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.     

Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.     

Enterohaemorrhagic Escherichia coli O157:H7 Shiga‐like toxin 1 is required for full pathogenicity and activation of the p38 mitogen‐activated protein kinase pathway in Caenorhabditis elegans

Enterohaemorrhagic Escherichia coli (EHEC) causes life‐threatening infections in humans as a consequence of the production of Shiga‐like toxins. Lack of a good animal model system currently hinders in vivo study of EHEC virulence by systematic genetic methods. Here we applied the genetically tractable animal, Caenorhabditis elegans, as a surrogate host to study the virulence of EHEC as well as the host immunity to this human pathogen. Our results show that E. coli O157:H7, a serotype of EHEC, infects and kills C. elegans. Bacterial colonization and induction of the characteristic attaching and effacing (A/E) lesions in the intact intestinal epithelium of C. elegans by E. coli O157:H7 were concomitantly demonstrated in vivo. Genetic analysis indicated that the Shiga‐like toxin 1 (Stx1) of E. coli O157:H7 is a virulence factor in C. elegans and is required for full toxicity. Moreover, the C. elegans p38 mitogen‐activated protein kinase (MAPK) pathway, anevolutionarily conserved innate immune and stress response signalling pathway, is activated in the regulation of host susceptibility to EHEC infection in a Stx1‐dependent manner. Our results validate the EHEC–C. elegans interaction as suitable for future comprehensive genetic screens for both novel bacterial and host factors involved in the pathogenesis of EHEC infection.     

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.     

Enterohaemorrhagic and enteropathogenic Escherichia coli use different mechanisms for actin pedestal formation that converge on N-WASP

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), two closely related diarrhoeagenic pathogens, induce actin rearrangements at the surface of infected host cells resulting in the formation of pseudopod-like structures termed pedestals beneath intimately attached bacteria. We have shown previously that N-WASP, a key integrator of signalling pathways that regulate actin polymerization via the Arp2/3 complex, is essential for pedestal formation induced by EPEC using N-WASP-defective cell lines. Here we show that actin pedestal formation initiated by EHEC also depends on N-WASP. Amino acid residues 226-274 of N-WASP are both necessary and sufficient to target N-WASP to sites of EHEC attachment. The recruitment mechanism thus differs from that used by EPEC, in which amino-terminal sequences of N-WASP mediate recruitment. For EPEC, recruitment of N-WASP downstream of Nck has been postulated to be mediated by WIP. However, we find a direct interaction of N-WASP with WIP to be dispensable for EPEC-induced pedestal formation and present data supporting an F-actin-dependent localization of WIP to actin pedestals induced by both EPEC and EHEC. In summary, our data show that EPEC and EHEC use different mechanisms to recruit N-WASP, which is essential for actin pedestal formation induced by both pathogens.     

Enterohemorrhagic E. coli Requires N-WASP for Efficient Type III Translocation but Not for EspFU-Mediated Actin Pedestal Formation

Upon infection of mammalian cells, enterohemorrhagic E. coli (EHEC) O157:H7 utilizes a type III secretion system to translocate the effectors Tir and EspF_U (aka TccP) that trigger the formation of F-actin-rich ‘pedestals’ beneath bound bacteria. EspF_U is localized to the plasma membrane by Tir and binds the nucleation-promoting factor N-WASP, which in turn activates the Arp2/3 actin assembly complex. Although N-WASP has been shown to be required for EHEC pedestal formation, the precise steps in the process that it influences have not been determined. We found that N-WASP and actin assembly promote EHEC-mediated translocation of Tir and EspF_U into mammalian host cells. When we utilized the related pathogen enteropathogenic E. coli to enhance type III translocation of EHEC Tir and EspF_U, we found surprisingly that actin pedestals were generated on N-WASP-deficient cells. Similar to pedestal formation on wild type cells, Tir and EspF_U were the only bacterial effectors required for pedestal formation, and the EspF_U sequences required to interact with N-WASP were found to also be essential to stimulate this alternate actin assembly pathway. In the absence of N-WASP, the Arp2/3 complex was both recruited to sites of bacterial attachment and required for actin assembly. Our results indicate that actin assembly facilitates type III translocation, and reveal that EspF_U, presumably by recruiting an alternate host factor that can signal to the Arp2/3 complex, exhibits remarkable versatility in its strategies for stimulating actin polymerization.     

Actin and alpha-actinin dynamics in the adhesion and motility of EPEC and EHEC on host cells

Two pathogenic Escherichia coli, Enteropathogenic E. coli (EPEC) and Enterohemorrhagic E. coli (EHEC), adhere to the outside of host cells and induce cytoskeletal rearrangements leading to the formation of membrane-encased pedestals comprised of actin filaments and other associated proteins beneath the bacteria. The structure of the pedestals induced by the two pathogens appears similar, although those induced by EHEC are shorter in length. Fluorescence Recovery After Photobleaching (FRAP) was used to determine potential differences of actin polymerization in EPEC and EHEC induced pedestals in cultured PtK2 cells expressing either Green or Yellow Fluorescent Protein (GFP or YFP) fused to actin or alpha-actinin. When all the fluorescent actin in a pedestal on EPEC-infected cells was photobleached, fluorescence recovery first occurred directly beneath the bacterium in a band that grew wider at a rate of one micron/minute. Consistently observed in all EPEC-induced pedestals, whether they were stationary, lengthening, or translocating, the rate of actin polymerization that occurred at the pedestal tip was approximately 1 mum/min. Overall, a much slower rate of actin polymerization was measured in long EHEC-induced pedestals. In contrast to the dynamics of GFP-actin, recovery of GFP-alpha-actinin fluorescence was not polarized, with the actin cross-linking protein exchanging all the length of the EPEC/EHEC induced pedestals. Surprisingly, the depolymerization and retrograde flow of pedestal actin, as well as pedestal translocations, were inhibited reversibly by either 2,3-butanedione monoxime (BDM) or by a combination of sodium azide and 2-deoxy D-glucose, leading to an increase in the lengths of the pedestals. A simple physical model was developed to describe elongation and translocation of EPEC/EHEC pedestals in terms of actin polymerization and depolymerization dynamics.     

Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection in mice

Enterohemorrhagic Escherichia coli (EHEC) is an important food-borne pathogen, which causes a wide spectrum of diseases ranging from hemorrhagic colitis to life-threatening hemolytic uremic syndrome (HUS). Currently, insufficient measures to prevent and treat EHEC infection make a vaccine against EHEC in great demand. EspA (E. coli secreted protein A), intimin, and Stx2 (Shiga toxin 2) are three predominant virulence factors of EHEC, and each of them has proved to be capable of inducing partial protective immunity. In this study, we constructed a trivalent recombinant protein designated EIS that is composed of EspA (E), C-terminal 300 amino acids of intimin (I) and B subunit of Stx2 (S), and tested it as vaccine using a mouse model. Our results showed that immunization of EIS induced strong humoral response to EspA, intimin and Stx2 and protected mice against the challenges with live EHEC or EHEC sonicated lysate. Moreover, it enhanced clearance of intestinally colonized bacteria. This work suggests that for EHEC vaccines using a combination of EspA, intimin and Stx2 antigens appears to be more effective than using any of these immunogens alone.     

Distribution of additional virulence factors related to adhesion and toxicity in Shiga toxin‐producing Escherichia coli isolated from raw products in Argentina

A total of 73 Shiga toxin‐producing Escherichia coli (STEC) isolates, belonging to 25 serotypes and isolated from raw products in Argentina, were examined for the occurrence of genes responsible for bacterial adhesions to intestine, ehaA (EHEC autotransporter), lpfAO113 (long polar fimbriae), sab (STEC autotransporter [AT] contributing to biofilm formation), ecpA (E. coli common pilus), hcpA (haemorrhagic coli pilus), elfA (E. coli laminin‐binding fimbriae), sfpA (sorbitol‐fermenting EHEC O157 fimbriae plasmid‐encoded) and of the toxigenic gene cdt‐V (cytolethal distending toxin). Our study showed different adhesin profiles that are not linked to one specific serotype and that all analysed isolates possess, besides stx genes, some adherence genes. Several of the isolates contained also multiple toxin genes. The results of the present work alert the presence of genes coding for additional adhesins and cdt‐V toxin in LEE‐negative STEC strains that occur in foods, and this traits could increase their pathogenic potential.

Significance and Impact of the Study

Meat products are one of the main vehicles of Shiga toxin‐producing E. coli, and the presence of genes coding for additional adhesins and toxins could increase their pathogenic potential. There is a need for a more detailed characterization of the strains in regard to these extra virulence factors.     

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.     

Cortactin is essential for F-actin assembly in enteropathogenic Escherichia coli (EPEC)- and enterohaemorrhagic E. coli (EHEC)-induced pedestals and the alpha-helical region is involved in the localization of cortactin to bacterial attachment sites

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are important human pathogens. Upon attachment to host cells, EPEC and EHEC are able to induce actin polymerization, which accumulates, forming a pedestal-like structure beneath the attached bacteria. Using siRNA, we show here that EPEC- and EHEC-induced pedestals are dependent on cortactin, an F-actin-binding protein found in the mammalian cell cortex. Knock-down of cortactin by siRNA resulted in a dramatic reduction of the pedestal formation induced by both pathogens. We also show that disruption of the Src homology 3 (SH3) domain of cortactin, or its downregulation by specific point mutations, negatively affects pedestal formation, suggesting that this domain is important for regulation of F-actin assembly by EPEC and EHEC. Green fluorescent protein (GFP) fused with the SH3 domain (GFP-SH3), proline-rich region (GFP-PRR) or alpha-helical region of cortactin markedly reduced the amount of F-actin at the bacterial attachment sites. Interestingly, neither GFP-SH3 nor GFP-PRR was recruited to the vicinity of the bacterial adherence sites; however, GFP fused to the alpha-helical region was efficiently recruited and colocalized with the attached bacteria. These results demonstrate that cortactin is a requirement for pedestal formation and suggest a novel function for the predicted alpha-helical region of cortactin in actin assembly induced by EPEC and EHEC.     

Changes in Pulsed-Field Gel Electrophoresis Patterns in Clinical Isolates of Enterohemorrhagic Escherichia coli O157:H7 Associated with Loss of Shiga Toxin Genes

Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments of enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains showed disappearance of a 70- or 80-kb fragment in their patterns associated with loss of Shiga toxin genes during maintenance or subcultivation. Hybridization experiments with a DNA probe complementary to Shiga toxin sequences revealed that the Shiga toxin genes in the parental strain were located on fragments the same size as the lost fragments from the toxin-negative derivatives. The evidence indicates that PFGE pattern of EHEC O157:H7 may change due to loss of Shiga toxin genes, which is likely to be associated with curing of Shiga toxin gene carrying phages in vitro. Received: 4 May 1998 / Accepted: 19 August 1998     

EspFU, a type III-translocated effector of actin assembly, fosters epithelial association and late-stage intestinal colonization by E. coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 induces filamentous actin-rich 'pedestals' on intestinal epithelial cells. Pedestal formation in vitro requires translocation of bacterial effectors into the host cell, including Tir, an EHEC receptor, and EspF_U, which increases the efficiency of actin assembly initiated by Tir. While inactivation of espF _U does not alter colonization in two reservoir hosts, we utilized two disease models to explore the significance of EspF_U-promoted actin pedestal formation. EHECΔ espF _U efficiently colonized the rabbit intestine during co-infection with wild-type EHEC, but co-infection studies on cultured cells suggested that EspF_U produced by wild-type bacteria might have rescued the mutant. Significantly, EHECΔ espF _U by itself was fully capable of establishing colonization at 2 days post inoculation but unlike wild type, failed to expand in numbers in the caecum and colon by 7 days. In the gnotobiotic piglet model, an espF _U deletion mutant appeared to generate actin pedestals with lower efficiency than wild type. Furthermore, aggregates of the mutant occupied a significantly smaller area of the intestinal epithelial surface than those of the wild type. Together, these findings suggest that, after initial EHEC colonization of the intestinal surface, EspF_U may stabilize bacterial association with the epithelial cytoskeleton and promote expansion beyond initial sites of infection.     

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.     

TLR9 limits enteric antimicrobial responses and promotes microbiota‐based colonisation resistance during Citrobacter rodentium infection

Mammalian cells express an array of toll‐like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human‐specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C. rodentium. Infected Tlr9^?/? mice suffered exaggerated intestinal damage and carried significantly higher (10–100 fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C. rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF‐κB signalling in the intestines of Tlr9^?/? mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9^?/? mice as compared with WT mice. Notably, antibiotic‐based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9^?/? mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota‐mediated colonisation resistance against C. rodentium infection.     

EspH,a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42-dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.     

A Family of Indoles Regulate Virulence and Shiga Toxin Production in Pathogenic E. coli

Enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and enteroaggregative E. coli (EAEC) are intestinal pathogens that cause food and water-borne disease in humans. Using biochemical methods and NMR-based comparative metabolomics in conjunction with the nematode Caenorhabditis elegans, we developed a bioassay to identify secreted small molecules produced by these pathogens. We identified indole, indole-3-carboxaldehyde (ICA), and indole-3-acetic acid (IAA), as factors that only in combination are sufficient to kill C. elegans. Importantly, although lethal to C. elegans, these molecules downregulate several bacterial processes important for pathogenesis in mammals. These include motility, biofilm formation and production of Shiga toxins. Some pathogenic E. coli strains are known to contain a Locus of Enterocyte Effacement (LEE), which encodes virulence factors that cause “attaching and effacing” (A/E) lesions in mammals, including formation of actin pedestals. We found that these indole derivatives also downregulate production of LEE virulence factors and inhibit pedestal formation on mammalian cells. Finally, upon oral administration, ICA inhibited virulence and promoted survival in a lethal mouse infection model. In summary, the C. elegans model in conjunction with metabolomics has facilitated identification of a family of indole derivatives that broadly regulate physiology in E. coli, and virulence in pathogenic strains. These molecules may enable development of new therapeutics that interfere with bacterial small-molecule signaling.     

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.     

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.