A nanobody targeting the translocated intimin receptor inhibits the attachment of enterohemorrhagic E. coli to human colonic mucosa.

Enterohemorrhagic E. coli (EHEC) is a human intestinal pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. No vaccines or specific therapies are currently available to prevent or treat these infections. EHEC tightly attaches to the intestinal epithelium by injecting the intimin receptor Tir into the host cell via a type III secretion system (T3SS). In this project, we identified a camelid single domain antibody (nanobody), named TD4, that recognizes a conserved Tir epitope overlapping the binding site of its natural ligand intimin with high affinity and stability. We show that TD4 inhibits attachment of EHEC to cultured human HeLa cells by preventing Tir clustering by intimin, activation of downstream actin polymerization and pedestal formation. Furthermore, we demonstrate that TD4 significantly reduces EHEC adherence to human colonic mucosa in in vitro organ cultures. Altogether, these results suggest that nanobody-based therapies hold potential in the development of much needed treatment and prevention strategies against EHEC infection.

The Intriguing Interaction of Escherichia coli with the Host Environment and Innovative Strategies To Interfere with Colonization: a Summary of the 2019 E. coli and the Mucosal Immune System Meeting.

The third E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from 2 to 5 June 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease and covering a broad range of pathotypes, including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic Escherichia coli As it is well established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host.

Oxygen and contact with human intestinal epithelium independently stimulate virulence gene expression in enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are important intestinal pathogens causing acute and persistent diarrhoeal illness worldwide. Although many putative EAEC virulence factors have been identified, their association with pathogenesis remains unclear. As environmental cues can modulate bacterial virulence, we investigated the effect of oxygen and human intestinal epithelium on EAEC virulence gene expression to determine the involvement of respective gene products in intestinal colonisation and pathogenesis. Using in vitro organ culture of human intestinal biopsies, we established the colonic epithelium as the major colonisation site of EAEC strains 042 and 17-2. We subsequently optimised a vertical diffusion chamber system with polarised T84 colon carcinoma cells for EAEC infection and showed that oxygen induced expression of the global regulator AggR, aggregative adherence fimbriae, E. coli common pilus, EAST-1 toxin, and dispersin in EAEC strain 042 but not in 17-2. Furthermore, the presence of T84 epithelia stimulated additional expression of the mucinase Pic and the toxins HlyE and Pet. This induction was dependent on physical host cell contact and did not require AggR. Overall, these findings suggest that EAEC virulence in the human gut is modulated by environmental signals including oxygen and the intestinal epithelium.

Attaching and effacing (A/E) lesion formation by enteropathogenic E. coli on human intestinal mucosa is dependent on non-LEE effectors.

Enteropathogenic E. coli (EPEC) is a human pathogen that causes acute and chronic pediatric diarrhea. The hallmark of EPEC infection is the formation of attaching and effacing (A/E) lesions in the intestinal epithelium. Formation of A/E lesions is mediated by genes located on the pathogenicity island locus of enterocyte effacement (LEE), which encode the adhesin intimin, a type III secretion system (T3SS) and six effectors, including the essential translocated intimin receptor (Tir). Seventeen additional effectors are encoded by genes located outside the LEE, in insertion elements and prophages. Here, using a stepwise approach, we generated an EPEC mutant lacking the entire effector genes (EPEC0) and intermediate mutants. We show that EPEC0 contains a functional T3SS. An EPEC mutant expressing intimin but lacking all the LEE effectors but Tir (EPEC1) was able to trigger robust actin polymerization in HeLa cells and mucin-producing intestinal LS174T cells. However, EPEC1 was unable to form A/E lesions on human intestinal in vitro organ cultures (IVOC). Screening the intermediate mutants for genes involved in A/E lesion formation on IVOC revealed that strains lacking non-LEE effector/s have a marginal ability to form A/E lesions. Furthermore, we found that Efa1/LifA proteins are important for A/E lesion formation efficiency in EPEC strains lacking multiple effectors. Taken together, these results demonstrate the intricate relationships between T3SS effectors and the essential role non-LEE effectors play in A/E lesion formation on mucosal surfaces.

Intestinal Colonization Traits of Pandemic Multidrug-Resistant Escherichia coli ST131.

Background: Epidemiological studies point to the gut as a key reservoir of multidrug resistant Escherichia coli multilocus sequence type 131 (ST131), a globally dominant pathogenic clone causing urinary tract and bloodstream infections. Here we report a detailed investigation of its intestinal lifestyle. Methods: Clinical ST131 isolates and type 1 fimbriae null mutants were assessed for colonization of human intestinal epithelia and in mouse intestinal colonization models. Mouse gut tissue underwent histologic analysis for pathology and ST131 localization. Key findings were corroborated in mucus-producing human cell lines and intestinal biopsy specimens. Results: ST131 strains adhered to and invaded human intestinal epithelial cells more than probiotic and commensal strains. The reference ST131 strain EC958 established persistent intestinal colonization in mice, and expression of type 1 fimbriae mediated higher colonization levels. Bacterial loads were highest in the distal parts of the mouse intestine and did not cause any obvious pathology. Further analysis revealed that EC958 could bind to both mucus and underlying human intestinal epithelia. Conclusions: ST131 strains can efficiently colonize the mammalian gut and persist long term. Type 1 fimbriae enhance ST131 intestinal colonization, suggesting that mannosides, currently developed as therapeutics for bladder infections and Crohn's disease, could also be used to limit intestinal ST131 reservoirs.

Shiga toxin 2 translocation across intestinal epithelium is linked to virulence of Shiga toxin-producing Escherichia coli in humans.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the release of potent Shiga toxins (Stx), which are associated with severe intestinal and renal disease. Although all STEC strains produce Stx, only a few serotypes cause infection in humans. To determine which virulence traits in vitro are linked to human disease in vivo, 13 Stx2a-producing STEC strains of seropathotype (SPT) A or B (associated with severe human intestinal disease and outbreaks) and 6 strains of SPT D or E (rarely or not linked to human disease) were evaluated in a microaerobic human colonic epithelial infection model. All SPT strains demonstrated similar growth, colonization of polarized T84 colon carcinoma cells and Stx release into the medium. In contrast, Stx translocation across the T84 cell monolayer was significantly lower in SPT group DE compared to SPT group AB strains. Further experiments showed that Stx penetration occurred via a transcellular pathway and was independent of bacterial type III secretion and attaching and effacing lesion formation. These results suggest that the extent of Stx transcytosis across the gut epithelium may represent an important indicator of STEC pathogenicity for humans.

The StcE metalloprotease of enterohaemorrhagic Escherichia coli reduces the inner mucus layer and promotes adherence to human colonic epithelium ex vivo.

Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen and tightly adheres to human colonic epithelium by forming attaching/effacing lesions. To reach the epithelial surface, EHEC must penetrate the thick mucus layer protecting the colonic epithelium. In this study, we investigated how EHEC interacts with the intestinal mucus layer using mucin-producing LS174T colon carcinoma cells and human colonic mucosal biopsies. The level of EHEC binding and attaching/effacing lesion formation in LS174T cells was higher compared to mucin-deficient colon carcinoma cell lines, and initial adherence was independent of the presence of flagellin, Escherichia coli common pilus, or long polar fimbriae. Although EHEC infection did not affect gene expression of secreted mucins, it resulted in reduced MUC2 glycoprotein levels. This effect was dependent on the catalytic activity of the secreted metalloprotease StcE, which reduced the inner mucus layer and thereby promoted EHEC access and binding to the epithelium in vitro and ex vivo. Given the lack of efficient therapies against EHEC infection, StcE may represent a suitable target for future treatment and prevention strategies.

Enterohemorrhagic Escherichia coli colonization of human colonic epithelium in vitro and ex vivo.

Enterohemorrhagic Escherichia coli (EHEC) is an important foodborne pathogen causing gastroenteritis and more severe complications, such as hemorrhagic colitis and hemolytic uremic syndrome. Pathology is most pronounced in the colon, but to date there is no direct clinical evidence showing EHEC binding to the colonic epithelium in patients. In this study, we investigated EHEC adherence to the human colon by using in vitro organ culture (IVOC) of colonic biopsy samples and polarized T84 colon carcinoma cells. We show for the first time that EHEC colonizes human colonic biopsy samples by forming typical attaching and effacing (A/E) lesions which are dependent on EHEC type III secretion (T3S) and binding of the outer membrane protein intimin to the translocated intimin receptor (Tir). A/E lesion formation was dependent on oxygen levels and suppressed under oxygen-rich culture conditions routinely used for IVOC. In contrast, EHEC adherence to polarized T84 cells occurred independently of T3S and intimin and did not involve Tir translocation into the host cell membrane. Colonization of neither biopsy samples nor T84 cells was significantly affected by expression of Shiga toxins. Our study suggests that EHEC colonizes and forms stable A/E lesions on the human colon, which are likely to contribute to intestinal pathology during infection. Furthermore, care needs to be taken when using cell culture models, as they might not reflect the in vivo situation.

Shiga toxin production and translocation during microaerobic human colonic infection with Shiga toxin-producing E. coli†O157:H7 and O104:H4.

Haemolytic uraemic syndrome caused by Shiga toxin-producing E. coli (STEC) is dependent on release of Shiga toxins (Stxs) during intestinal infection and subsequent absorption into the bloodstream. An understanding of Stx-related events in the human gut is limited due to lack of suitable experimental models. In this study, we have used a vertical diffusion chamber system with polarized human colon carcinoma cells to simulate the microaerobic (MA) environment in the human intestine and investigate its influence on Stx release and translocation during STEC O157:H7 and O104:H4 infection. Stx2 was the major toxin type released during infection. Whereas microaerobiosis significantly reduced bacterial growth as well as Stx production and release into the medium, Stx translocation across the epithelial monolayer was enhanced under MA versus aerobic conditions. Increased Stx transport was dependent on STEC infection and occurred via a transcellular pathway other than macropinocytosis. While MA conditions had a similar general effect on Stx release and absorption during infection with STEC O157:H7 and O104:H4, both serotypes showed considerable differences in colonization, Stx production, and Stx translocation which suggest alternative virulence strategies. Taken together, our study suggests that the MA environment in the human colon may modulate Stx-related events and enhance Stx absorption during STEC infection.

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier.

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS.

Development of a novel human intestinal model to elucidate the effect of anaerobic commensals on Escherichia coli infection.

The gut microbiota plays a crucial role in protecting against enteric infection. However, the underlying mechanisms are largely unknown owing to a lack of suitable experimental models. Although most gut commensals are anaerobic, intestinal epithelial cells require oxygen for survival. In addition, most intestinal cell lines do not produce mucus, which provides a habitat for the microbiota. Here, we have developed a microaerobic, mucus-producing vertical diffusion chamber (VDC) model and determined the influence of Limosilactobacillus reuteri and Ruminococcus gnavus on enteropathogenic Escherichia coli (EPEC) infection. Optimization of the culture medium enabled bacterial growth in the presence of mucus-producing T84/LS174T cells. Whereas L. reuteri diminished EPEC growth and adhesion to T84/LS174T and mucus-deficient T84 epithelia, R. gnavus only demonstrated a protective effect in the presence of LS174T cells. Reduced EPEC adherence was not associated with altered type III secretion pore formation. In addition, co-culture with L. reuteri and R. gnavus dampened EPEC-induced interleukin 8 secretion. The microaerobic mucin-producing VDC system will facilitate investigations into the mechanisms underpinning colonization resistance and aid the development of microbiota-based anti-infection strategies. This article has an associated First Person interview with the first author of the paper.

Dissecting the role of the Tir:Nck and Tir:IRTKS/IRSp53 signalling pathways in vivo.

Attaching and effacing (A/E) lesions and actin polymerization, the hallmark of enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) infections, are dependent on the effector Tir. Phosphorylation of Tir(EPEC/CR) Y474/1 leads to recruitment of Nck and neural Wiskott-Aldrich syndrome protein (N-WASP) and strong actin polymerization in cultured cells. Tir(EPEC/CR) also contains an Asn-Pro-Tyr (NPY(454/1)) motif, which triggers weak actin polymerization. In EHEC the NPY(458) actin polymerization pathway is amplified by TccP/EspF(U), which is recruited to Tir via IRSp53 and/or insulin receptor tyrosine kinase substrate (IRTKS). Here we used C. rodentium to investigate the different Tir signalling pathways in vivo. Following infection with wild-type C. rodentium IRTKS, but not IRSp53, was recruited to the bacterial attachment sites. Similar results were seen after infection of human ileal explants with EHEC. Mutating Y471 or Y451 in Tir(CR) abolished recruitment of Nck and IRTKS respectively, but did not affect recruitment of N-WASP or A/E lesion formation. This suggests that despite their crucial role in actin polymerization in cultured cells the Tir:Nck and Tir:IRTKS pathways are not essential for N-WASP recruitment or A/E lesion formation in vivo. Importantly, wild-type C. rodentium out-competed the tir tyrosine mutants during mixed infections. These results uncouple the Tir:Nck and Tir:IRTKS pathways from A/E lesion formation in vivo but assign them an important in vivo role.

Determining Shiga Toxin-Producing Escherichia coli Interactions with Human Intestinal Epithelium in a Microaerobic Vertical Diffusion Chamber.

The environment in the human intestine is low in oxygen. This affects virulence gene expression of enteropathogens including Shiga toxin-producing E. coli (STEC) and enables mucosal colonization by oxygen-sensitive commensal bacteria. To simulate the oxygen-restricted milieu at the intestinal epithelium, we have developed a vertical diffusion chamber model (VDC) which allows infection of polarized human intestinal epithelia under microaerobic conditions. In this chapter, we present a detailed protocol for performing STEC infections in the VDC system and subsequent analysis of STEC pathogenesis.

Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells.

Advances in the understanding of the pathogenesis of enterohaemorrhagic Escherichia coli (EHEC) have greatly benefited from the use of human epithelial cell lines under aerobic conditions. However, in the target site of EHEC infection, the human intestine, conditions are microaerobic. In our study we used polarized human colon carcinoma cells in a vertical diffusion chamber system to investigate the influence of reduced apical oxygen levels on EHEC colonization. While apical microaerobiosis did not affect cell integrity and barrier function, numbers of adherent bacteria were significantly increased under low compared with high apical oxygen concentrations. In addition, expression and translocation of EHEC type III secreted (T3S) effector proteins was considerably enhanced under microaerobic conditions and dependent on the presence of host cells. Increased colonization was mainly mediated via EspA as adherence levels of an isogenic deletion mutant were not influenced by low oxygen levels. Other potential adherence factors (E. coli common pilus and flagella) were only minimally expressed under high and low oxygen levels. Addition of nitrate and trimethylamine N-oxide as terminal electron acceptors for anaerobic respiration failed to further increase bacterial colonization or T3S under microaerobiosis. This study indicates that EHEC T3S and colonization are enhanced by the microaerobic environment in the gut and therefore might be underestimated in conventional aerobic cell culture systems.

The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection.

In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial-host cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC-negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.

Identification and characterisation of enteroaggregative Escherichia coli subtypes associated with human disease.

Enteroaggregative E. coli (EAEC) are a major cause of diarrhoea worldwide. Due to their heterogeneity and carriage in healthy individuals, identification of diagnostic virulence markers for pathogenic strains has been difficult. In this study, we have determined phenotypic and genotypic differences between EAEC strains of sequence types (STs) epidemiologically associated with asymptomatic carriage (ST31) and diarrhoeal disease (ST40). ST40 strains demonstrated significantly enhanced intestinal adherence, biofilm formation, and pro-inflammatory interleukin-8 secretion compared with ST31 isolates. This was independent of whether strains were derived from diarrhoea patients or healthy controls. Whole genome sequencing revealed differences in putative virulence genes encoding aggregative adherence fimbriae, E. coli common pilus, flagellin and EAEC heat-stable enterotoxin 1. Our results indicate that ST40 strains have a higher intrinsic potential of human pathogenesis due to a specific combination of virulence-related factors which promote host cell colonization and inflammation. These findings may contribute to the development of genotypic and/or phenotypic markers for EAEC strains of high virulence.

The Escherichia coli common pilus and the bundle-forming pilus act in concert during the formation of localized adherence by enteropathogenic E. coli.

Although the bundle-forming pilus (BFP) of enteropathogenic Escherichia coli (EPEC) mediates microcolony formation on epithelial cells, the adherence of BFP-deficient mutants is significantly abrogated, but the mutants are still adherent due to the presence of intimin and possibly other adhesins. In this study we investigated the contribution of the recently described E. coli common pilus (ECP) to the overall adherence properties of EPEC. We found that ECP and BFP structures can be simultaneously observed in the course (between zero time and 7 h during infection) of formation of localized adherence on cultured epithelial cells. These two pilus types colocalized at different levels of the microcolony topology, tethering the adhering bacteria. No evidence of BFP disappearance was found after prolonged infection. When expressed from a plasmid present in nonadherent E. coli HB101, ECP rendered this organism highly adherent at levels comparable to those of HB101 expressing the BFP. Purified ECP bound in a dose-dependent manner to epithelial cells, and the binding was blocked with anti-ECP antibodies, confirming that the pili possess adhesin properties. An ECP mutant showed only a modest reduction in adherence to cultured cells due to background expression levels of BFP and intimin. However, isogenic mutants not expressing EspA or BFP were significantly less adherent when the ecpA gene was also deleted. Furthermore, a DeltaespA DeltaecpA double mutant (unable to translocate Tir and to establish intimate adhesion) was at least 10-fold less adherent than the DeltaespA and DeltaecpA single mutants, even in the presence of BFP. A Delta bfp DeltaespA DeltaecpA triple mutant showed the least adherence compared to the wild type and all the isogenic mutant strains tested, suggesting that ECP plays a synergistic role in adherence. Our data indicate that ECP is an accessory factor that, in association with BFP and other adhesins, contributes to the multifactorial complex interaction of EPEC with host epithelial cells.

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models.

Interactions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei. Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host-anaerobe and pathogen-commensal interaction studies in vitro.

Experimental models to study intestinal microbes-mucus interactions in health and disease.

A close symbiotic relationship exists between the intestinal microbiota and its host. A critical component of gut homeostasis is the presence of a mucus layer covering the gastrointestinal tract. Mucus is a viscoelastic gel at the interface between the luminal content and the host tissue that provides a habitat to the gut microbiota and protects the intestinal epithelium. The review starts by setting up the biological context underpinning the need for experimental models to study gut bacteria-mucus interactions in the digestive environment. We provide an overview of the structure and function of intestinal mucus and mucins, their interactions with intestinal bacteria (including commensal, probiotics and pathogenic microorganisms) and their role in modulating health and disease states. We then describe the characteristics and potentials of experimental models currently available to study the mechanisms underpinning the interaction of mucus with gut microbes, including in vitro, ex vivo and in vivo models. We then discuss the limitations and challenges facing this field of research.

Cortactin recruitment by enterohemorrhagic Escherichia coli O157:H7 during infection in vitro and ex vivo.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important human pathogen that colonizes the gut mucosa via attaching and effacing (A/E) lesions; A/E lesion formation in vivo and ex vivo is dependent on the type III secretion system (T3SS) effector Tir. Infection of cultured cells by EHEC leads to induction of localized actin polymerization, which is dependent on Tir and a second T3SS effector protein, TccP, also known as EspF(U). Recently, cortactin was shown to bind both the N terminus of Tir and TccP via its SH3 domain and to play a role in EHEC-triggered actin polymerization in vitro. In this study, we investigated the recruitment of cortactin to the site of EHEC adhesion during infection of in vitro-cultured cells and mucosal surfaces ex vivo (using human terminal ileal in vitro organ cultures [IVOC]). We have shown that cortactin is recruited to the site of EHEC adhesion in vitro downstream of TccP and N-WASP. Deletion of the entire N terminus of Tir or replacing the N-terminal polyproline region with alanines did not abrogate actin polymerization or cortactin recruitment. In contrast, recruitment of cortactin to the site of EHEC adhesion in IVOC is TccP independent. These results imply that cortactin is recruited to the site of EHEC adhesion in vitro and ex vivo by different mechanisms and suggest that cortactin might have a role during EHEC infection of mucosal surfaces.