Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis:: Functional analysis of mutant enzymes
Novel gyrase mutations in quinolone-resistant and -hypersusceptible clinical isolates of Mycobacterium tuberculosis:: Functional analysis of mutant enzymes
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DOI：
10.1128/aac.50.1.104-112.2006
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发表时间：
2006-01-01
影响因子：
4.9
通讯作者：
Fisher, LM
中科院分区：
文献类型：
--
作者：
Aubry, A;Veziris, N;Fisher, LM
DNA旋切酶GyrA(2)GyrB(2)复合物的突变与结核分枝杆菌对喹诺酮类药物的耐药性有关。随着氟喹诺酮类药物越来越多地用于结核病的治疗，我们鉴定了几种耐多药结核分枝杆菌临床分离株，这些分离株携带与喹诺酮类药物耐药或超敏感相关的GyrA或GyrB亚基编码基因突变。除了报道的GyrA的喹诺酮类药物耐药突变，即A90V、D94G和D94H外，我们还发现GyrB N510D突变也与氧氟沙星耐药有关。令人惊讶的是，一些具有gyrA T80A和A90G新组合变化的分离株对氧氟沙星敏感。结核分枝杆菌GyrA和GyrB亚基（野生型[WT]和突变型）在大肠杆菌中过表达，纯化至均匀性，用于重建高活性的gyrase复合物。通过诱变从工程gyrA和gyrB等位基因中产生相似的突变蛋白。测定莫西沙星、加替沙星、氧氟沙星、左氧氟沙星和依诺沙星的mic、酶抑制和药物诱导的DNA切割。含有GyrA A90V、D94G、D94H和GyrB N510D的突变型gyrase复合物对喹诺酮类药物的抑制具有抗性（mic和50%抑制浓度[IC(50)s]至少是WT浓度的3.5倍），并且除GyrB突变体外，所有突变体作为喹诺酮类裂解复合物的捕获效率都较低。与此形成鲜明对比的是，携带GyrA T80A或A90G基因的gyrase复合物对许多喹诺酮类药物的作用敏感，这种效应在携带两种突变的复合物（mic和IC(50)s比WT值低14倍）中得到强化。这是首次对结核分枝杆菌的超敏感和耐药进行详细的酶分析。
Mutations in the DNA gyrase GyrA(2)GyrB(2) complex are associated with resistance to quinolones in Mycobacterium tuberculosis. As fluoroquinolones are being used increasingly in the treatment of tuberculosis, we characterized several multidrug-resistant clinical isolates of M. tuberculosis carrying mutations in the genes encoding the GyrA or GyrB subunits associated with quinolone resistance or hypersusceptibility. In addition to the reported putative quinolone resistance mutations in GyrA, i.e., A90V, D94G, and D94H, we found that the GyrB N510D mutation was also associated with ofloxacin resistance. Surprisingly, several isolates bearing a novel combination of gyrA T80A and A90G changes were hypersusceptible to ofloxacin. M. tuberculosis GyrA and GyrB subunits (wild type [WT] and mutants) were overexpressed in Escherichia coli, purified to homogeneity, and used to reconstitute highly active gyrase complexes. Mutant proteins were produced similarly from engineered gyrA and gyrB alleles by mutagenesis. MICs, enzyme inhibition, and drug-induced DNA cleavage were determined for moxifloxacin, gatifloxacin, ofloxacin, levofloxacin, and enoxacin. Mutant gyrase complexes bearing GyrA A90V, D94G, and D94H and GyrB N510D were resistant to quinolone inhibition (MICs and 50% inhibitory concentrations [IC(50)s] at least 3.5-fold higher than the concentrations for the WT), and all, except the GyrB mutant, were less efficiently trapped as a quinolone cleavage complex. In marked contrast, gyrase complexes bearing GyrA T80A or A90G were hypersusceptible to the action of many quinolones, an effect that was reinforced for complexes bearing both mutations (MICs and IC(50)s up to 14-fold lower than the values for the WT). This is the first detailed enzymatic analysis of hypersusceptibility and resistance in M. tuberculosis.