Quinolone Action During Mycobacterial Growth Arrest

描述(申请人提供)：本项目的目标是了解喹诺酮类药物对结核分枝杆菌的作用。对几种细菌的研究表明，细菌生长缓慢和/或停止生长会极大地降低这类抗菌剂的致死活性。最近对大肠杆菌的研究证实，氟喹诺酮类药物存在一种微小的杀灭途径，可以绕过生长抑制的负面影响。通过改变喹诺酮类药物的结构来利用这一途径，原则上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结核分枝杆菌“休眠”的潜伏期，因此结核病有望成为评估轻微喹诺酮类药物致死途径相关性的良好模型。本提案的重点是假定在感染后不久发生的细菌生长停滞，因为在模型系统中，生长停滞会大大降低包括氟喹诺酮类在内的抗菌剂的活性。对大肠杆菌的研究表明，在药物-酶-DNA复合体形成和DNA断裂之后，微小的杀伤途径涉及旋转酶亚基的解离。DNA旋转酶突变体已被鉴定为增强这一次要途径。在分枝杆菌菌株中发现了氨基酸序列变化方面相同的突变体。这些突变体将被用来确定在生长停滞期间，微小致死途径的增强是否会增加氟喹诺酮类药物对结核分枝杆菌的总体致死率。旋转酶突变、喹诺酮类药物结构和致死作用之间的关系有望更好地理解药物-酶复合体在染色体DNA上形成时发生的分子相互作用。将研究两种生长停滞的模型系统：小鼠的低剂量气溶胶感染和低氧分压下的结核分枝杆菌液体培养。编码优势抗原的基因转录图谱将被用来评估体外系统与小鼠模型的相关性。这项研究有望提供1)对喹诺酮类药物致死性的新看法和2)新的喹诺酮类药物活性检测方法。这种类型的工作可能最终会导致新的药物，有效地杀死生长受阻的分枝杆菌，并迅速清除感染。

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.