Pseudomonas aeruginosa persists as a major cause of life-threatening infections for individuals with the following conditions: cystic fibrosis, burns, wounds, cancer (leukemias), those receiving immunosuppressive therapy, diabetics, as well as intravenous drug users. The pili of this bacterium are protein filaments that extend from the ends of the cell. These structures serve as adhesion factors, and so are important virulence factors. Work from my laboratory has shown that the pill of P. aeruginosa 1244 are glycosylated with the O-antigen repeating unit of this organism. This trisaccharide moiety originates in the lipopolysaccharide O-antigen biosynthetic pathway. The long-term objectives of this project are to determine the role of pilus glycosylation in P. aeruginosa pathogenesis and to ascertain the importance of the glycan in pilus vaccine design. The work described in this proposal aims to determine the glycan and the pilin substrates in the P. aeruginosa 1244 pilin glycosylation reaction. In addition, the subcellular location of PilO, the enzyme that catalyzes pilin glycosylation in P. aeruginosa will be established. The membrane topology of this enzyme will also be determined. Finally, the subcellular location of the P. aeruginosa 1244 pilin glycosylation reaction established. [unreadable] [unreadable]