Epithelial cell adherence mediated by the enterotoxigenic Escherichia coli tia protein.
In vitro studies have shown that enterotoxigenic Escherichia coli (ETEC) strains are capable of invading cultured epithelial cells derived from the human ileum and colon. Two separate invasion loci (tia and tib) have previously been isolated from the classical ETEC strain H10407. The tia locus has been shown to direct the synthesis of Tia, a 25-kDa outer membrane protein. Tia is sufficient to confer the adherence and invasion phenotypes on laboratory stains of E. coli, suggesting that this protein is an adhesin and invasin. Here we report the purification of Tia and characterize its biological activity. Tia was purified by electroelution of outer membrane proteins that had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified Tia was labeled with biotin and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Polyclonal anti-Tia antiserum blocked this binding. These results show that Tia acts as an adhesin. Polyclonal anti-Tia antiserum also inhibited invasion of recombinant E. coli bearing tia clones, indirectly suggesting that Tia may also act as an invasin. We predict Tia to contain eight transmembrane amphipathic beta-sheets with four loops that are exposed on the surface of the bacterial cell. A peptide corresponding to 19 residues in one of the four predicted surface-exposed loops inhibits Tia-mediated epithelial cell invasion. Seeding HCT8 cells on wells coated with purified Tia reduced Tia-mediated epithelial cell invasion. Together, these results indicate that Tia is an invasin and adhesin that binds a specific receptor on HCT8 cells.
Mammarappallil
J G
JG
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045-2106, USA.
Elsinghorst
E A
EA
D018830:Adhesins, Escherichia coli / Q000737:chemistry / Q000302:isolation & purification / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D004926:Escherichia coli / Q000472:pathogenicity*; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007413:Intestinal Mucosa / Q000382:microbiology*; D008969:Molecular Sequence Data; D011817:Rabbits
D018830:Adhesins, Escherichia coli; D007106:Immune Sera