喹诺酮药物杀菌的两条分子途径研究

Bacterial resistance to antimicrobial therapy is a major challenge to the clinical management of infectious disease. One approach for controlling the emergence of resistance is to find new ways to reduce bacterial tolerance to antimicrobials and to markedly improve bactericidal activity. The present proposal focuses on understanding how the quinolones kill bacteria. These broad-spectrum agents, which are highly effective and have an excellent safety record, are now among the most widely used antimicrobials. However, the molecular mechanisms underlying quinolone-mediated killing are poorly understood and are therefore a potential source of novel perturbations that will make the agents effective enough to overcome resistance. Our recent work reveals the existence of two distinct quinolone-mediated killing pathways, each of which is controlled by unidentified proteins. Mutations in genes encoding some of these unidentified proteins are expected to neither grow nor die when exposed to quiniolones. Three such quinolone-toleratant mutants have already been obtained in our preliminary work. In the present application, we propose to dissect the two pathways of quinolone killing by obtaining drug tolerant mutant via enrichment and selection procedures that we recently developed. Mutants obtained, along with their isogenic wild-type parental strain, will be subjected to whole-genome DNA sequence analysis to identify genes encoding "lethal proteins" involved in the killing pathway that requires de novo protein synthesis and genes encoding "auxiliary proteins" contributing to the protein synthesis-insensitive pathway of killing. We expect to gain insight on how these "lethal protein(s)" and "auxiliary protein(s)" interact with quinoloe-topoisomerase-DNA ternary complex, process the complex, and release double-stranded DNA breaks from the complex. Such work will eveutually elucidate molecular mechanisms of quinolone lethality, which would in turn help devise high-throughput screening systems for identification of quinolone lethality potentiators and new quinolone derivatives that are highly active in killing non-growing, dormant pathogens. Successful completion of the present program will guide new antimicroboal development and help contain development of clinical resistance.

细菌耐药性是困扰感染性疾病临床治疗的难题之一；通过新策略降低细菌的药物耐受力、大幅提高药物杀菌活性，有望遏制耐药性产生。喹诺酮是临床广泛使用的广谱抗菌药，但其杀菌的分子机理尚不清楚。我们新近证明喹诺酮可通过2条不同途径进行杀菌，各途径分别由不同的未知关键蛋白控制；并筛选获得喹诺酮压力下既不生长也不死亡的药物耐受突变体。申请项目拟以喹诺酮杀菌的2条途径为研究对象，利用已建立的药物耐受自发突变体富集/筛选技术、结合基因组测序，破译"蛋白从头合成依赖型"杀菌途径的关键效应调节"致死蛋白"和"不依赖蛋白从头合成"途径中的关键"辅助蛋白"，获悉它们与"药物-拓扑异构酶-DNA"三联复合物相互作用、促进复合物释放断裂DNA的分子加工规律，从而加深对喹诺酮分子杀菌机理的理解，为降低临床耐药和促进新药研发提供指导。

抗生素耐药是困扰感染性疾病临床治疗的难题之一；通过新策略降低细菌的药物耐受力、大幅提高药物杀菌活性，有望遏制耐药性产生。喹诺酮是临床广泛使用的广谱抗菌药，但其杀菌的分子机理尚不清楚。本项目对基于文献及前期工作基础上提出的喹诺酮杀菌的两条不同分子通路进行了鉴定与分子机制研究。获得并鉴定出3个蛋白从头合成依赖型杀菌途径的突变体；筛选并获得1不依赖蛋白从头合成途径的突变体。深入研究表明：所有突变体不仅对喹诺酮杀菌产生耐受，而且同时对多种不同抗生素乃至非抗生素杀菌剂产生交叉耐受，耐受机制与降低细菌体内活性氧生成与积累相关，活性氧水平的降低与细胞ATP合成降低以及参与氧化还原应激反应相关酶的表达上调存在因果关系。这些研究不仅加深了我们对喹诺酮杀菌分子机制的了解，而且还确立了氧化还原应激在所有抗生素杀菌中的通用功能，为研究细菌凋亡的共有机制开辟了新的生长点，为开发广谱抗生素杀菌增效剂打下了坚实理论基础并提供了必要的技术储备，对克服、抑制抗生素耐药，有效治疗多种细菌感染具有重大指导作用。