The immediate goal of this research is to determine whether exoenzyme S contributes to severe P. aeruginosa infections by promoting the spread of the bacterium from epithelial colonization sites to the bloodstream. Exoenzyme S is an extracellular ADP-ribosyltransferase whose expression correlates with fatal sepsis in acute lung and burn animal model infections. This proposal focuses on the construction of P. aeruginosa strains that express or fail to express catalytic forms of exoenzyme S for testing in both acute and chronic animal model infection systems. These experiments are designed specifically to test the relevance of the 49 kDa enzymatically active form of exoenzyme S in vivo. The long-term goals are to define the pathway required for both exoenzyme S synthesis and secretion in P. aeruginosa. The Research Career Development Award will permit relief from the applicant s teaching and academic committee responsibilities at the Medical College to allow her to commit >80 percent effort to research. The award of an RCDA would occur at a critical point in her research career when her research is poised to determine the mechanisms contributing to epithelial damage and dissemination mediated by P. aeruginosa; her studies will further provide a valuable basis for designing vaccine candidates that may prevent sepsis and epithelial damage. The environment provided by the Department of Microbiology and the 13 productive and well funded faculty is collegial, productive, interactive, and highly conducive to collaboration. The greatest concentration of molecular biologist on campus are within the Department of Microbiology where the disciplines of bacteriology, immunology, and virology are represented. The applicant's department chairman and dean will reduce the current teaching and committee work, safeguard her research time,help subsidize her equipment purchases, graduate students, and foreign and domestic travel.The research project proposes to define the role of the 49- kDa enzymatically active form of exoenzyme S and coordinately regulated virulence determinants in P. aeruginosa epithelial damage and dissemination. The approaches that will be used involve the mutagenesis of exo S and allelic replacement, defining the structural domains for ADP-ribosyltransferase activity and secretion, and identifying the role of loci homologous to the ysc genes of Yersinia and FliI of Bacillus and Salmonella in synthesis and secretion of exoenzyme S. The analysis of the exoenzyme S pathway will contribute to defining the mechanisms of bacterial pathogenesis and invasiveness and aid in designing new therapeutic agents or vaccines for patients with burns, cystic fibrosis, and neoplastic disease.
