Enterotoxigenic Escherichia coli TibA glycoprotein adheres to human intestine epithelial cells

Abstract

Enterotoxigenic Escherichia coli (ETEC) is capable of invading epithelial cell lines derived from the human ileum and colon. Two separate invasion loci (tia and tib) that direct noninvasive E. coli strains to adhere to and invade cultured human intestine epithelial cells have previously been isolated from the classical ETEC strain H10407. The tib locus directs the synthesis of TibA, a 104-kDa outer membrane glycoprotein. Synthesis of TibA is directly correlated with the adherence and invasion phenotypes of the tib locus, suggesting that this protein is an adhesin and invasin. Here we report the purification of TibA and characterization of its biological activity. TibA was purified by continuous-elution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified TibA was biotin labeled and then shown to bind to HCT8 human ileocecal epithelial cells in a specific and saturable manner. Unlabeled TibA competed with biotin-labeled TibA, suggesting the presence of a specific TibA receptor in HCT8 cells. These results show that TibA acts as an adhesin. Polyclonal anti-TibA antiserum inhibited invasion of ETEC strain H10407 and of recombinant E. coli bearing tib locus clones, suggesting that TibA also acts as an invasin. The ability of TibA to direct epithelial cell adhesion suggests a role for this protein in ETEC pathogenesis.

FIG. 1

Purification of TibA. (A) Coomassie blue-stained SDS-PAGE (7.5% polyacrylamide). Lanes: 1, outer membranes (20 μg of protein) from E. coli DH5α(pET109); 2, 5 μg of purified TibA. (B) Coomassie blue-stained SDS-PAGE (7.5% polyacrylamide) of outer membranes prepared from E. coli DH5α bearing either the TibA-expressing plasmid pET109 (lane 1, 20 μg of protein) or pHG165 (the vector for plasmid pET109) (lane 2, 40 μg of protein). (C) SDS-PAGE (7.5% polyacrylamide) gel of the indicated samples was transferred to nitrocellulose and then stained for glycoprotein as described in Materials and Methods. Lanes: 1, purified TibA (5 μg); 2, outer membranes prepared from E. coli DH5α(pHG165) (20 μg of protein). The positions of molecular mass standards (in kilodaltons) are indicated to the left of each panel. The migration of TibA is indicated by an arrowhead to the right of each panel.

FIG. 2

Binding of purified and biotinylated TibA to HCT8 monolayers. Bound TibA was detected by peroxidase-coupled streptavidin. Color development in each well was measured at 405 nm. Symbols: ♦, saturation curve performed with 0.0, 1.0, 2.4, 4.8, 9.6, 19.2, and 48.0 pmol of TibA per well; ▴, competition curve performed with 4.8 pmol of labeled TibA plus 0.0, 1.0, 4.8, 9.6, or 48.0 pmol of unlabeled TibA. Every experiment was performed at least three times. The extent of biotin labeling varied from day to day, resulting in significant differences in color development. Therefore, both curves shown here represent the average of two experiments performed in duplicate.

FIG. 3

Characterization of anti-TibA antiserum. SDS-PAGE (7.5% polyacrylamide) of the indicated samples was transferred to nitrocellulose and then probed with the indicated sera. (A) Immunoblot performed with preabsorbed and affinity-purified rabbit polyclonal anti-TibA IgG (1:1,000 dilution) as the primary antibody. Lanes: 1, 20 μg of outer membranes from E. coli DH5α(pHG165); 2, 20 μg of outer membranes from E. coli DH5α(pET109); 3, 1 μg of purified TibA. (B) Immunoblot performed with unabsorbed rabbit polyclonal anti-TibA antiserum (1:1,000 dilution) as the primary antibody. Lanes: 1, whole-cell lysate of E. coli DH5α(pET109); 2, whole-cell lysate of E. coli DH5α(pHG165). For all blots, the nitrocellulose membrane was stained with Ponceau S after transfer and before blocking to ensure that transfer was uniform. The migration of molecular mass standards (in kilodaltons) is shown on the left of each panel. The migration of TibA is indicated by an arrowhead to the right of each panel.

FIG. 4

Inhibition of TibA-mediated invasion by anti-TibA antiserum. Invasion assays were performed in the absence of antibodies (▪) or in the presence of preabsorbed and affinity-purified IgG from rabbit preimmune serum or polyclonal anti-TibA antiserum. (A) Invasion of HCT8 cells relative to E. coli DH5α(pET109), representing 100% (actual invasion of this strain in the absence of antibody was 1.30% ± 0.05%). Statistically significant effects of antibody treatment on the invasion efficiency of DH5α(pET109) are indicated by ∗ (P < 0.01) or ∗∗ (P < 0.005) as determined by analysis of variance of three experiments, each performed in triplicate. (B) Invasion of HCT8 cells relative to ETEC strain H10407, representing 100% (actual invasion of this strain in the absence of antibody was 0.24% ± 0.02%). TIB3 is a tib locus deletion mutant of H10407. Data are shown as averages for three replicates. Statistically significant effects of antibody treatment on the invasion efficiency of H10407 are indicated by ∗ (P < 0.01) as determined by analysis of variance of three experiments, each performed in triplicate.