Staphylococcal Fibronectin-Binding Protein A Engages Heat Shock Cognate Protein 70 Through a Fibronectin-Independent Interaction in Mammary Epithelial Cells

Staphylococcal mastitis is a clinically important inflammatory disease of the mammary gland across mammalian hosts, including women and dairy cattle. Using bovine mammary epithelial cells as a model, we investigated which protein(s) could function as a direct binding partner of fibronectin-binding protein A (FnBPA). A proteomics screen using GST-FnBPA (N2N3) pull-downs from MAC-T lysates followed by LC-MS revealed heat shock cognate protein 70 (Hsc70/HSPA8) as a candidate binding partner for FnBPA. The result was first confirmed by Western blot, with endogenous Hsc70 captured by GST-FnBPA but not by GST alone. Purified His-Hsc70 bound GST-FnBPA in a reconstituted system. ELISA revealed a concentration-dependent, saturable interaction with an apparent Kd of 515 ± 83.93 nM. Confocal imaging showed partial co-localization of FnBPA and Hsc70 in MAC-T cells, predominantly in the cytoplasm with limited overlap at the plasma membrane. Docking (HDOCK) and MM/GBSA analysis (HawkDock) predicted an interaction interface involving the FnBPA N2N3 trench and Hsc70 residues within the nucleotide-binding and substrate-binding domains, highlighting hotspot residues consistent with a stable complex. Together, these data support a fibronectin-independent route in which FnBPA engages Hsc70 as a direct host-associated binding partner in the bovine mammary epithelial context, operating in parallel with or as an alternative to the canonical Fn–integrin α5β1 pathway. Targeting the FnBPA–Hsc70 interaction may offer a path to anti-adhesion strategies for controlling S. aureus mastitis in the bovine setting and also provide a comparative framework for investigating alternative mammary infection pathways in other mammalian hosts.