Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: Role in internalization by epithelial cells

We reported previously that internalization of Staphylococcus aureus by non professional phagocytes involves an interaction between fibronectin (Fn) bi nding protein (FnBP) and the host cell, resulting in signal transduction, t yrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J . M. Patti, C. F. Deobald, K. W. Bayles, W. R, Trumble, and G, A, Bohach, i nfect, Immun, 67:4673-4678, 1999), The goal of the present study was to ide ntify the host molecules responsible for uptake of the organism through an interaction with FnBP, First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp -2 cells, which are deficient in Fn synthesis, Fn antibodies blocked intern alization of the organism by MAC-T cell monolayers, a bovine epithelial cel l line which expresses Fn, Second, a monoclonal antibody (MAb) specific for beta (1) integrins dramatically reduced S. aureus invasion, suggesting tha t the formation of a Fn bridge linking the host cell beta (1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane pr oteins with a functional fragment of FnBP consistently identified an additi onal similar to 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as h eat shock protein 60 (Hsp60), The interaction between FnBP and Hsp60 also o ccurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell memb rane, Pretreatment of epithelial cells with a MAb specific for eukaryotic H sp60 significantly reduced internalization of S. aureus, Combined, these re sults suggest that the FnBP binds directly to both Hsp60 and Fn and is link ed to beta (1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta (1) integrins and Hsp60, suggests tha t FnBP is a multifunctional adhesin that mediates internalization in a mann er similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolo g (J. P. M, van Putten, T. D. Duensing, and R. L. Cole, Mel. Microbiol, 29: 369-379, 1998).