PSEUDOMONAS DEFICIENT IN PROTEASE-IV HAS SIGNIFICANTLY REDUCED CORNEAL VIRULENCE

Purpose. The role of protease IV in the pathogenesis of Pseudomonas ae ruginosa keratitis was investigated by comparing a mutant strain compl etely deficient in protease IV activity with its protease IV activity- producing parent. Methods. A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which p roduces protease IV, through transposon insertion. Protease ni activit y was determined by a casein agar assay, zymography, and cleavage of t he chromogenic substrate, Chromozym FL. Corneal virulence was evaluate d by slit lamp examination and bacterial cultures in both a rabbit int rastromal model and a mouse topical model of keratitis. Results. The p rotease IV-deficient strain PA103-29::Tn9 had significantly reduced co rneal virulence relative to its parent strain PA103-29 in both a rabbi t intrastromal model and a mouse topical model of infection. In the ra bbit model, ocular damage (slit lamp examination score) mediated by th e parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infe ction. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model , eyes inoculated with the protease IV-producing parent strain had sig nificant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant cornea l damage during 6 days of infection. Conclusions. The ability to produ ce active protease IV was the determining factor in the severity of co rneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications des igned to minimize corneal damage during Pseudomonas keratitis.