Corneal virulence of Pseudomonas aeruginosa elastase B and alkaline protease produced by Pseudomonas putida

Purpose: To measure the specific virulence contributions of two Pseudomonas aeruginosa proteases, elastase B and alkaline protease, when expressed separately by Pseudomonas putida in a rabbit model of bacterial keratitis. Methods: P. putida KT2440 was transformed with plasmids that enabled the extracellular production of either elastase or alkaline protease. Protease expression was confirmed by zymography and immunoblotting. P. putida expressing elastase, alkaline protease, or vector alone was injected intrastromally (10(3) colony forming units [CFU]) into rabbit corneas (n = 6). Infected eyes were graded by slit-lamp examination (SLE) at 20, 24, 28, and 32 hr postinfection (PI). Rabbits were sacrificed at 33 hr PI, and the log CFU (+/- SEM) per cornea was determined. Results: SLE scores for eyes infected with P. putida producing elastase were significantly higher than those infected with vector alone at all time points (p <= 0.008). SLE scores for eyes infected with P putida producing alkaline protease were not significantly higher than the control (p >= 0.1), but small erosions formed in 33% of corneas. At both 24 and 28 hr PI, the SLE scores for corneas infected with P putida producing elastase were significantly higher than those infected with P. putida producing alkaline protease (p <= 0.002). Conclusions: Elastase production by R putida caused significant increases in SLE scores whereas expression of alkaline protease caused limited corneal erosions. This suggests that the production of elastase during P aeruginosa keratitis enhances ocular pathology, whereas alkaline protease production contributes to limited corneal erosion.