Infectious ulcerative keratitis is one of the most common bacterial infectious diseases of the eye, with tens of thousands of cases being treated annually in the U.S. An increase in the incidence of infectious keratitis has been associated with the increased use of contact lenses; and new refractive therapies that disrupt corneal surface integrity, a key step in the pathogenesis of infectious keratitis, have recently gained approval. Relatively little is known of the pathogenesis of Staphylococcus aureus keratitis, one of the most common etiologies of the disease. Preliminary studies indicate that surface proteins contribute to the attachment of S. aureus to human corneal epithelial cells in vitro, and that the binding of S. aureus to these cells is effected by proteins negatively regulated by the staphylococcal global regulatory systems, agr and sar. Removal of S. aureus surface proteins by protease treatment significantly reduces corneal epithelial cell binding. Moreover, preliminary results indicate that mutants in select surface proteins exhibit reduced binding, but not reduced to the extent of protease-treated staphylococci. These results suggest that binding is likely a multifactorial process mediated by as yet unidentified adhesin combinations or other surface factors. We propose to identify the staphylococcal corneal adhesins by assessing the relative binding of isogenic mutants in an in vitro model, using a newly developed fluorescence-based binding assay, with an independent reassessment and confirmation in vivo. A modified signature tagged mutagenesis system will also be used to identify mutants selectively unable to bind human corneal epithelial cells. Complementation will be used to confirm that reductions in binding are the direct result of the corresponding mutation. We also propose to explore the contribution of the host to staphylococcal corneal adherence by identifying corneal receptors involved in staphylococcal adherence and examining cytokine and cellular adhesin expression in response to staphylococcal binding. Finally, keratitis clinical isolates will be compared at the genetic level (by CHEF genomic analysis and PCR identification of putative virulence genes) to determine whether strains associated with corneal disease represent subsets of the species.
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