Properties of PASP: a Pseudomonas protease capable of mediating corneal erosions.

PURPOSE: To analyze PASP in terms of its gene distribution and expression, its corneal pathologic effects, its enzymatic properties, and the protectiveness of the immune response to this protease. METHODS: Twenty-five strains of P. aeruginosa were analyzed for the PASP gene and secreted protein by PCR and Western blot analysis, respectively. Active recombinant (r)PASP (10 microg/20 microL) or heat-inactivated rPASP was intrastromally injected into rabbit corneas. Pathologic changes were monitored by slit lamp examination (SLE) and histopathology. Purified rPASP was assayed for cleavage of collagens and susceptibility to TLCK. Rabbit antibody to rPASP was produced and tested for enzyme inactivation, and actively immunized rabbits were challenged by intrastromal injection of active rPASP (5 microg). RESULTS: All 25 strains of P. aeruginosa analyzed were positive for the PASP gene and protein. SLE scores of eyes injected with active rPASP were significantly higher than control eyes at all postinjection times (PI; P or= 10,000) was produced, but this antibody did not protect against active rPASP challenge. CONCLUSIONS: PASP is a commonly produced Pseudomonas protease that can cleave collagens and cause corneal erosions.

Age-related differences in rabbits during experimental Staphylococcus aureus keratitis.

PURPOSE: To analyze age-related changes in susceptibility to experimental Staphylococcus aureus keratitis and purified alpha-toxin in rabbits. METHODS: Intrastromal injection of S. aureus (100 colony-forming units [CFUs]) induced keratitis in young (6-8 weeks) and aged (approximately 30 months) New Zealand White rabbits. Bacteria and polymorphonuclear leukocytes (PMNs) per cornea were quantified. Purified alpha-toxin at 1, 10, 25, or 50 hemolytic units (HU) or heat-inactivated alpha-toxin was intrastromally injected into corneas, and pathologic changes were determined by slit lamp examination (SLE) and histopathologic analysis. alpha-Toxin hemolysis assays were performed using erythrocytes from young and aged rabbits. RESULTS: S. aureus keratitis produced significantly higher SLE scores in young rabbits than in aged rabbits at 15, 20, and 25 hours postinfection (PI; P < or = 0.001); aged rabbits essentially recovered from S. aureus keratitis by 7 days PI. At 25 hours PI, numbers of CFUs and PMNs in corneas of young and aged rabbits were equivalent (P > or = 0.6); the bacterial burden in aged rabbits declined by 5 logs per cornea from day 1 to day 7 PI. Intrastromal injection of > or =10 HU alpha-toxin also produced significantly more disease in young than in aged rabbit corneas (P < or = 0.05), whereas 1 HU or heat-inactivated toxin yielded negligible pathologic changes in either group. Hemolysis assays of erythrocytes from young rabbits demonstrated greater susceptibility to alpha-toxin compared with those from aged rabbits. CONCLUSIONS: Corneas and erythrocytes of young rabbits, relative to aged rabbits, are significantly more susceptible to S. aureus keratitis and to alpha-toxin.