title: Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic Escherichia coli during ex vivo colonization of human intestinal mucosa is different to cell culture models.
abstract: Tir, the translocated intimin receptor of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) and Citrobacter rodentium, is translocated into the host cell by a filamentous type III secretion system. Epithelial cell culture has demonstrated that Tir tyrosine phosphorylation is necessary for attaching effacing (A/E) lesion formation by EPEC and C. rodentium, but is not required by EHEC O157:H7. Recent in vivo work on C. rodentium has reported that Tir translocation, but not its phosphorylation, is necessary for colonization of the mouse colon. In this study we investigated the involvement of Tir and its tyrosine phosphorylation in EPEC and EHEC human intestinal colonization, N-WASP accumulation and F-actin recruitment using in vitro organ culture (IVOC). We showed that both EPEC and EHEC Tir are translocated into human intestinal epithelium during IVOC and that Tir is necessary for ex vivo intestinal colonization by both EPEC and EHEC. EPEC, but not EHEC, Tir is tyrosine phosphorylated but Tir phosphorylation-deficient mutants still colonize intestinal explants. While EPEC Tir recruits the host adaptor protein Nck to initiate N-WASP-Arp2/3-mediated actin polymerization, Tir derivatives deficient in tyrosine phosphorylation recruit N-WASP independently of Nck indicating the presence of a tyrosine phosphorylation-independent mechanism of A/E lesion formation and actin recruitment ex vivo by EPEC in man.
authors.affiliation: Centre for Paediatric Gastroenterology, Royal Free and University College Medical School, London, UK.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Schüller
authors.forename: Yuwen
authors.initials: Y
authors.lastname: Chong
authors.forename: Jackie
authors.initials: J
authors.lastname: Lewin
authors.forename: Brendan
authors.initials: B
authors.lastname: Kenny
authors.forename: Gad
authors.initials: G
authors.lastname: Frankel
authors.forename: Alan D
authors.initials: AD
authors.lastname: Phillips
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D004927:Escherichia coli Infections / Q000378:metabolism / Q000382:microbiology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007413:Intestinal Mucosa / Q000378:metabolism / Q000382:microbiology* / Q000648:ultrastructure; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D008954:Models, Biological; D015513:Oncogene Proteins / Q000378:metabolism*; D009924:Organ Culture Techniques; D010766:Phosphorylation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000378:metabolism*
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D029968:Escherichia coli Proteins; C078332:Nck protein; D015513:Oncogene Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; D051316:Wiskott-Aldrich Syndrome Protein