抑制N-乙酰葡糖胺-1-磷酸转移酶（WecA）有效增强分枝杆菌利福平敏感性的作用与机制研究

Drug resistance is a bottleneck of tuberculosis treatment. It is of great significance in the development of anti-tuberculosis drugs based on new targets. N-acetylglucosamine-1-phosphate transferase (WecA) as a key enzyme which participates the synthesis of the cell wall of Mycobacterium tuberculosis, is a potential drug target. In previous study we used Mycobacterium smegmatis as a model mycobacterial strain and found that knockdown of WecA decreased the growth rate of mycobacteria, and caused the strains to exhibit dramatically morphological alterations. Further study showed that down-regulation of WecA increased the sensitivity of mycobacteria against rifampin. In addition, the transcriptome profiling data showed that the expression of MIPS and MshA, enzymes participated the synthesis of mycothiol which acted as a primary reducing agent for the detoxification of antibiotics in mycobacteria, were down-expressed along with the knockdown of WecA. Based on these findings, we suppose that WecA might affect the sensitivity of mycobacteria against rifampin through regulating the synthesis and detoxification pathway of mycothiol. Moreover, inhibiting the activity of WecA together with rifampin treatment might effectively prevent mycobacterial growth. In order to validate the hypothesis, the effect of mycothiol synthetic pathway mediated by MIPS and MshA on the WecA inhibition sensitizing mycobacteria to rifampin will be clarified through the establishment of mycobacterial MIPS and MshA over-expression as well as knockdown strain model. The rifampin sensitivity-related proteins regulated by WecA will be explored throuth the proteomic analysis. M. tuberculosis H37Ra, the avirulent strain of M. tuberculosis will be used to further validate the results obtained from M. smegmatis above. In order to screen the specific inhibitors of WecA, WecA protein of M. tuberculosis will be over-expressed in E. coli expression system and purified through affinity chromatography. For WecA activity analysis, a high-throughput assay will be established based on the detection of UMP, the product of WecA enzymatic reaction. By using this assay, the kinetic properties of WecA enzyme will be determined and potential inhibitors will be screened from the secondary metabolite library of marine actinomycetes. This study will lay a theoretical foundation for the elucidation of WecA catalytic mechanism and the development of WecA inhibitors, as well as provide new approaches for the treatment of tuberculosis and improving the effevtiveness of rifampin treatment.

耐药是结核病治疗瓶颈，基于新靶点的药物研发意义重大。细胞壁合成关键酶WecA是潜在抗结核药物靶标。申请人前期以耻垢分枝杆菌为模型，发现敲减WecA导致菌体对利福平敏感性增强，转录组学分析进一步发现，此时小分子解毒剂分枝硫醇合成相关酶MIPS与MshA表达下调。由此我们假设：抑制WecA可能因减少分枝硫醇合成影响其解毒功能而有效增强分枝杆菌利福平敏感性。为证实假说，本项目拟研究MIPS、MshA介导的分枝硫醇生物合成对下调WecA增强分枝杆菌利福平敏感性的影响，通过蛋白质组学探寻与WecA调控利福平敏感性相关蛋白，并利用结核分枝杆菌无毒菌株模型加以验证；同时应用大肠杆菌表达体系诱导并纯化WecA蛋白，建立高通量酶活性测定方法并获得酶促反应动力学参数，为特异性WecA酶抑制剂筛选创造条件。本项目将为阐明WecA催化机制和研发抗结核新药奠定理论基础，为对抗结核病、提高利福平治疗有效性提供新思路。

本项目以结核分枝杆菌细胞壁合成关键酶之一——N-乙酰葡糖胺-1-磷酸转移酶（WecA）为研究靶标，旨在通过获得WecA纯酶并阐明其酶促反应动力学特征，为酶抑制剂筛选、抗结核新药研发奠定基础，同时基于“抑制WecA有效增强分枝杆菌对利福平的敏感性”这一前期发现，利用组学和分子生物学技术阐明其分子机制，旨在为对抗结核病、提高利福平治疗有效性提供新思路。.由于WecA是一个具有11个跨膜结构域的膜蛋白，结构十分复杂，高表达及纯化难度极大。在本项目实施过程中，我们尝试了多种方法（包括使用不同的表达载体、表达菌株、表达标签、诱导表达条件以及体外表达等），但结果均不理想，WecA蛋白无表达或表达量极低。项目后期课题组使用T7溶菌酶可调控的E.coli Lemo21(DE3)菌株，构建WecA-GFP融合表达载体，通过GFP的检测确定WecA的表达情况，优化表达条件。最终我们从菌株膜蛋白组分中获得了高表达的WecA蛋白。我们纯化了WecA蛋白并利用质谱技术进行了定性。WecA酶学研究工作即将开始，我们将建立高通量WecA酶活性检测方法，最终获得WecA酶促反应动力学参数。.另一方面，我们基于申请书中提出的科学假说——抑制WecA因减少MSH合成影响其解毒功能而有效增强分枝杆菌对利福平的敏感性，通过构建MSH合成关键酶MIPS、MshA的过表达和敲减菌株，初步证实分枝杆菌对利福平的敏感性与MIPS及MshA的表达呈负相关。为避免原方案中质粒、诱导剂等处理因素的影响，课题后期调整了研究方案，即分析分枝杆菌在利福平、衣霉素（WecA天然抑制剂）单药及两药联用作用下的蛋白组学变化，进而探索抑制WecA表达能够有效增强分枝杆菌对利福平敏感性的分子机制，达到本项目的研究目的。目前，本项目已使用1/2 MIC浓度的两药单独及联合处理耻垢分枝杆菌，菌体已送生物公司进行定量蛋白组分析（4D Label-free），结果尚未返回。未来的研究工作中，我们将根据组学结果及信号通路分子富集情况提出新的科学假说，并加以验证，以期阐明抑制WecA有效增强分枝杆菌利福平敏感即衣霉素与利福平联合应用能够增强对分枝杆菌抑菌效果的分子机制。.此外，本项目还对绿色荧光蛋白（GFP）在原核表达系统中的作用、GAPDH作为原核表达系统内参的应用进行了探索，期待其为生物化学及分子生物学学科的科研与教学工作提供新方法、新工具。