The roles of the Pseudomonas aeruginosa proteases LasB (elastase) and LasA and the transcriptional activator LasR, which regulates the expression of these proteases, were evaluated in a murine model of P. aeruginosa corneal infection. In scarified corneas, P. aeruginosa PAO-A1 (LasA negative) or PAO-B1A1 (LasB and LasA negative) at a dose of 10(8) CFU per eye caused very mild or no disease following infection; however, the defect in PAO-A1 could not be complemented by supplying a functional copy of lasA either on a plasmid or inserted into the chromosome. In contrast, PAO-B1 (LasB negative) colonized the cornea and caused disease equal in severity to disease caused by the parental strain, PAO1-I. Although LasR is a known regulator of lasA expression, PAO-R1, a lasR-negative derivative of PAO1-I, was as virulent as the parental strain during corneal infection. When transcriptional fusion plasmids were used to quantify the expression of the lasB and lasA genes in P. aeruginosa PAO1-I and PAO-R1, the lasB::lacZ fusion in PAO-R1 showed only 3.5% as much activity as it did in PAO1-I, while the activity of the lasA::lacZ fusion in PAO-R1 was 27.8% of that in PAO1-I. Coadministration of 5 microg of purified LasA protease with PAO-A1 did not reconstitute a wild-type infection. This treatment produced an acute toxic reaction leading to prolonged eyelid closure without inflammatory destruction of the cornea that was similar to that observed when LasA was administered alone. These results indicate that insertional inactivation of lasA renders P. aeruginosa avirulent in a murine model of keratitis and that neither LasR nor elastase production is required for the establishment and maintenance of corneal infection. However, the lack of virulence of the LasA-deficient strains cannot be ascribed with certainty to the deficiency of LasA from the available data.

The lasA gene was the first of the Pseudomonas aeruginosa genes involved in proteolysis and elastolysis to be cloned and sequenced. Its function and significance have been studied by genetic approaches (D. S. Toder, M. J. Gambello, and B. H. Iglewski, Mol. Microbiol. 5:2003-2010, 1991) and by attempts to purify an active fragment of the protein (J. E. Peters and D. R. Galloway, J. Bacteriol. 172:2236-2240, 1990). To further study LasA in vivo, we have constructed and characterized an insertional mutant in the lasA gene in strain PAO1 (PAO-A1) and in the lasB insertional mutant, PAO-B1. Analysis of these isogenic strains demonstrates that the lasA lesion diminished elastolysis more than proteolysis and that LasA is required for staphylolytic activity. Despite previous suggestions that lasB elastase cleaves the LasA protein, the size of the LasA protein was the same whether or not lasB elastase was present. Expression of lasA in a lasR-negative mutant, PAO-R1, demonstrated that the LasA protein is produced in an active form in the absence of (lasB) elastase or alkaline protease and is itself a protease with elastolytic activity. We also observed that PAO-A1 was closer to the parental phenotype, with respect to elastolytic and proteolytic activities, than the previously characterized, chemically induced lasA mutant PAO-E64. Quantification of promoter activity with lasA::lacZ and lasB::lacZ fusions suggests that PAO-E64 harbors a mutation in a gene which regulates expression of both lasA and lasB.

The full elastolytic phenotype of Pseudomonas aeruginosa requires lasB, the structural gene for elastase, its transcriptional activator lasR, and lasA. The lasB gene was insertionally inactivated with the omega fragment and this mutated gene introduced into the P. aeruginosa chromosome. Replacement of the wild-type gene with the inactivated gene was verified by Southern analysis and confirmed by lack of elastase antigen on Western blots and lack of activity in liquid assays. The mutant did, however, retain elastolytic activity on elastin plates. This residual activity was abolished by inactivation of lasB in PAO-E64, a lasA-deficient mutant, demonstrating that it was due to the lasA gene product. Northern analysis demonstrated that, like lasB, lasA is transcriptionally controlled by the lasR gene product.