Ocular Pathology of a Staphylococcus aureus Mutant Lacking a Recently Discovered Virulence Factor

Abstract

Purpose: : Ocular virulence of S. aureus was previously shown to be mediated by the cytolytic alpha- and gamma-toxins. A mutant lacking both toxins was defective in ocular virulence, but retained the ability to cause limited pathology. The purpose of this project was to identify the factor responsible, to isolate a mutant defective in its expression in a strain already defective in alpha- and gamma-toxins, and to measure virulence in a rabbit model of keratitis.

Methods: : The gene encoding the novel putative virulence factor was deleted by allelic replacement using a temperature sensitive vector plasmid in strain Newman already defective in alpha- and gamma-toxins. As a control to demonstrate that the difference in phenotype of the triple mutant strain compared to the double mutant was due to the isolated mutation and not due to polarity or an unselected regulatory mutation, the deleted gene was restored to wild-type by allelic exchange (rescue strain). Clones were screened by PCR and by Western Blott using rabbit antibody against the new toxin. Strains were tested for corneal virulence by intrastromal injection into rabbit corneas. Slit lamp examination (SLE) was performed at 24 hours PI. Corneas were cultured for bacterial numbers (log CFU ± SEM).

Results: : S. aureus

Conclusions: : The gene for the newly discovered protein must be functional to achieve maximal corneal virulence.

Keywords: Staphylococcus • bacterial disease • gene/expression