DNA GYRASE AND QUINOLONE RESISTANCE IN TUBERCULOSIS

结核分枝杆菌DNA旋转酶的研究将更好地 了解抗生素耐药性的获得和决定因素 分枝杆菌的胞内DNA结构。结果表明，M. 结核病将被克隆，它们的核苷酸序列将被 确定提供分析所需的分子信息 氟喹诺酮类耐药。那么来自氟喹诺酮的旋转酶基因- 将对耐药临床菌株进行突变检查，以确定 这是抵抗的原因。从这些菌株中克隆的旋转酶基因将是 用于表达旋转酶，然后进行纯化和体外研究 以获得阻力特性。以确定是否需要旋转酶 抗性，遗传互补将用克隆的， 野生型旋转酶基因导入临床对喹诺酮类药物耐药 菌株。对通透性细胞DNA合成的抑制将是 检查以确定耐药菌株是非旋转酶所致 决定因素。旋转酶抑制剂也将用于获得更多的 已经改变了超螺旋的稳态水平的突变体。这些遗嘱 用于询问超卷的扰动是否干扰 通过检测突变体抑制分枝杆菌的能力来确定毒力 培养的巨噬细胞的存活和繁殖。获取的信息 将定义结核分枝杆菌对喹诺酮类药物的耐药性 将开始描述DNA超卷曲和 结核分枝杆菌的毒力。预计这项工作将有助于指导努力 开发新的、更有效的抗结核药物。

DNA gyrase of Mycobacterium tuberculosis will be studied to better understand acquisition of antibiotic resistance and factors that determine intracellular DNA structure in mycobacteria. The two gyrase genes of M. tuberculosis will be cloned, and their nucleotide sequences will be determined to provide molecular information needed for analysis of fluoroquinolone resistance. Then the gyrase genes from fluoroquinolone- resistant clinical strains will be examined for mutations that will account for resistance. Gyrase genes cloned from these strains will be used to express gyrase, which will then be purified and studied in vitro for resistance properties. To determine whether gyrase is required for resistance, genetic complementation will be carried out using cloned, wild-type gyrase genes introduced into quinolone-resistant clinical strains. Inhibition of DNA synthesis in permeabilized cells will be examined to identify strains in which resistance is due to non-gyrase determinants. Gyrase inhibitors will also be used to obtain additional mutants that have altered steady-state levels of supercoiling. These will be used to ask whether perturbation of supercoiling interferes with virulence by examining the ability of mutants to inhibit mycobacterial survival and reproduction in cultured macrophages. Information obtained from this program will define quinolone resistance in M. tuberculosis and will begin to describe relationships between DNA supercoiling and virulence in M. tuberculosis. The work is expected to help guide efforts to develop new, more effective anti-tuberculosis agents.