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

Abstract

We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify 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 internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).

FIG. 1

Ligand blotting for FnBP receptors on epithelial cells. Proteins in MAC-T or HEp-2 cell membrane preparations were resolved by SDS-PAGE and transferred to membranes. The proteins were probed with Du-D4 peptide followed by Du-D4 rabbit antiserum (A) or with Hsp60 MAb (B).

FIG. 2

Affinity purification of potential host cell receptors on FnBP–Affi-Gel 102. Solubilized membrane fractions from MAC-T cells, HEp-2 cells, or Caco-2 cells were incubated with FnBP–Affi-Gel 102. The gel was then washed extensively. Proteins on the gel were eluted, analyzed by SDS-PAGE, stained, and in some experiments subjected to N-terminal sequence determinations. Control BSA–Affi-Gel 102 conjugates did not retain proteins from cell lysates (results not shown). std, protein standards.

FIG. 3

Immunoblot of biotin-tagged and nontagged Hsp60. Intact MAC-T or HEp-2 cells were biotinylated with sulfo-NHS-LC-biotin (lanes 2 and 3) or treated with sulfo-NHS-acetate (for MAC-T cells only) as a negative control (lane 1). Cell lysates were resolved by SDS-PAGE and probed with an Hsp60 MAb.

FIG. 4

FnBP-Hsp 60 interaction on cell surfaces. MAC-T cells were incubated for 30 min with no bacteria (lane 1), S. aureus DU5883 (lane 2), S. aureus DU5883(pFNBPA4) preincubated with Du-D4 rabbit antiserum (lane 3), or S. aureus DU5883(pFNBPA4) (lane 4). After lysis of the MAC-T cells, the residual bacterial cell suspension and attached proteins were recovered by centrifugation and the pellets were solubilized in SDS-PAGE sample buffer, resolved by SDS-PAGE, transferred to a membrane, and probed with Hsp60 MAb (A) or Du-D4 followed by Du-D4 antiserum (B). The protein bands shown in the figure correspond to Hsp 60 (apparent size, ∼55 kDa) (A and B) and S. aureus FnBPA (104 kDa) (B).

FIG. 5

Effect of Hsp60 MAb (A) or bovine Fn rabbit antiserum (B) on internalization of S. aureus DU5875 by MAC-T cells. MAC-T cell monolayers were preincubated for 30 min with serum or ascites proteins at the dilution or quantities indicated (MOI = 20). The number of internalized bacteria was determined in a standard internalization assay, and the results are presented as a percentage of the number in identical control cultures treated with nonimmune ascites (A) or normal rabbit serum (B).

FIG. 6

Effect of Fn on internalization of staphylococci by HEp-2 or MAC-T cells. Monolayers were incubated for 30 min with Fn, at the concentrations shown, prior to infection with the bacterial suspension (MOI = 25). The number of internalized bacteria was determined by the standard invasion assay, and the results are presented as the percentage of the inoculum used.

FIG. 7

Effect of β₁ integrin MAb on internalization of S. aureus DU5875 by HEp-2 cells. HEp-2 monolayers were preincubated with or without 5 nM Fn plus nonimmune ascites (1:200) or β₁ integrin MAb (1:200) for 30 min prior to infection with the bacterial suspension (MOI = 17). The number of internalized bacteria was determined by the standard invasion assay, and the results are presented as the percentage of inoculum used.

FIG. 8

Potential models for FnBP receptor binding leading to internalization. (A) FnBP interacts with integrin and Hsp60 coreceptors through a Fn linkage. (B) FnBP interacts independently with Hsp60 and the Fn-integrin complex.