Epithelial Cell Adherence Mediated by the Enterotoxigenic Escherichia coli Tia Protein

Abstract

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 ␤-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.Diarrheal disease is a persistent problem in developing countries. Enterotoxigenic Escherichia coli (ETEC) is a leading cause of this disease, and is responsible for about 600 million cases of diarrhea worldwide, annually resulting in over 1.2 million deaths, 700,000 of which occur in children under the age of 5 years (35). ETEC infections are initiated by the consumption of contaminated food or drink. The bacteria then colonize the small intestine via fimbrial colonization factor antigens. Subsequent to colonization, the bacteria release heat-stable and/or heat-labile enterotoxins that lead to a net secretion of fluid and diarrhea (28). However, human and animal challenge studies performed with ETEC strains that no longer produce heat-stable or heat-labile enterotoxins indicate that enterotoxins may not be exclusively required for diarrhea (21,30,31,34). This result suggests the presence of previously uncharacterized enterotoxins or other virulence factors in ETEC strains.Many diarrheal pathogens are capable of penetrating intestine epithelial cells. Although there is currently no direct evidence that epithelial cell invasion occurs during ETEC pathogenesis in humans, it has been found that ETEC strains are capable of invading epithelial cell lines derived from the human ileocecum and colon (10). Additionally, intestinal biopsy samples taken from ETEC-infected piglets have shown intracellular bacteria ...

Interaction of an Outer Membrane Protein of Enterotoxigenic Escherichia coli with Cell Surface Heparan Sulfate Proteoglycans

“…When added to HCT-8 epithelial cell monolayers immediately prior to inoculation with bacteria, sp.tia76-94 (final concentration, 100 g/ml) reduced invasion of DH5␣(pET185) by 80% Ϯ 3.4%. This peptide is nearly identical to the peptide used in the study of Tia topology of Mammarappallil and Elsinghorst, which showed that this region participates in the Tia-mediated interaction with epithelial cells (36). sp.tia76-94 (Fig.…”

The Different Strategies Used by Salmonella to Invade Host Cells

“…Tia and Hek are predicted to have eight transmembrane regions, four long exposed extracellular loops and three short periplasmic turns (Mammarappallil & Elsinghorst, 2000;Fagan et al, 2008). Thus, PagN probably adopts a similar conformation as that of Hek and Tia.…”

Cytotoxic and Apoptotic Effects of Recombinant Subtilase Cytotoxin Variants of Shiga Toxin-Producing Escherichia coli

“…subAB 1 ) operon, two new chromosomal variants have been described. The subAB operon is located on the chromosomal pathogenicity island SEPAI, close to the tia gene encoding an invasin in enterotoxigenic E. coli (ETEC) (23,24). The subAB 2-1 genes are about 90% homologous to the plasmid-carried subAB 1 with the catalytic triad being present in subA 2-1 , but the subB 2-1 open reading frame is one triplet shorter than that of subB 1 .…”