Host focal adhesion protein domains that bind to the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC).
Enteropathogenic Escherichia coli (EPEC) attach to the plasma membrane of infected host cells and induce diarrhea in a variety of farm animals as well in humans. These bacteria inject a three-domain protein receptor, Tir (translocated intimin receptor), that is subsequently inserted into the plasma membrane. EPEC induce the host cell to form membrane-covered actin-rich columns called pedestals. Focal adhesion constituents, alpha-actinin, talin, and vinculin, are localized along the length of the pedestals and we have previously reported they bind the two cytoplasmic domains of Tir, (Tir I and Tir III) [Freeman et al., 2000: Cell Motil. Cytoskeleton 47:307-318]. In the present study, various constructs were made expressing different regions of these three focal adhesion proteins to determine which domains of the proteins bound Tir I. Three different assays were used to detect Tir I/host protein domain interactions. In co-precipitation assays, His-Tir I bound to the 27-kDa region of alpha-actinin; to four different domains of talin; and to the N-terminal domain of the vinculin head and the vinculin tail domain. A yeast two-hybrid analysis of Tir I and the various focal adhesion fusion proteins revealed a region near the C-terminus of talin was the only domain to interact with Tir I. Finally, to assess direct binding interactions, biotinylated Tir I was used in overlay assays and confirmed the binding of Tir I with the 27-kDa region of alpha-actinin, the four regions of talin, and the vinculin tail. These binding interactions between hostfocal adhesion proteins and EPEC Tir may facilitate the adhesion of EPEC to the host cell surface.
Huang
Lily
L
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6058, USA.
Mittal
Balraj
B
Sanger
Joseph W
JW
Sanger
Jean M
JM
D000185:Actinin / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016608:Talin / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D016596:Vinculin / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D016608:Talin; C109287:Tir protein, E coli; D000185:Actinin; D016596:Vinculin