The goal of this research is to carry out a detailed genetic and biochemical analysis of the elastase of Pseudomonas aeruginosa. This enzyme is capable of degrading or inactivating a variety of biologically important substrates such as elastin, IgG, fibrin and complement. While existing data suggests that elastase contributes to the virulence of P. aeruginosa, the roles of its various activities in Pseudomonas infections is unknown. We have succeeded in cloning the P. aeruginosa elastase gene (las) into both E. coli and P. aeruginosa. We intent to characterize the las gene(s) using restriction enzymes and in vivo and in vitro mutagenesis. Selected mutant genes will be introduced into P. aeruginosa. These elastase mutants will be compared to isogenic elastase positive pairs in appropriate animal models. Altered elastase proteins produced by selected mutants will be purified and compared to native P. aeruginosa elastase. The proposed studies will help elucidate the structure and function of elastase and clarify the role of this enzyme in the pathogenesis of P. aeruginosa infections.
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