MECHANISMS OF ACTION AND RESISTANCE TO QUINOLONE AGENTS

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Our objectives are to understand the mechanisms of action of and bacterial resistance to quinolone antimicrobial agents, a class of agents antagonizing the activities of the essential bacterial enzyme DNA gyrase. The newer quinolone agents show great promise for the treatment of a variety of human infections. Understanding the mechanisms of action and of bacterial resistance to these drugs may help identify resistance mechanisms in bacteria isolated from infected patients and thereby provide epidemiologic tools. In addition, understanding how quinolones kill bacteria may help clarify how best to use these agents in infections in which bacterial killing is necessary for cure. Our specific aims include (1) correlation of antimicrobial activity with the effect of several quinolones on several activities of DNA gyrase in vitro, (2) determination of quinolone binding to purified DNA, DNA gyrase, and DNA gyrase-DNA complexes, (3) determination of the molecular requirements for bacterial killing by quinolones, (4) determination of the genetic loci responsible for resistance to newer, more potent quinolone agents, and (5) determination of the mechanisms of quinolone uptake by bacterial cells. Methods to be used to accomplish these goals include (1) assays of quinolone inhibition of gyrase-mediated DNA supercoiling and decatenation and assays of drug stabilization of a complex between gyrase and DNA; (2) assays of radiolabeled quinolone binding to DNA and DNA gyrase using glycerol gradients under conditions that assure enzyme stability; (3) assays of quinolone killing of bacteria with mutations in genes whose expression is stimulated by quinolone treatment and direct identification and mapping of mutants in which drug-induced bacterial killing is selectively blunted (tolerant mutants); (4) selection, mapping and characterization of quinolone-resistant mutants; and (5) assays of radiolabeled quinolone binding to wildtype and putative permeation-deficient mutant bacteria.