SummarySubversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans -membrane receptor protein -translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir EPEC requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir EHEC is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Ncklike coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit a a a a -actinin, Arp3, N-WASP and actin to the site of bacterial adhesion.When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo . Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro . These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP ؉ . Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.

Enterohemorrhagic Escherichia coli, enteropathogenic E. coli, and Citrobacter rodentium are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions. These pathogens utilize a type III secretion system (TTSS) apparatus, encoded by the locus of enterocyte effacement, to translocate bacterial effector proteins into epithelial cells. Here, we report the identification of EspJ (E. coli-secreted protein J), a translocated TTSS effector that is carried on the 5 end of the cryptic prophage CP-933U. Infection of epithelial cells in culture revealed that EspJ is not required for A/E lesion activity in vivo and ex vivo. However, in vivo studies performed with mice demonstrated that EspJ possesses properties that influence the dynamics of clearance of the pathogen from the host's intestinal tract, suggesting a role in host survival and pathogen transmission. Enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC) (35), andCitrobacter rodentium (29) are highly adapted enteropathogens that successfully colonize their host's gastrointestinal tract via the formation of attaching and effacing (A/E) lesions (reviewed in references 13 and 28). These lesions are characterized by the localized destruction (effacement) of intestinal epithelial microvilli, an intimate attachment between the bacterium and the host cell apical membrane, and the formation of pedestal-like structures containing high concentrations of actin (26) and intermediate filaments (2) directly beneath sites of bacterial attachment. Formation of A/E lesions is mediated by a filamentous type III secretion system (FTTSS) apparatus (20, 42) responsible for the

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Using a DNA microarray, we determined changes in enterohemorrhagic Escherichia coli O157:H7 gene expression during binding to plasma membranes. Analysis of the complete transcriptomes of the bound bacteria revealed increased levels of stress-associated mRNAs and decreased levels of mRNA encoding proteins involved in translation and type III secretion.Enterohemorrhagic Escherichia coli (EHEC) is an important cause of acute gastroenteritis in humans (26). EHEC causes a wide spectrum of illnesses ranging from mild diarrhea to severe diseases, such as hemorrhagic colitis and hemolytic-uremic syndrome; hemolytic-uremic syndrome is the leading cause of acute renal failure in children and is associated with the production of potent Shiga toxins (Stx) (26). Strains of EHEC belonging to serogroup O157 are most commonly associated with severe human disease.Adhesion of EHEC to intestinal epithelial cell results in induction of a histopathological feature known as attaching and effacing lesions (8), which are characterized by localized destruction of brush border microvilli and intimate attachment of the bacteria to actin-rich pedestal-like structures that are formed on the apical membrane underneath attached bacteria. The capacity to form attaching and effacing lesions is encoded on a pathogenicity island termed the locus of enterocyte effacement (LEE) (21). The LEE can be divided into three functional regions (6); the 5Ј end consists of three operons (LEE1 to LEE3) encoding a positive regulator, Ler (23), and the main structural components of the bacterial type III secretion system (TTSS). The central part of the LEE harbors an operon (LEE5) encoding the outer membrane adhesin intimin (15) and the translocated intimin receptor (Tir) (17). The 3Ј end of the LEE (LEE4) encodes additional TTSS structural proteins, translocators, and effectors.TTSSs are commonly found in pathogenic, gram-negative bacteria (13) and are used to transfer effector proteins directly into eukaryotic cells, where the normal cellular functions are subverted for the benefit of the pathogen. Both the organization and the composition of TTSSs are broadly conserved in different bacteria, and TTSSs contain homologues of many flagellar components (1). However, the TTSS of EHEC is unique in that, in addition to the conserved cell wall-associated needle complex, it contains a filamentous extension to the needle formed by one of the secreted proteins, EspA (20, 27).The three-dimensional structure of the EspA filament shows that it consists of a helical tube with an outer diameter of ca. 120 Å containing a hollow central channel that is ca. 25 Å in diameter and has a continuous wall, which is likely to contain the proteins within the channel during transfer (2). Evidence suggests that EspA is the major component of this filamentous structure, while EspD, in complex with a second translocator protein, EspB, is predicted to form a translocation pore in the host cell membrane that facilitates subsequent entry of effector proteins (Tir, EspF, Map, EspG, EspH, Cif,...