demonstrate that a specific and high affinity association between EspF and SNX9 induces membrane remodeling in host cells. These membrane-remodeling events are directly coupled to N-WASP/Arp2/3-mediated actin nucleation. In addition to providing a biochemical mechanism of EspF function, we find that EspF dynamically localizes to membrane-trafficking organelles in a spatiotemporal pattern that correlates with SNX9 and N-WASP activity in living cells. Thus, our findings suggest that the EspF-dependent assembly of SNX9 and N-WASP represents a novel form of signaling mimicry used to promote EPEC pathogenesis and gastrointestinal disease.     

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC ΔespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild‐type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two‐hybrid, proteomics, immunofluorescence and co‐immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC‐mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.