五肽重复蛋白MfpAMt介导的结核分枝杆菌喹诺酮抗性机制研究

Tuberculosis, caused by the pathogen Mycobacterium tuberculosis, is one of the most serious infectious diseases in the world. The prevalence of Multi-drug and Extensively drug-resistant tuberculosis (MDR and XDR-TB) worsen the situation and the treatment of drug-resistant tuberculosis becomes more complicated and expensive. The fluoroquinolones (FQ) are the cornerstone of the treatment for patients with MDR or XDR. There are several FQ-resistant mechanisms reported and only a genetic target gyrase was confirmed in both clinical and lab. But mutations on gyrase coding genes only present in 30-70% of resistant clinical isolates thus make it difficult to diagnose FQ-resistance in M. tuberculosis. Several previous studies showed that the regulator of gryase involves into drug-resistance, such as Mycobacterial FQ-resistant pentapeptide in Mycobacterium tuberculosis, MfpAMt. We reasoned that the interaction proteins with MfpAMt might regulate DNA gyrase activity and take an effect on the FQ resistance. In preliminary data, we identified that its upstream gene Rv3362c (MfpBMt) is essential for MfpAMt-conferred protection of DNA gyrase from drug interference. In this project, we plan to study the interaction of MfpAMt and Rv3362c and how the complex regulates the interplay of DNA gyrase and FQ by using genetics and biochemistry methods. Furthermore, we would characterize the interactions between the proteins within MfpAMt operon (including Rv3361c-Rv3365c) and the influence on the FQ resistance both in vivo and in vitro. Moreover, to further explore the mechanisms of the MfpA-mediated FQ resistance, the Co-IP and MS methods are chosen to fish the interaction proteins with MfpAMt and MfpBMt within the whole cell and figure out the roles of candidate proteins in FQ resistance. Our results will give an insight of the mechanisms of FQ resistance in Mycobacterium tuberculsis and the knowledge should lead to develop MDR/XDR diagnosis methods and provide the possible targets for drug screening.

耐药结核分枝杆菌的流行是目前结核病防治的世界难题。氟喹诺酮(fluoroquinolones, FQ)类药物是治疗耐药结核病的核心药物。FQ的作用靶标是DNA旋转酶。MfpAMt是最新发现的与DNA旋转酶互作的蛋白。我们认为对MfpAMt进行深入研究将会加深对细菌FQ抗性的了解。我们的前期实验发现MfpAMt受其上游蛋白Rv3362c的调控。本课题在基础上，首先利用遗传方法和生化方法研究MfpAMt与Rv3362c相互作用的方式和体外系统检测二者对DNA旋转酶活性和对DNA旋转酶与FQ互作的影响；然后研究MfpAMt操纵子内蛋白间的互作关系以及这些互作对MfpAMt介导的FQ抗性的影响；再利用免疫共沉淀和质谱连用的方法发掘更多细胞内与MfpAMt互作的蛋白和研究这些蛋白在FQ抗性形成中的作用。此项研究将深入理解结核分枝杆菌抗氟喹诺酮的机制，为耐药结核的防治提供理论基础。

氟喹诺酮(fluoroquinolones, FQ)类药物是治疗耐药结核病的核心药物，其作用靶标为DNA 旋转酶。MfpA通过影响DNA 旋转酶调控细菌的FQ抗性。本项目探讨了调控MfpA的蛋白及其作用机制。主要分析了（1）Rv3362c（MfpB）调控MfpA活性的作用机制以及（2）MfpE, MfpD, MfpC, MfpB及MfpA的互作及调控MfpB和MfpA活性的特征。发现了MfpB能够与MfpA直接互作并影响MfpA与旋转酶的互作，进而影响旋转酶的活性。MfpB作为一个小GTP酶，以GTP-和GDP结合的形式调控其同MfpA的互作，进而影响MfpA的功能。MfpB的GTP-和GDP-结合形式的转化可能受到MfpD和MfpC的调控，另外MfpE执行ATP酶活性，其分解ATP将影响MfpD或MfpC的活性，从而影响MfpB活性（GTP结合）及非活性（GDP结合）形式的转换。本研究揭示了结核分枝杆菌FQ抗性调控的新机制，MfpE可能通过感应外界或胞内信号启动其ATP酶活性，通过MfpD和 MfpC的传导影响 MfpB的GTP酶活性，进而调控MfpA与旋转酶的互作并影响细菌的FQ抗性。本研究暗示，通过干扰MfpA参与的信号传导通路，可以有效地抑制细菌的FQ抗性，为结核病的治疗提供新的方式和辅助途径。