DNA GYRASE AND QUINOLONE RESISTANCE IN TUBERCULOSIS

对结核分枝杆菌DNA旋转酶的研究， 了解抗生素耐药性的获得和决定因素 分枝杆菌细胞内DNA结构。M. 结核病将被克隆，它们的核苷酸序列将被克隆。 确定提供分析所需的分子信息， 氟喹诺酮耐药。氟喹诺酮的促旋酶基因- 将检查耐药临床菌株的突变， 抵抗的原因。从这些菌株中克隆的促旋酶基因将被 用于表达促旋酶，然后将其纯化并在体外研究 用于电阻特性。为了确定是否需要促旋酶， 抗性，遗传互补将使用克隆， 将野生型促旋酶基因引入喹诺酮类耐药的临床 菌株在透性化细胞中抑制DNA合成将是重要的。 检查以鉴定其中抗性是由于非旋转酶引起的菌株 决定因素促旋酶抑制剂也将用于获得额外的 改变超螺旋稳态水平的突变体。这些将 用来询问超螺旋的扰动是否会干扰 通过检测突变体抑制分枝杆菌的能力 在培养的巨噬细胞中存活和繁殖。获得的信息 将定义M.结核病和 将开始描述DNA超螺旋和 毒力M.结核预计这项工作将有助于指导 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.