High Throughput Screening of Inhibitors of Pyoverdine Production

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DESCRIPTION (provided by applicant): Pseudomonas aeruginosa, a gram-negative bacterial species, is a human pathogen that is particularly problematic for immunocompromised patients and is a common cause of nosocomial infections. The well-publicized prevalence of drug resistant bacteria demonstrates a need for the characterization of new targets and for the identification of novel inhibition strategies that may be developed into new antibiotic therapies. Pseudomonas aeruginosa uses a non-ribosomal peptide synthetic cluster to produce a peptide siderophore that is necessary for iron uptake. Iron in the host organism is a limiting nutrient and numerous animal and plant studies have demonstrated the important role that pyoverdine plays in virulence. Thus, any of the 14 proteins responsible for the synthesis of pyoverdine are viable targets for inhibition to prevent the establishment of an infection. We have developed a biochemical assay for use with PvdQ, the periplasmic acylase that is responsible for a late step in the chemical maturation of pyoverdine. Secondary and tertiary assays will validate that results from the primary screening assay and assess the viability of compounds in a cell growth assay. Preliminary screening with a small chemical library demonstrates very good signal to noise and reproducibility statistics and screening at the Molecular Libraries and Probe Production Centers Network will likely identify novel compounds that block PvdQ. Compounds identified in this effort will be used to further our understanding of the role of PvdQ and related proteins in Pseudomonas pathogenesis with a long term goal of optimizing these compounds as specific inhibitors of pyoverdine production. PUBLIC HEALTH RELEVANCE: Pseudomonas aeruginosa is a human pathogen that requires biochemical systems for the acquisition of iron to establish an infection. An assay has been developed to identify chemical probes that are able to block this process. High-throughput screening will facilitate the identification of these inhibitory compounds as an initial step towards the development of novel antibiotics.