PSEUDOMONAS-AERUGINOSA PROTEASE-IV PRODUCES CORNEAL DAMAGE AND CONTRIBUTES TO BACTERIAL VIRULENCE

PURPOSE. A Pseudomonas mutant deficient in protease IV has significant ly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augmen t the virulence of protease-IV-deficient bacteria were anayzed. METHOD S. The toxicity of purified protease IV was determined by intrastromal ly injecting the exoenzyme (20 - 200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient s train, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV o r 200 ng heat-inactivated protease IV. Changes in ocular disease, dete rmined by slit-lamp examination, were measured at 3, 16, 22, and 27 ho urs after infection. Colony-forming units per cornea were quantified a t 27 hours after infection. RESULTS. Purified protease IV at doses fro m 50 to 200 ng induced epithelial defects within 3 hours of injection. injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the pro tease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P les s than or equal to 0.0001). Neither active nor heat-inactivated protea se IV altered the growth of bacteria in the cornea (6 log units; P = 0 .81). CONCLUSIONS. The important role of protease IV in corneal virule nce was demonstrated by direct toxicity and by its ability to signific antly augment the virulence of protease-IV-deficient Pseudomonas.