P. aeruginosa is an important opportunistic pathogen responsible for a wide range of infections. Antibiotic resistance is fairly common, which hampers effective treatment, especially in chronic infections of CF patients. There is a need to identify new drug targets, which may include primary biosynthetic pathways required for bacterial survival and virulence factor synthesis. The goal of this research is to establish the pathway of fatty acid (Fab) and autoinducer (PAI) biosynthesis and explore their usefulness as novel antimicrobial targets. This linked pathway is an excellent candidate for drug development, because it is responsible for the synthesis of essential cellular components and is also involved in synthesis of conserved central regulatory elements for virulence factor synthesis (quorum sensing). As part of this proposal, an in vitro Fab system will be established using purified recombinant proteins in order to define enzymes leading to synthesis of acyl-acyl carrier protein, which is the substrate for PAI synthesis. Second, in vitro PAI synthesis will be demonstrated using purified PAI synthases, leading to the production of biologically-active PAI. Various Fab and PAI synthesis inhibitors, including thiolactomycin (TLM) and its analogs will be evaluated. The effects of these inhibitors will be studied in vitro and in vivo. The mechanism of TLM resistance will be addressed using clinical isolates of P. aeruginosa, as well as resistant mutants isolated in the laboratory. The successful outcome of this line of investigation should result in the identification of the entire pathway of AI synthesis. These studies should provide a better understanding of the quorum sensing pathways and lead to the identification of novel anti microbial targets and antimicrobials effective against gram-negative pathogens.
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