結(jié)核病是由結(jié)核分枝桿菌引起的人類(lèi)慢性傳染性疾病,，是導(dǎo)致人類(lèi)死亡最多的傳染病之一，并且全球約有1／3人口為結(jié)核分枝桿菌感染者,。我國(guó)是全世界22個(gè)結(jié)核病高負(fù)擔(dān)國(guó)家之一,，感染人數(shù)超過(guò)4億，每年新發(fā)傳染性病人150萬(wàn),，約有13萬(wàn)人死于結(jié)核病,。新近的統(tǒng)計(jì)數(shù)據(jù)顯示大于10%的病例至少對(duì)一種藥物有抗性，1%病例為多耐藥菌感染,，在某些地方已經(jīng)分離出目前尚無(wú)藥可治的結(jié)核分枝桿菌耐藥菌株,。因此研究結(jié)核分枝桿菌抗藥機(jī)制對(duì)結(jié)核病的防治非常重要,。
氟喹諾酮類(lèi)藥物具有半衰期長(zhǎng),、殺菌活性強(qiáng)、可與其他抗結(jié)核藥聯(lián)合使用等特點(diǎn),，成為縮短療程的候選藥物,，同時(shí)還有殺傷持留菌的功效,，其通過(guò)抑制DNA促旋酶活性來(lái)抑制細(xì)菌生長(zhǎng)。盡管如此,，臨床大量應(yīng)用也導(dǎo)致很多耐藥菌的形成,。一般認(rèn)為細(xì)菌氟喹諾酮抗性的獲得與促旋酶的突變相關(guān)，但臨床上這兩者的相關(guān)性較低,。最近研究發(fā)現(xiàn)結(jié)核分枝桿菌五肽重復(fù)蛋白MfpA可以模擬DNA結(jié)構(gòu),，能直接與促旋酶互作從而阻斷氟喹諾酮的結(jié)合。MfpA突變可以降低結(jié)核分枝桿菌氟喹諾酮抗性,；這表明MfpA可保護(hù)DNA旋轉(zhuǎn)酶免受氟喹諾酮的影響。但在體外的無(wú)細(xì)胞體系中MfpA并不能保護(hù)氟喹諾酮對(duì)DNA促旋酶活性的影響,。這說(shuō)明在結(jié)核分枝桿菌中可能存在其他因素影響MfpA的功能,。
中國(guó)科學(xué)院微生物研究所米凱霞課題組及其合作者通過(guò)基因敲除、遺傳互補(bǔ),、過(guò)表達(dá)以及生物化學(xué)等分析鑒定了一個(gè)影響MfpA功能的蛋白MfpB,。MfpB作為一個(gè)小GTP酶，以GTP結(jié)合的形式與MfpA直接互作,。MfpB通過(guò)抑制MfpA的活性來(lái)調(diào)控DNA促旋酶的活性和保護(hù)促旋酶免受氟喹諾酮的影響,。該研究解釋了MfpA體內(nèi)和體外實(shí)驗(yàn)結(jié)果差異性的原因并首次表明了小GTP酶在結(jié)核分枝桿菌氟喹諾酮抗性調(diào)控中非常重要的功能，為進(jìn)一步了解氟喹諾酮抗性形成的機(jī)制和開(kāi)發(fā)新的氟喹諾酮藥物和藥物靶標(biāo)提供了理論基礎(chǔ),。
研究成果已在線(xiàn)發(fā)表在國(guó)際生物學(xué)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),。米凱霞課題組的工作人員陶均為論文第一作者。該項(xiàng)研究得到了國(guó)家973項(xiàng)目,，國(guó)家自然科學(xué)基金和中國(guó)科學(xué)院重要方向性項(xiàng)目等資助,。（生物谷Bioon.com）
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.
