Objective
We have demonstrated differences in the proteome of a virulent clinical isolate and an avirulent laboratory strain 8325-4 and identified four possible virulence factors: fibronectin binding protein (FnBp), extracellular adherence protein (Eap), GAPDH and staphopain. We investigated the role of these virulence factors in the pathogenesis of microbial keratitis using a range of mutant strains and inhibitors to these proteins.

Methods
S. aureus strains (Staph 38, NCTC 8325-4, ΔFnBpA , ΔFnBpB, ΔFnBpAB, FnBpA+, FnBp B+, Newman, ΔEap) were used in in vitro bacterial invasion of cells and corneal infections in mice. Staph 38 and 8325-4 strains were also incubated with antibodies and inhibitors to virulence factors prior to infection. Clinical scores, PMNs and bacterial numbers were determined.

Results
All FnBp and Eap mutants showed significant reduction in the ability to invade corneal epithelial cells. In the animal model FnBpB and double mutant FnBpAB demonstrated reduced clinical scores, PMNs and bacterial numbers. The FnBpA mutant showed less bacterial numbers but no change in clinical score or PMN numbers. Corneal infection with Eap mutants resulted in reduced bacterial numbers and clinical scores. Inhibition of staphopain and GAPDH significantly reduced both bacterial numbers and clinical scores and to a greater extent than FnBp.

Conclusions These results show that the identified virulence factors are important in the development of corneal infection by S. aureus particularly with respect to staphopain and GAPDH. Our findings also suggest that there may be modulation of the PMN response by FnBp.