Interaction of the enteropathogenic Escherichia coli protein, translocatedintimin receptor (Tir), with focal adhesion proteins

When enteropathogenic Escherichia coli (EPEC) attach and infect host cells, they induce a cytoskeletal rearrangement and the formation of cytoplasmic columns of actin filaments called pedestals. The attached EPEC and pedestal s move over the surface of the host cell in an actin-dependent reaction [Sa nger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the mem brane of host cells, where it binds the EPEC outer membrane protein, intimi n [Kenny et al., 1997: Cell 91:511-520], suggests Tir serves two functions: tethering the bacteria to the host cell and providing a direct connection to the host's cytoskeleton. The sequence of Tir predicts a protein of 56.8 kD with three domains separated by two predicted trans-membrane spanning re gions. A GST-fusion protein of the N-terminal 233 amino acids of Tir (Tir1) binds to alpha-actinin, talin, and vinculin from cell extracts. GST-Tir1 a lso coprecipitates purified forms of alpha-actinin, talin, and vinculin whi le GST alone does not bind these three focal adhesion proteins. Biotinylate d probes of these three proteins also bound Tir1 cleaved from GST. Similar associations of alpha-actinin, talin, and vinculin were also detected with the C-terminus of Tir, i.e., Tir3, the last 217 amino acids. Antibody stain ing of EPEC-infected cultured cells reveals the presence of focal adhesion proteins beneath the attached bacteria. Our experiments support a model in which the cytoplasmic domains of Tir recruit, a number of focal adhesion pr oteins that can bind actin filaments to form pedestals. Since pedestals als o contain villin, tropomyosin and myosin IT [Sanger et al., 1996: Cell Moti l. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure s haring properties of both focal adhesions and microvilli. Cell Motil. Cytos keleton 47:307-318, 2000. (C) 2000 Wiley-Liss, Inc.