Retrospective analysis of Pseudomonas aeruginosa clinical isolates for secreted PrpL protease: a key virulence factor associated with corneal tissue damage

Abstract

BackgroundPseudomonas aeruginosa is a ubiquitous organism that adapts well in different environments. It is an opportunistic bacterial pathogen that produces a wide range of virulence factors, colonizes lungs to cause pneumonia, causes non-healing wounds especially in burn victims, and is a major culprit in destructive keratitis. It can reach the cornea through reusable, extended-use contact lenses and by contaminated eyedrops and artificial tears. Secreted proteases of P. aeruginosa together with pyocyanin metabolite, which inhibits Serine Protease Inhibitors (Serpins) activity contribute to severe tissue damage during infection. MethodsP. aeruginosa strains isolated from different clinical sites were obtained from different researchers and clinicians for this study. We examined P. aeruginosa strains and secretion defective and prpL knockout mutants in PA64481 strain for lysyl endopeptidase (PrpL, a serine protease that cleaves after a lysine residue) activity using serine protease specific D-Val-Leu-Lys-p-nitroanalide substrate. We also determined pyocyanin production in these strains. ResultsExamination of secreted milieu from P. aeruginosa showed that 41 corneal isolates had detectable lysyl endopeptidase activity associated with PrpL at levels significantly higher than by 27 non-corneal isolates. We found that PrpL is secreted by the xcp-based type II secretion system. Bacterial culture supernatants displaying higher PrpL activity and not low secreted PrpL levels disrupted corneal epithelial cell monolayers in vitro, which is consistent with a role of this protease in destructive keratitis. Many examined strains also produced high levels of pyocyanin. ConclusionsThis retrospective examination of clinical P. aeruginosa suggests that high levels of PrpL and pyocyanin-producing isolates are more prevalent among corneal isolates and could enhance tissue damage during infection. Supporting this premise, corneal epithelial cell monolayers disrupted by high PrpL-producing strains but remained intact after treatment with P. aeruginosa mutants culture supernatants that lack or have reduced secreted PrpL.