Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa

The expression of many virulence factors in Pseudomonas aeruginosa is depen dent upon environmental conditions, including iron levels, oxygen, temperat ure, and osmolarity. The virulence of A aeruginosa PAO1 is influenced by th e iron- and oxygen-regulated gene encoding the alternative sigma factor Pvd S, which is regulated through the ferric uptake regulator (Fur). We observe d that overexpression of PvdS in strain PAO1 and a Delta pvdS::Gm mutant re sulted in increased pyoverdine production and proteolytic activity compared to when PvdS was not overexpressed. To identify additional PvdS-regulated genes, we compared extracellular protein profiles from PAO1 and the Delta p vdS::Gm mutant grown under iron-deficient conditions. A protein present in culture supernatants from PAO1 but not in supernatants from Delta pvdS::Gm was investigated. Amino acid sequence analysis and examination of the genom ic database of PAO1 revealed that the N terminus of this 27-kDa protein is identical to that of protease IV of P. aeruginosa strain PA103-29 and is ho mologous to an endoprotease produced by Lysobacter enzymogenes. In this stu dy, the gene encoding an endoprotease was cloned from PAO1 and designated p rpL (PvdS-regulated endoprotease, lysyl class). All (n = 41) but one of the strains of P. aeruginosa, including clinical and environmental isolates, e xamined carry prpL. Moreover, PrpL production among these strains was highl y variable. Analysis of RNase protection assays identified the transcriptio n initiation site of prpL and confirmed that its transcription is iron depe ndent. In the Delta pvdS::Gm mutant, the level of prpL transcription was ir on independent and decreased relative to the level in PAO1. Furthermore, tr anscription of prpL was independent of PtxR, a PvdS-regulated protein. Fina lly, PrpL cleaves casein, lactoferrin, transferrin, elastin, and decorin an d contributes to PAO1's ability to persist in a rat chronic pulmonary infec tion model.