Pseudomonas aeruginosa produces a variety of extracellular products which may play a significant role in the pathogenesis of Pseudomonas infections in experimental animal models and in humans. In addition we and others have shown that the virulence of Pseudomonas is dependent upon its ability to acquire iron (Fe) or inorganic phosphate (Pi) from the environment and jthat production of some virulence determinants is regulated by these ions. Of the extracellular products of P. aeruginosa, two toxins along with protease have been mentioned prominently in the Pseudomonas literature as likey candidates for major virulence determinants. They are Exotoxin A which has a mechanism of action like diphtheria toxin and the Heat-labile Hemolysin (Phospholipase C) which has a mechanism of action like the alpha toxin of Clostridium perfringens. However, the actual importance of these toxins in the pathogenesis of even on kind of Pseudomonas infection is not yet fully understood. In addition, the specific mechanism by which this opportunistic pathogen is able to acquire Fe and Pi from the environment and the importance of these mechanisms in the virulence of Pseudomonas have not been elucidated. The overall goal of this research is to elucidate the genetic and biochemical bases for the virulence of P. aeruginosa. These studies may ultimately lead to the rational development of immunotherapy and/or immunoprophylaxis for infections caused by this opportunistic pathogen. The immediate objectives are as follows: 1) we will isolate well-defined mutants whih are altered in their ability to produce virulence factors (toxins) or which are altered in their ability to acquire Fe or Pi from the environment. To accomplish this objective, we will use in vitro recombinant DNA techniques or we will use in vivo classical microbial genetic techniques (e.g., generation of R' factors) to clone genes for virulence determinants. 2) We will generate mutations in those cloned genes by in vitro or in vivo methods and then transfer the mutant genes to wild-type Pseudomonas strains. 3) We will characterize the mutants in detail by genetic and biochemical methods. 4) We will compare the virulence of these well-defined, well-characterized mutants to the isogenic parental strains in appropriate experimental animal models.
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