Internalization of Staphylococcus aureus by human corneal epithelial cells: role of bacterial fibronectin-binding protein and host cell factors

Abstract

Wild-type Staphylococcus aureus was observed to be capable of invading human corneal epithelial cells (HCEC) in vitro. Internalization of S. aureus required expression of fibronectin-binding proteins (FnBPs); the capacity of an FnBP-deficient isogenic strain to invade HCEC was reduced by more than 99%. The binding of S. aureus to HCEC did not require viable bacteria, since UV-killed cells were observed to adhere efficiently. Invasion of HCEC by S. aureus involved active host cell mechanisms; uptake was nearly completely eliminated by cytochalasin D and genistein. These data suggest that FnBPs play a key role in host-parasite interactions and may serve as an important adhesin or invasin in ulcerative keratitis caused by S. aureus.

FIG. 1.

Adhesion of S. aureus to HCEC. HCEC monolayers were incubated for 1 h in the presence of S. aureus 8325-4 (wild type) (A), UV-killed 8325-4 (B), ALC135 (agr sar) (C), or DU5883 (fnbAB) (D). Bacteria (arrows) were labeled with FITC prior to inoculation. Monolayers were stained with Alexa-Fluor 488 phalloidin and ethidium bromide. Magnification, ×20.

FIG. 2.

Invasion of HCEC by S. aureus. HCEC monolayers were incubated for 1 h in the presence of S. aureus. Monolayers were washed, and extracellular bacteria were killed by the addition of gentamicin. Monolayers were homogenized, serially diluted, and plated for enumeration of viable intracellular bacteria. Data represent mean values + standard errors of the means for duplicate experiments, each performed in triplicate. Asterisk indicates statistically significant reduction in internalization (P < 0.05, Student's t test) compared to wild-type internalization.

FIG. 3.

Inhibition of S. aureus invasion. HCEC monolayers were incubated for 1 h in the presence of inhibitors. Monolayers were incubated for an additional 1 h in the presence of inhibitors and wild-type S. aureus. Monolayers were washed, and extracellular bacteria were killed by the addition of gentamicin. Monolayers were homogenized, serially diluted, and plated for enumeration of viable intracellular bacteria. Data represent mean values + standard errors of the means for duplicate experiments, each performed in triplicate. Asterisks indicate statistically significant reductions in internalization (P < 0.05, Student's t test) compared to wild-type internalization.