MECHANISMS OF ACTION AND RESISTANCE TO QUINOLONE AGENTS

我们的目标是了解和细菌的作用机制。 对喹诺酮类抗菌药的耐药性 拮抗细菌必需酶DNA旋转酶的活性。 较新的喹诺酮类药物在治疗非霍奇金淋巴瘤方面前景看好。 各种各样的人类感染。了解行动机制和 细菌对这些药物的耐药性可能有助于确定耐药性机制 从受感染的病人身上分离出的细菌，从而提供 流行病学工具。此外，了解喹诺酮类药物是如何杀死 细菌可能有助于阐明如何最好地使用这些制剂来治疗感染 细菌杀灭是治愈疾病所必需的。 我们的具体目标包括(1)抗菌活性与 几种喹诺酮类药物对鼻咽癌DNA旋转酶几种活性的影响 体外，(2)喹诺酮类药物与纯化的DNA，DNA旋转酶， 和DNA旋转酶-DNA复合体，(3)分子测定 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座 以及(5)喹诺酮类药物摄取机制的测定。 细菌细胞。 用于实现这些目标的方法包括(1)分析 喹诺酮类药物对旋转酶介导的DNA超螺旋和脱链的抑制作用 旋转酶与DNA形成的络合物的药物稳定性测定； 放射性标记喹诺酮类药物与DNA和DNA旋转酶结合的检测 保证酶稳定性的甘油梯度；(3) 喹诺酮类药物对基因突变细菌的杀灭试验 喹诺酮类药物治疗和直接鉴定刺激表达 以及药物诱导的细菌死亡的突变体的图谱 选择性钝化(耐受突变体)；(4)选择、定位和 喹诺酮类耐药突变株的特征；和(5) 放射性标记的喹诺酮类药物与野生型和推定的结合 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.