FIG. 6.

YadA-mediated invasion of epithelial cells via fibronectin (Fn) molecules. About 5 × 10⁴ HEp-2 cells were preincubated for 30 min at 37°C in RPMI 1640 medium supplemented with 0.4% bovine serum albumin and 20 mM HEPES (pH 7.0) without or with human cellular fibronectin (Fn) and for 30 min with certain dilutions of MAb (Ab) P4C10 directed against β₁ integrins (A) or 3E1 directed against human fibronectin (B). Subsequently, about 5 × 10⁶ bacteria of strains CC118/pAM226 (inv⁺), grown in LB medium, and CC118/pPD284 (yadA⁺), grown in LB medium supplemented with 0.2% arabinose, were added to the cell monolayer, which was incubated for 1 h at 20°C to determine cell attachment or at 37°C to monitor invasion efficiency. The total number of adherent bacteria was determined by plating the bacteria after separation from cells, and the number of intracellular bacteria was determined by the gentamicin protection assay as described in reference 17. Each value represents the mean of three independent assays done in triplicate.