Abstract
Abstract: :
Purpose: Pseudomonas aeruginosa is a major cause of bacterial keratitis, primarily due to its production of secreted proteases that destroy host defenses and degrade the cornea. Strain PA103 is deficient in elastase, LasA, and alkaline protease. A PA103 mutant deficient in protease IV has been shown to retain reduced, yet significant, corneal virulence. These results suggest that another protease could exist, providing an additional mechanism of corneal virulence. Methods: P. aeruginosa strain PA103 was grown to stationary phase and its secreted products were analyzed for protease activity. Analyses included determination of caseinase activity by measuring the lysis of a fluorescence–labeled casein substrate in the presence and absence of potential inhibitors, and gelatin zymography to visualize the molecular mass of the protease. A protease with a unique molecular mass on zymograms was purified by ion–exchange chromatography, and mass spectrometry and amino–terminal sequencing were performed. Results: The culture supernatant of PA103 contained a protease that caused degradation of casein in the presence of a serine protease inhibitor, but was inhibited by EDTA. Gelatin zymograms showed protease activity at >200 kDa (protease IV) and at about 80 kDa. The protease with zymogram activity at 80 kDa, after purification, was determined by mass spectrometry to have a native mass of 18.5 kDa. The amino–terminal sequence of this protease matched the sequence of a hypothetical protein in the P. aeruginosa genome database (www.pseudomonas.com). Conclusion: P. aeruginosa PA103, in addition to protease IV, produces a protease that is susceptible to an inhibitor of metalloproteases.
Keywords: bacterial disease • keratitis • Pseudomonas