Pseudomonas keratitis: protease IV gene conservation, distribution, and production relative to virulence and other Pseudomonas proteases

Abstract

Purpose: To determine the distribution of the protease IV gene, the production of this and other proteases by multiple strains of Pseudomonas, and the virulence of a mutant specifically deficient in protease IV.

Methods: The protease IV gene was cloned, its sequence analyzed, and its chromosomal location determined by pulse-field gel electrophoresis. Three PCR reactions were used to detect the protease IV gene in 30 Pseudomonas isolates and protease production was determined by Western blot analysis, colorimetric assay, and zymography. An allelic replacement mutant deficient in the protease IV gene was analyzed for enzyme production, corneal growth, and corneal virulence.

Results: The protease IV gene was identified in all P. aeruginosa, but none of the non-aeruginosa strains tested. The protease IV genes of strains PA103-29 and PAO1 were in a common chromosomal site and had 98.5% sequence identity with variations occurring mainly in the promoter region. The protease IV activity of the 23 wild-type P. aeruginosa strains tested varied from 2.3 to 221.5 x 10(-3) U/mg protein in the culture supernatant. Protease IV was produced by all P. aeruginosa wild-type strains. A protease IV-deficient mutant derived from strain PA103-29 had reduced virulence compared with its parent strain and unexpectedly produced alkaline protease.

Conclusions: The protease IV gene and its product are common to P. aeruginosa, but not to other Pseudomonas species. Protease IV activity varies among P. aeruginosa strains, and a mutant specifically deficient in this activity produced alkaline protease and had reduced corneal virulence.