Quinolone Action During Mycobacterial Growth Arrest

描述（由申请人提供）：本项目的目的是了解喹诺酮类药物在结核分枝杆菌中的作用。对几种细菌物种的研究表明，缓慢的细菌生长和/或生长的停止大大降低了这类抗菌剂的致死活性。最近对大肠杆菌的研究已经确定了氟喹诺酮类药物杀死大肠杆菌的次要途径，该途径可能会绕过生长停滞的负面影响。原则上，通过改变喹诺酮结构来开发这一途径可以使喹诺酮类药物成为更有效的抗菌剂。由于结核病通常包括一个潜伏期，其中M。由于结核病是“休眠的”，因此结核病有望成为评估次要喹诺酮类药物杀伤途径相关性的良好模型。本提案的重点是假定感染后不久发生的细菌生长停滞，因为在模型系统中，生长停滞大大降低了抗菌剂（包括氟喹诺酮类）的活性。研究了E.大肠杆菌的研究表明，次要杀伤途径涉及药物-酶-DNA复合物形成后促旋酶亚基的解离和DNA的断裂。DNA促旋酶突变体已被确定，增强这一次要途径。在分枝杆菌菌株中发现了氨基酸序列改变方面的相同突变体。这些突变体将用于确定是否增强次要致死途径增加氟喹诺酮类药物的总体致死性与M。生长停滞期的肺结核旋转酶突变，喹诺酮类药物的结构和致死作用之间的关系，预计将提供一个更好的了解发生的分子相互作用时，药物-酶复合物形成染色体DNA。研究了两种生长停滞模型系统：小剂量气溶胶感染小鼠和液体培养M。低氧张力下的肺结核编码显性抗原的基因的转录谱分析将用于评估体外系统与鼠模型的相关性。该研究有望提供1）喹诺酮致死性的新观点和2）喹诺酮活性的新测定方法。这种类型的工作最终可能导致新的药物，有效地杀死生长停滞的分枝杆菌和快速清除感染。

DESCRIPTION (provided by applicant): The goal of this project is to understand quinolone action in Mycobacterium tuberculosis. Studies with several bacterial species have shown that slow bacterial growth and/or cessation of growth drastically reduces the lethal activity of this class of antibacterial agent. Recent work with Escherichia coli has established the existence of a minor pathway of killing by fluoroquinolones that may bypass the negative effect of growth arrest. Exploitation of this pathway through changes in quinolone structure could, in principle, make the quinolones much more effective anti-bacterial agents. Since tuberculosis often includes a latent stage in which M. tuberculosis is "dormant", tuberculosis is expected to serve as a good model for assessing the relevance of the minor quinolone killing pathway. The present proposal focuses on the bacterial growth arrest presumed to occur shortly after infection, since in model systems growth arrest drastically reduces the activity of antimicrobial agents, including fluoroquinolones. Studies with E. coli indicate that the minor killing pathway involves the dissociation of gyrase subunits following drug-enzyme-DNA complex formation and breakage of DNA. DNA gyrase mutants have been identified that enhance this minor pathway. Identical mutants, with respect to amino acid sequence change, have been found among mycobacterial strains. These mutants will be used to determine whether enhancement of the minor lethal pathway increases the overall lethality of fluoroquinolones with M. tuberculosis during growth arrest. Relationships between gyrase mutations, quinolone structure, and lethal action are expected to provide a better understanding of the molecular interactions occurring when drug-enzyme complexes form on chromosomal DNA. Two model systems of growth arrest will be studied: low-dose aerosol infection of mice and liquid culture of M. tuberculosis under low oxygen tension. Transcription profiling of genes encoding dominant antigens will be used to assess the relevance of the in vitro system to the murine model. The study is expected to provide 1) a new view of quinolone lethality and 2) novel assays for quinolone activity. This type of work may eventually lead to new agents that effectively kill growth-arrested mycobacteria and clear infection rapidly.