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. aureusinvades 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.

Bacteria have developed various mechanisms for inducing internalization into nonprofessional phagocytes (8,9). A shared requirement is that a molecular interaction must occur between a bacterial surface adhesin and a host ligand in the cytoplasmic membrane. This interaction must be of sufficiently high affinity to induce signal transduction through the membrane, resulting in cytoskeletal rearrangements and uptake of the organism (14, 37). For some organisms, a simple model involving the binding of a bacterial adhesin to its host receptor is sufficient to induce uptake. Yersinia species and Listeria monocytogenes are examples of organisms with this pattern of uptake. The adhesins on these facultative intracellular pathogens bind directly to integrins or E-cadherin, respectively, with an affinity that is sufficient to induce internalization (15,24).A number of reports have described bacterial surface adhesins, which adhere to host extracellular matrix (ECM) proteins. The ECM binding proteins are termed MSCRAMMS, and Staphylococcus aureus expresses several of these proteins with different ligand specificities (17,18,27). We demonstrated previously that the S. aureus surface adhesin responsible for stimulating signal transduction upon uptake by nonprofessional phagocytes is one of its MSCRAMMs, fibronectin (Fn) binding protein (FnBP) (6). Our data were confirmed in two additional publications (22,28). This finding raised several questions regarding the nature of the molecular interactions at the host cell surface.Fn is the ECM protein commonly associated with integrins. It is known that Fn is bivalent and can serve as a bridging molecule between FnBP and the host cell integrins (17,26, 39). Although other bacteria use Fn as a link to adhere to host tissues, the mechanisms by which this linkage could induce internalization are less clear. For example, Tran Van Nhieu and Isberg showed that coating of S. aureus with Fn did not lead to efficient internalization (37). It was proposed that the binding affinity between Fn and integrins was not sufficient to induce uptake. Interestingly, at least one organism, Neisseria gonorrhoeae has overcome this limitation by employing a heparin-containing accessory coreceptor, which is necessary to induce maximal internalization by HEp-2 cells (39).The present study was initiated to identify potential cellular ligands and other molecular requirements for uptake of S. aureus by nonprofessional phagocytes. Using a variety of methods, we found that FnBP binds directly to heat shock protein 60 (Hsp60) on the membranes of human and bovine epithelial cells. Fn and ␤ 1 integrins are also required for maximal uptake. Based on these combined results, a potential model to explain the molecular interactions leading to uptake of S. aureus is proposed.

SummaryStaphylococcus aureus invades a variety of mammalian cells and escapes from the endosome to multiply in the cytoplasm. We had previously hypothesized that the molecular events leading to escape of S. aureus from the endosome involved the Agr virulence factor regulatory system. In this report we demonstrate that temporal changes in intracellular activation of the Agr regulon correlates with expression of membrane active toxins. Also, the initial expression of Agr by even small numbers of staphylococci resulted in the permeabilization of the endosomal membrane and the eventual escape of bacteria into the cytoplasm by 3 h post invasion. After Agr downregulation, a second peak of expression coincided with increased permeability of the host cell membrane. In contrast to the parental strain, an Agrmutant was unable to escape into the cytoplasm and was observed in intact endosomes as late as 5 h post invasion. These data provide evidence that staphylococcal virulence factor production during invasion of host cells is mediated by an Agr-dependent process that is most accurately described in the context of diffusion sensing.

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