Pseudomonas aeruginosa LasA Protease as a Therapeutic Agent in Staphylococcal Keratitis

Abstract

Abstract: : Purpose: To study the effectiveness of LasA protease, a staphylolytic endopeptidase secreted by Pseudomonas aeruginosa, in the management of experimental Staphylococcal keratitis, induced by methicillin–sensitive (MSSA) and methicillin–resistant (MRSA) isolates. Methods: LasA protease was purified from the culture filtrate of P. aeruginosa by cation–exchange chromatography. Keratitis was induced in New Zealand white rabbits by intrastromal injections of 10³ S. aureus cells. Eyes were treated with LasA protease or bovine serum albumin (control). Drops were applied every 30 min from 4 to 9 h post–infection, then hourly for 4 additional hours (early onset) or from from 10 to 15 h post–infection (late onset). Rabbits were sacrificed 1 h after cessation of treatment and their corneas were excised and homogenized for bacterial quantification. In another experiment, eyes with MSSA–induced keratitis were treated every 30 min from 4 to 9 h post–infection and evaluated clinically at 1 and 84 h after termination of therapy. Some rabbits were sacrificed at the same time points and the eyes enucleated for histopathological analysis. Results: In the early–onset therapy group essentially all of the MSSA– and MRSA–infected corneas were sterilized by LasA protease whereas all of the control corneas were heavily infected, with median values of 4.5x10⁶ (MSSA) and 5x10⁵ (MRSA) CFU/cornea (P values <0.001). In the late–onset therapy group, LasA protease reduced the CFU values in both MSSA and MRSA–infected corneas by 3–4 orders of magnitude as compared to controls (median values 1380 and 30 CFU/cornea as compared to 1.2x10⁶ and 5x10⁵ CFU/cornea in the respective controls; P=0.001). The mean clinical scores of the LasA protease–treated eyes at 1 and 84 h after cessation of treatment were 1.5 and 1.25, respectively, as compared to 3.5 and 15 in the corresponding control eyes. Histopathological analysis of the LasA protease–treated eyes showed only mild inflammatory signs, as compared to bacterial clusters, stromal vascularization and abscesses in the respective control eyes. Conclusions: LasA protease is effective in treating keratitis caused by either methicillin–sensitive or methicillin–resistant S. aureus, supporting its potential use as a new broad–range therapeutic tool for Staphylococcal keratitis.

Keywords: antibiotics/antifungals/antiparasitics • bacterial disease • enzymes/enzyme inhibitors