Interaction of the enteropathogenic Escherichia coli protein, translocated intimin 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 pedestals move over the surface of the host cell in an actin-dependent reaction [Sanger et al., 1996: Cell Motil Cytoskeleton 34:279-287]. The discovery that EPEC inserts the protein, translocated intimin receptor (Tir), into the membrane of host cells, where it binds the EPEC outer membrane protein, intimin [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 regions. 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 also coprecipitates purified forms of alpha-actinin, talin, and vinculin while GST alone does not bind these three focal adhesion proteins. Biotinylated 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 staining 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 proteins that can bind actin filaments to form pedestals. Since pedestals also contain villin, tropomyosin and myosin II [Sanger et al., 1996: Cell Motil. Cytoskeleton 34:279-287], the pedestals appear to be a novel structure sharing properties of both focal adhesions and microvilli.
Freeman
N L
NL
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6058, USA.
Zurawski
D V
DV
Chowrashi
P
P
Ayoob
J C
JC
Huang
L
L
Mittal
B
B
Sanger
J M
JM
Sanger
J W
JW
D000185:Actinin / Q000378:metabolism; D000595:Amino Acid Sequence; D019921:Biotinylation; D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D022001:Focal Adhesions / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D016608:Talin / Q000378:metabolism; D016596:Vinculin / 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; D005982:Glutathione Transferase