Abstract:
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.
P. putida KT2660 was transformed with one of two plasmid constructs encoding either elastase B (lasB) or alkaline protease (aprA) along with the accessory proteins required for its secretion (aprD, aprE, aprF, and aprI). Protease expression was confirmed by zymography and Western blotting. P. putida expressing elastase B (Put:plasBT), alkaline protease (Put:pAP), or vector alone (Put:p20) was injected intrastromally (103 colony forming units [CFU] in 10 µl) into rabbit corneas (n=6). Infected corneas were graded by slit lamp examination (SLE) at 20, 24, 28, and 32 hours (h) post infection (PI). Rabbits were sacrificed at 32 h PI and the log CFU (± SEM) per cornea were determined.
SLE scores for each group at 20, 24, 28, and 32 h PI are shown in the table below.
SLE scores for corneas infected with Put:plasBT were significantly higher than those infected with vector alone at all time points (p ≤ 0.008). SLE scores for corneas infected with Put:pAP were not significantly higher than those infected with vector alone at any time point (p ≥ 0.1). At both 24 and 28 h PI, the SLE scores for corneas infected with Put:lasBT were significantly higher than those infected with Put:pAP (p ≤ 0.002). At 32 h PI, the mean log CFU for corneas infected with Put:lasBT (5.51 ± 0.48) was significantly lower (p ≤ 0.02) than those infected with Put:p20 (6.80 ± 0.13) or Put:pAP (6.66 ± 0.12). The expression of elastase B, but not alkaline protease, in the P. putida model of keratitis significantly increased SLE scores at all time points measured. This suggests that elastase B contributes to the pathology observed during P. aeruginosa keratitis.
Keywords: Pseudomonas • keratitis • pathology: experimental