Infections caused by Pseudomonas aeruginosa are a significant problem in human healthcare. This bacterial pathogen is the principle agent of sepsis in burn patients;of persistent lung infections and mortality in cystic fibrosis patients;of nosocomial infections in HIV and other immune-suppressed patients;and of the outbreak of deleterious multi-drug resistant infections in hospitals. The long-term goal of this proposal is to provide effective and selective therapies that reduce the incidence and complications of human P. aeruginosa infections. This study proposes this goal can be achieved by drugs that prevent or limit the activation of the MvfR/HAQ pathway, and the development of such anti-infective compounds is the immediate goal of this application. This proposal is based on the hypothesis that MvfR, a P. aeruginosa transcriptional regulator, is a candidate target for anti-infective drugs, as it plays a central role in modulating the expression of many QS-controlled virulence-associated factors;and its activation is mediated by its binding to a specific ligand, which is essential for its function. To identify MvfR-pathway inhibitors, and demonstrate their in vivo anti-infective activity, this study proposes three Specific Aims: 1) to confirm the identity of the MvfR ligand, identify its binding site, and determine its mechanism of action;2) to identify compounds that inhibit the MvfR/HAQ pathway;and 3) to determine the in vivo efficacy and potential feasibility of these inhibitors to limit P. aeruginosa infection in mammals.
These aims will be accomplished via three sets of experiments. First, biochemical, mass spectrometric, and molecular genetic analyses will confirm the identity of the P. aeruginosa MvfR-ligand;define the MvfR ligand binding domain;and determine the pqsA promoter sequence recognized and bound by MvfR and the MvfR-ligand complex. Second, biochemical and mass spectrometric analyses will identify compounds that prevent ligand-mediated MvfR activation by limiting the synthesis and/or binding of its ligand, and that are metabolically stable in P. aeruginosa. Third, each identified inhibitor will be tested in the Drosophila melanogaster, the mouse full-thickness skin thermal injury, and the mouse neonatal respiratory model, to determine their toxicity, their in vivo efficacy to limit P. aeruginosa infection;and their """"""""immunity"""""""" to the development of bacterial resistance. Specific inhibition of a pathway that directly mediates virulence is less likely to generate selective pressure to develop resistance to the inhibitor, than for drugs, including most antibiotics, that reduce bacterial viability. Such targeted inhibitors could significantly enhance the long-term prognosis of burn, cystic fibrosis, and HIV patients. To this end, the results here should enable novel therapies to treat and/or prevent P. aeruginosa-human infections.
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