结核分枝杆菌耐多药/广泛耐药机制研究及新药靶基因的鉴定

The emergence and spread of multidrug-resistant and extensively drug-resistant mycobacterium tuberculosis brought serious challenges to the global TB control. The mechanism of Multidrug-resistant/extensively drug-resistant mycobacterium tuberculosis is still not very clear, a large number of studies suggest that the lipid of cell walls of mycobacterium tuberculosis may be involved in the pathogenesis. Lipid is an important factor of toxicity and survival mycobacterium tuberculosis, is also a potential new drug targets. This research team perform the whole genome sequencing of 2 strains extensively drug-resistant mycobacterium tuberculosis in the early stage of the work, The results found 284 new mutations, Based on the biological function of mutated genes encoding proteins and amino acids and the analysis of the software, 20 new mutations genes most likely related to multi-drug resistance/extensively drug-resistant was screened out， Further analysis that a unique code of the cell walls of mycobacterium tuberculosis two sets of fatty acid structure domain polyketone synthase (pks12).Pks12 owns the largest of open reading frame (ORF) of mycobacterium tuberculosis ，According to a report in the literature that pks12 is one of the largest protein coding mycobacterium tuberculosis in molecular weight.The purpose of this study is to detect pks12 in sensitive with the variation of drug-resistant mycobacterium tuberculosis, This study intends to detect pks12 in sensitive and variation of drug-resistant mycobacterium tuberculosis, And pks12 gene overexpression、 knockout and covering will be applied in standard strain H37Rv， It was observed that the related strains on pks12 gene mutations influence the minimum inhibitory concentration (MIC) , Further understand the role of pks12 gene of mycobacterium tuberculosis in drug resistance mechanism, to provide new drug enzymes target gene screening for multidrug-resistant/xdr-tb treatment .

耐多药及广泛耐药结核分枝杆菌（MDR-TB/XDR-TB）的出现和传播给全球结核病防治带来严峻挑战。结核分枝杆菌耐多药/广泛耐药机制至今未明，研究提示结核分枝杆菌细胞壁脂类可能参与发病机制,是其毒力和生存的重要因素，也是潜在的新药靶点。本课题组前期对2株XDR-TB进行了全基因组测序，发现284个新突变基因,根据突变基因编码蛋白的生物学功能和氨基酸特性以及软件分析，筛选出与MDR/XDR-TB最可能相关的20个新突变基因,进一步分析获得1个独特的编码结核分枝杆菌细胞壁两套脂肪酸结构域的聚酮合酶（pks12）。pks12拥有结核分枝杆菌中最大的开放阅读框（ORF），预测是编码结核分枝杆菌中分子量最大的蛋白质。本研究拟检测pks12在敏感与耐药结核分枝杆菌中的变异情况，并利用pks12基因在H37Rv标准菌株中过表达、敲除、回补等技术，观察pks12突变对相应菌株最低抑菌浓度（MIC）的影响。

研究结核分枝杆菌(MTB)耐药机制具有公共卫生意义和临床价值，前期研究中发现Rv2048c（pks12）基因与MTB耐药相关，但具体功能及致病机制不明。本项目以Rv2048c基因为研究对象，验证Rv2048c基因敲除株H37RvΔRv2048c对比MTB标准菌株H37Rv-WT对抗结核药物敏感性的影响，分析Rv2048c基因突变与药物敏感性的对应关系；其次是验证目的蛋白Rv2048c对巨噬细胞内存活的影响。通过构建Rv2048c基因敲除株H37RvΔRv2048c，液体培养标准菌株H37Rv-WT及敲除株H37RvΔRv2048c，通过测定生长曲线、抗酸染色、扫描电镜观察标准株与敲除株生长情况及菌落形态变化；H37Rv-WT及H37RvΔRv2048c进行12种抗结核药物的最低抑菌浓度（MIC）试验，筛选出敲除株H37RvΔRv2048c中MIC值有明显差异的药物；收集MDR-TB临床分离菌，提取DNA送全基因组测序（WGS），检测出单核苷酸多态性（SNP）位点在Rv2048c基因序列范围存在内14个非同义突变位点。筛选的MDR-TB临床分离菌进行12种抗结核药物MIC试验，发现Rv2048c基因可增加MTB对ETH的耐受性；62例MDR-TB临床分离菌的MIC检测显示ETH与PTH的药敏表型完全一致；通过全基因组测序分析Rv2048c基因突变与ETH/PTH药敏表型的关系，发现3个突变基因未见报道的新突变位点。在探索Rv2048c基因对MTB在巨噬细胞内存活的影响及其机制时还发现Rv2048c通过NF-κB信号通路调节巨噬细胞炎症细胞因子的表达，抑制细胞因子TNF-α与IL-6的分泌，增加细菌的毒力，增强MTB在巨噬细胞内的存活能力。本研究进一步了解Rv2048c参与结核病的发病机制，为探索MTB突变位点与药物耐受性的关系提供新的理论依据。