Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells

Abstract

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

FIG. 1

Internalization of S. aureus 8325-4 and DU5883 by MAC-T cells. A dose-response internalization assay was performed by inoculating MAC-T cell monolayers with various numbers of bacterial cells to generate the MOI indicated. After 1 h of incubation, extracellular bacteria were killed with gentamicin and the number of intracellular bacteria was determined. Data points represent the means of results from three separate but identically processed infected MAC-T cultures. Results of one representative experiment are shown in this figure. The experiment was performed 10 times.

FIG. 2

Effect of D3 peptide on internalization of S. aureus 8325-4 by MAC-T cells. D3 peptide was added to MAC-T cells 15 min prior to infection and remained in the medium during the experiment. Data are presented as percentage of bacteria (mean and standard error of the mean) internalized in the absence of D3 peptide and represent the mean of triplicate values. An MOI of 5 was used for these experiments, and 100% internalization was equivalent to 4 × 10⁴ CFU.

FIG. 3

Comparison of internalization and adherence of S. aureus 8325-4 and DU5883 by MAC-T cells. Monolayers were incubated with bacteria for 1 h at an MOI of 20. Thereafter, either extracellular bacteria were killed by adding gentamicin or the wells were washed and the adherent and internalized bacteria were quantified. The number of adherent bacteria was calculated by subtracting the internalized bacteria from the total count. To assess the effect of Fn, bacterial cells were pretreated with Fn at the concentration shown and washed before being added to the monolayers. Data are presented as the percentage of bacteria adherent or internalized compared to the original inoculum and represent the means and standard errors of the mean of three independent experiments.

FIG. 4

Effect of genistein on internalization of S. aureus 8325-4 by MAC-T cells. MAC-T cells were pretreated with genistein (250 μM) for 15 min immediately prior to inoculation with bacteria (MOI = 11). Control cultures had no genistein added. Genistein was retained in all cultures for 60 min. After 60 min, the genistein was removed by washing the monolayers and internalized bacteria were quantified immediately (0) or after an additional period of incubation (minutes) indicated on the abscissa. Data are presented as the percentage of bacteria internalized in the absence of genistein and are means and standard errors of the mean of three independent experiments.