Quinolone Resistance Mechanisms in Staphylococcus aureus

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DESCRIPTION (provided by applicant): Quinolone Resistance Mechanisms in Staphylococcus aureus. Quinolone antimicrobials act on two topoisomerases and are used widely in human infections. Their use for common S. aureus infections has been limited by emerging resistance. Resistance mechanisms include altered topoisomerases and increased expression of multidrug resistance (MDR) efflux pumps. Defining these mechanisms is key to strategies to avoid resistance. Long-term objectives of the project are to use quinolones and resistant mutants as a model system to study the control and function of topoisomerases and efflux pumps found in many bacteria. Specific aims are (1) to define the roles of novel mutations in topoisomerase IV in effecting resistance and altering enzyme function. Mutant enzymes will be purified and studied for their catalytic functions and binding of DNA and quinolones as well as formation of quinolone-induced DNA cleavage. Aim (2) is to identify the sites of quinolone interaction with complexes of topoisomerase IV, DNA, and quinolone using x-ray crystallography of wildtype and mutant enzymes complexed with DNA and drug. Aim (3) is to identify the mechanism by which the NorR protein regulates expression of the Nora efflux pump and possibly other related pumps using DNA footprinting with norA promoter DNA and purified NorR. We will also perform transcriptional profiling of the expression of genes encoding efflux pumps with DNA microarrays hybridized with RNA prepared from strains with mutations in norR and arlS, both of which affect norA expression. Aim (4) is to identify additional factors regulating norA expression by purification of a 28-kd protein that in addition to NorR binds upstream of norA, identification of the gene encoding, and generating mutants in this gene. Aim (5) is to identify the complement of multidrug resistance pumps and analyze their expression in an abscess model. This work will be done by analysis of genes related to those of known MDR pumps and their regulators and selected cloning and overexpression of these genes in S. aureus. Overall patterns of expression of the genes will be compared by transcriptional profiling in DNA microarrays using RNA prepared from bacteria surviving in a subcutaneous abscess in rats and RNA from bacteria grown in vitro.