结核杆菌RNA降解酶GpsI介导吡嗪酰胺耐药的分子机制

Drug resistance to pyrazinamide (PZA) poses a significant challenge for tuberculosis (TB) treatment. The genes involved in PZA resistance and their mechanisms are unknown, except for pncA and rpsA. We have recently found novel GpsI (involved in mRNA dehydrogenase ) mutation (K702Q) in some PZA-resistant MDR-TB isolates without common pncA and rpsA mutations. Our preliminary data in vitro has revealed that K702Q mutation is directly related to PZA resistance and that this mutant site is located in the S1-like motif of GpsI protein. Based on our previous study, we have obtained GpsI wild-type and mutant protein and constructed the gpsI site-directed mutant and complement strains successfully, we intend to identify the the molecular mechanism of how gpsI regulates PZA resistance by using in vitro and in vivo experiments. These studies will shed new light on the molecular basis of PZA in M. tuberculosis and improve our understanding of tuberculosis pathogenesis with implications of rational design of new vaccines and therapeutic agents.

结核分枝杆菌对一线药物吡嗪酰胺（PZA）耐药机制是目前研究热点。申请人前期发现PZA耐药临床结核菌株中存在mRNA降解酶GpsI（K702Q）突变，且该突变导致细菌特异性耐受PZA。结构分析发现K702Q位于S1-like功能域中，推测POA（PZA体内活性形式）通过竞争GpsI该功能域与反式翻译核心分子tmRNA结合而杀菌，K702Q突变导致POA结合GpsI能力减弱，引起PZA耐受。为详尽阐明以上假设，本项目拟采用应激表达量检测和凝胶迁移滞后实验明确GpsI通过S1-like功能域与tmRNA结合参与反式翻译；利用酶活抑制实验证明POA竞争该功能域，GpsI K702Q突变影响POA结合GpsI能力等；动物体内模型进一步明确该突变对结核菌存活及抵抗PZA能力的影响，最终明确GpsI在结核菌耐受PZA中具体分子机制。本研究成果将为寻找抗结核药物新靶标及建立耐药结核感染的治疗方法奠定基础。

吡嗪酰胺（PZA）是抗结核治疗的重要一线药物之一，其优势尤其在于缩短结核治疗疗程。PZA作用机制是非常复杂的，其在体内的活性形式POA在结核杆菌内有多个耐药靶标（RpsA、PanD和CLPC1等）。最近的研究发现结核杆菌GpsI（Rv2783c）双功能核酸降解酶是POA的新靶标，然而POA是如何作用于Rv2783的具体机制尚不清楚。我们研究发现一个新的PZA耐药位点Rv2783cA2104C，突变基因的表达会增加PZA在临床菌株中的耐药性。并且，酶活实验证明相应的Rv2783K702Q突变蛋白的降解酶活性会被POA显著抑制，而野生型Rv2783蛋白的酶活没有改变。另外，凝胶迁移实验证实POA会和tmRNA竞争性结合Rv2783蛋白，而无法结合Rv2783K702Q突变蛋白。因此我们的研究阐明结核分枝杆菌Rv2783双功能酶结合tmRNA，参与反式翻译的功能，进一步补充和完善结核分枝杆菌对PZA耐受的分子机制，为耐药结核分枝杆菌在PZA药物治疗中的耐药调节通路提供新的实验依据。