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Staphylococcus aureus is a common wound-infecting organism which can interact with cells via the extracellular matrix protein fibronectin (FN). The aim of this study was to determine the effect of the FN-binding protein (FnBP) of S. aureus on the behaviour of a human skin keratinocytes cell line (UP). FN-coated plates and Transwell membranes were exposed to a recombinant protein encompassing the D1-D4 repeat region of S. aureus FnBPB (rFnBPBD1-D4) before cell adhesion and migration assays. The influence of this protein on migration into a wounded area, cell cycle progression and endogenous cellular FN assembly was also assessed. The rFnBPBD1-D4 protein potently inhibited S. aureus entry into UP keratinocytes but had no effect on cell adhesion to FN substrate. It inhibited UP keratinocyte Transwell migration by 17% (P=0.04) and 31% (P=0.02) at 10 and 100 μg/ml, respectively. In the wound assay, 100 μg/ml of rFnBPBD1-D4 protein reduced the migration area by approximately 30%. No differences in cell cycle progression were observed. In the presence of rFnBPBD1-D4, most of the cellular FN matrix on the cell surface and along the cell filopodia seen in untreated cells was absent. Interaction of S. aureus FnBPs with FN may influence cell behaviour and thus play a role in delayed epithelial closure in infected healing wounds.