title: Binding of intimin from enteropathogenic Escherichia coli to Tir and to host cells.
abstract: Enteropathogenic Escherichia coli (EPEC) induce characteristic attaching and effacing (A/E) lesions on epithelial cells. This event is mediated, in part, by binding of the bacterial outer membrane protein, intimin, to a second EPEC protein, Tir (translocated intimin receptor), which is exported by the bacteria and integrated into the host cell plasma membrane. In this study, we have localized the intimin-binding domain of Tir to a central 107-amino-acid region, designated Tir-M. We provide evidence that both the amino- and carboxy-termini of Tir are located within the host cell. In addition, using immunogold labelling electron microscopy, we have confirmed that intimin can bind independently to host cells even in the absence of Tir. This Tir-independent interaction and the ability of EPEC to induce A/E lesions requires an intact lectin-like module residing at the carboxy-terminus of the intimin polypeptide. Using the yeast two-hybrid system and gel overlays, we show that intimin can bind both Tir and Tir-M even when the lectin-like domain is disrupted. These data provide strong evidence that intimin interacts not only with Tir but also in a lectin-like manner with a host cell intimin receptor.
authors.affiliation: Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, UK.
authors.forename: E L
authors.initials: EL
authors.lastname: Hartland
authors.forename: M
authors.initials: M
authors.lastname: Batchelor
authors.forename: R M
authors.initials: RM
authors.lastname: Delahay
authors.forename: C
authors.initials: C
authors.lastname: Hale
authors.forename: S
authors.initials: S
authors.lastname: Matthews
authors.forename: G
authors.initials: G
authors.lastname: Dougan
authors.forename: S
authors.initials: S
authors.lastname: Knutton
authors.forename: I
authors.initials: I
authors.lastname: Connerton
authors.forename: G
authors.initials: G
authors.lastname: Frankel
mesh_terms: D018829:Adhesins, Bacterial; D001422:Bacterial Adhesion; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D002352:Carrier Proteins; D002460:Cell Line; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008957:Models, Genetic; D008958:Models, Molecular; D010957:Plasmids; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism*; D015003:Yeasts / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; C109287:Tir protein, E coli; C073819:eaeA protein, E coli; D001616:beta-Galactosidase