MECHANISMS OF ACTION AND RESISTANCE TO QUINOLONE AGENTS

我们的目标是了解和细菌的作用机制 对喹诺酮类抗菌药物的耐药性， 拮抗细菌必需酶DNA促旋酶的活性。 较新的喹诺酮类药物显示出治疗 各种人类感染。 了解其作用机制， 细菌对这些药物的耐药性可能有助于确定耐药机制 在从受感染的患者分离的细菌中， 流行病学工具。 此外，了解喹诺酮类药物如何杀死 细菌可能有助于阐明如何最好地使用这些药物治疗感染， 杀死细菌是治愈的必要条件 我们的具体目标包括（1）抗菌活性与 几种喹诺酮类药物对大肠杆菌DNA促旋酶活性的影响 体外，（2）测定喹诺酮与纯化DNA的结合，DNA促旋酶， 和DNA回旋酶-DNA复合物，（3）分子量的测定 喹诺酮类药物杀菌要求;（4） 对更新、更有效的喹诺酮类药物产生耐药性的基因位点 药物，和（5）确定喹诺酮摄取的机制， 细菌细胞 用于实现这些目标的方法包括（1）测定 喹诺酮类药物抑制螺旋酶介导的DNA超螺旋和去连环化 以及促旋酶和DNA之间的复合物的药物稳定性的测定;（2） 放射性标记的喹诺酮与DNA和DNA促旋酶结合的测定， 在确保酶稳定性的条件下的甘油梯度;（3） 喹诺酮类药物对具有突变基因的细菌的杀灭试验， 通过喹诺酮处理和直接鉴定来刺激表达 和绘制突变体，其中药物诱导的细菌杀伤是 选择性钝化（耐受突变体）;（4）选择，定位和 喹诺酮耐药突变体的表征;和（5）测定 放射性标记的喹诺酮与野生型和推定的 渗透缺陷突变细菌。

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.