Identification of the intimin-binding domain of Tir of enteropathogenic Escherichia coli.
Enteropathogenic Escherichia coli (EPEC) attaches intimately to mammalian cells via a bacterial outer membrane adhesion molecule, intimin, and its receptor in the host cell membrane, Tir. Tir is a bacterial protein translocated into the host cell membrane and tyrosine phosphorylated after insertion. Tir-intimin binding induces organized actin polymerization beneath the adherent bacteria, resulting in the formation of pedestal-like structures. A series of Tir deletion derivatives were constructed to analyse which Tir domains are involved in intimin binding. We have localized the intimin-binding domain (IBD) of Tir using a yeast two-hybrid system and a gel-overlay approach to a region of 109 amino acids that is predicted to be exposed on the surface of the plasma membrane. A truncated Tir protein lacking this domain was translocated to the host cell membrane and tyrosine phosphorylated, but failed to bind intimin or to induce either actin polymerization or Tir accumulation beneath the bacteria. These results indicate that only a small region of Tir is needed to bind intimin and support the predicted topology for Tir, with both N- and C-terminal regions in the mammalian cell cytosol. They also confirm that Tir-intimin interactions are needed for cytoskeletal organization. We have also identified N-terminal regions involved in Tir stability and Tir secretion to the media.
de Grado
M
M
Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Abe
A
A
Gauthier
A
A
Steele-Mortimer
O
O
DeVinney
R
R
Finlay
B B
BB
D000199:Actins / Q000378:metabolism; D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000382:microbiology* / Q000473:pathology; D004926:Escherichia coli / Q000737:chemistry / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005455:Fluorescent Antibody Technique; D017353:Gene Deletion; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
D000199:Actins; D018829:Adhesins, Bacterial; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C109287:Tir protein, E coli; C073819:eaeA protein, E coli; D014443:Tyrosine