title: The N-terminus of enteropathogenic Escherichia coli (EPEC) Tir mediates transport across bacterial and eukaryotic cell membranes.
abstract: Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to translocate into host cells several effector molecules that are required for virulence. One of these, the translocated intimin receptor, Tir, inserts into the host cell cytoplasmic membrane, where it functions as the receptor for intimin, an outer membrane adhesin expressed by EPEC. A chaperone for Tir, called CesT, is required for stability of Tir in the EPEC cytoplasm. In this study, the cyaA gene reporter system was used to identify domains in Tir that mediate secretion into the culture supernatant and translocation into host cells. A Tir-CyaA fusion containing the first 15 N-terminal residues of Tir was secreted and translocated into HeLa cells by a deltatirdeltacesT mutant; however, maximal secretion and translocation was observed with the first 26 N-terminal residues of Tir. Fusions containing progressively larger N-terminal sequences of Tir were also efficiently secreted and translocated into HeLa cells by the deltatirdeltacesT strain. However, in a deltatir mutant that expresses CesT, Tir26-CyaA and an additional fusion containing the first 69 N-terminal residues of Tir were not secreted or translocated, but fusions containing larger N-terminal Tir sequences were secreted and translocated by the deltatir mutant. Wild-type EPEC secreted and translocated the Tir15-CyaA fusion, whereas longer fusions, such as Tir26-CyaA and Tir69-CyaA, were translocated to higher levels, similar to what was observed with the deltatirdeltacesT mutant. A Tir-CyaA fusion containing the CesT binding domain was translocated into HeLa cells more rapidly in the presence of CesT compared with translocation in the absence of CesT. Collectively, these results suggest that an N-terminal domain of 26 amino acids functions as a CesT-independent signal that is capable of delivering Tir into both the culture supernatant and the cytosol of host cells. Furthermore, in addition to its role in the stability of Tir, CesT may function in translocation by mediating rapid delivery of Tir into host cells.
authors.affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors.forename: J Adam
authors.initials: JA
authors.lastname: Crawford
authors.forename: James B
authors.initials: JB
authors.lastname: Kaper
mesh_terms: D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D017077:Culture Media, Conditioned; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C122044:CesT protein, E coli; D017077:Culture Media, Conditioned; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C109287:Tir protein, E coli