Nucleic Acids Res：MfpB基因可能是结核分枝杆菌药物抗性的机制

结核病是由结核分枝杆菌引起的人类慢性传染性疾病，是导致人类死亡最多的传染病之一，并且全球约有1／3人口为结核分枝杆菌感染者。我国是全世界22个结核病高负担国家之一，感染人数超过4亿，每年新发传染性病人150万，约有13万人死于结核病。新近的统计数据显示大于10%的病例至少对一种药物有抗性，1%病例为多耐药菌感染，在某些地方已经分离出目前尚无药可治的结核分枝杆菌耐药菌株。因此研究结核分枝杆菌抗药机制对结核病的防治非常重要。

氟喹诺酮类药物具有半衰期长、杀菌活性强、可与其他抗结核药联合使用等特点，成为缩短疗程的候选药物，同时还有杀伤持留菌的功效，其通过抑制DNA促旋酶活性来抑制细菌生长。尽管如此，临床大量应用也导致很多耐药菌的形成。一般认为细菌氟喹诺酮抗性的获得与促旋酶的突变相关，但临床上这两者的相关性较低。最近研究发现结核分枝杆菌五肽重复蛋白MfpA可以模拟DNA结构，能直接与促旋酶互作从而阻断氟喹诺酮的结合。MfpA突变可以降低结核分枝杆菌氟喹诺酮抗性；这表明MfpA可保护DNA旋转酶免受氟喹诺酮的影响。但在体外的无细胞体系中MfpA并不能保护氟喹诺酮对DNA促旋酶活性的影响。这说明在结核分枝杆菌中可能存在其他因素影响MfpA的功能。

中国科学院微生物研究所米凯霞课题组及其合作者通过基因敲除、遗传互补、过表达以及生物化学等分析鉴定了一个影响MfpA功能的蛋白MfpB。MfpB作为一个小GTP酶，以GTP结合的形式与MfpA直接互作。MfpB通过抑制MfpA的活性来调控DNA促旋酶的活性和保护促旋酶免受氟喹诺酮的影响。该研究解释了MfpA体内和体外实验结果差异性的原因并首次表明了小GTP酶在结核分枝杆菌氟喹诺酮抗性调控中非常重要的功能，为进一步了解氟喹诺酮抗性形成的机制和开发新的氟喹诺酮药物和药物靶标提供了理论基础。

研究成果已在线发表在国际生物学知名期刊Nucleic Acids Research上(TAO Jun， HAN Jiao， WU Hanyu， HU Xinling， DENG Jiaoyu， Joy Fleming， Anthony Maxwell， BI Lijun*， and MI Kaixia*. 2012. Mycobacterium fluoroquinolone resistance protein B， a novel small GTPase， is involved in the regulation of DNA gyrase and drug resistance. dio： 10/1093/nar/gks1351)。米凯霞课题组的工作人员陶均为论文第一作者。该项研究得到了国家973项目，国家自然科学基金和中国科学院重要方向性项目等资助。

doi:10.1093/nar/gks1351
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Mycobacterium fluoroquinolone resistance protein B, a novel small GTPase, is involved in the regulation of DNA gyrase and drug resistance

Jun Tao1, Jiao Han1,2, Hanyu Wu1,3, Xinling Hu1, Jiaoyu Deng4, Joy Fleming5, Anthony Maxwell6, Lijun Bi5,* and Kaixia Mi1,*

DNA gyrase plays a vital role in resolving DNA topological problems and is the target of antibiotics such as fluoroquinolones. Mycobacterium fluoroquinolone resistance protein A (MfpA) from Mycobacterium smegmatis is a newly identified DNA gyrase inhibitor that is believed to confer intrinsic resistance to fluoroquinolones. However, MfpA does not prevent drug-induced inhibition of DNA gyrase in vitro, implying the involvement of other as yet unknown factors. Here, we have identified a new factor, named Mycobacterium fluoroquinolone resistance protein B (MfpB), which is involved in the protection of DNA gyrase against drugs both in vivo and in vitro. Genetic results suggest that MfpB is necessary for MfpA protection of DNA gyrase against drugs in vivo; an mfpB knockout mutant showed greater susceptibility to ciprofloxacin than the wild-type, whereas a strain overexpressing MfpA and MfpB showed higher loss of susceptibility. Further biochemical characterization indicated that MfpB is a small GTPase and its GTP bound form interacts directly with MfpA and influences its interaction with DNA gyrase. Mutations in MfpB that decrease its GTPase activity disrupt its protective efficacy. Our studies suggest that MfpB, a small GTPase, is required for MfpA-conferred protection of DNA gyrase.