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PASP is a protease that has not been previously identified. It causes corneal epithelial erosions, indicating its likely activity as a virulence-promoting factor in Pseudomonas keratitis.

The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease. The results also confirm a significant correlation between production of protease IV and corneal virulence.

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 μg/20 μL) 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 μg). 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 ≤ 0.004). Histopathologic studies documented the destruction of the corneal epithelial layer and portions of the corneal stroma at 9 hours PI, and polymorphonuclear (PMN) leukocyte infiltration into the cornea by 24 hours after active rPASP injection. PASP cleaved type I and IV collagens and was susceptible to TLCK inhibition. PASP was present in the cytoplasm and periplasm, but only secreted PASP was enzymatically active. A high antibody titer (ELISA titer ≥ 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.

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Bacterial keratitis is a disease of the cornea characterized by pain, redness, inflammation, and opacity. Common causes of this disease are Pseudomonas aeruginosa and Staphylococcus aureus. Animal models of keratitis have been used to elucidate both the bacterial factors and the host inflammatory response involved in the disease. Reviewed herein are animal models of bacterial keratitis and some of the key findings in the last several decades.

The protease IV gene and its product are common to P. aeruginosa, but not to other Pseudomonas species. Protease IV activity varies among P. aeruginosa strains, and a mutant specifically deficient in this activity produced alkaline protease and had reduced corneal virulence.