Purpose: The virulence contribution of Pseudomonas aeruginosa small protease (PASP) during experimental keratitis was studied by comparing a PASP-deficient mutant with its parent and rescue strains.
Methods: The pasP gene of P. aeruginosa was replaced with the tetracycline resistance gene via allelic exchange. A plasmid carrying the pasP gene was introduced into the PASP-deficient mutant to construct a rescue strain. The PASP protein in the culture supernatants was determined by Western blot analysis. Corneal virulence was evaluated in rabbit and mouse keratitis models by slit lamp examination (SLE), bacterial enumeration, and/or histopathological analysis. Various host proteins and the rabbit tear film were analyzed for their susceptibility to PASP degradation.
Results: The PASP-deficient mutant produced a significantly lower mean SLE score when compared with the parent or rescue strain (P ≤ 0.03) at 29 hours postinfection (PI). All of the strains grew equally in the rabbit cornea (P = 0.971). Corneas infected with the PASP-deficient mutant showed moderate histopathology compared with those infected with the parent or rescue strain, which produced severe pathology inclusive of epithelial erosions, corneal edema, and neutrophil infiltration. In the mouse model, eyes inoculated with the PASP-deficient mutant had a significantly lower mean SLE score at 24 hours PI than the eyes inoculated with the parent or rescue strain (P ≤ 0.007). PASP was found to degrade complement C3, fibrinogen, antimicrobial peptide LL-37, and constituents of the tear film.
Conclusions: PASP is a commonly secreted protease of P. aeruginosa that contributes significantly to the pathogenesis of keratitis.