ndh基因突变致结核分枝杆菌对氯法齐明耐药的分子机制研究

Clofazimine (Cfz) might shorten multiple drug resistant tuberculosis regimen, but the mechanisms associated with Cfz resistance of mycobacterium tuberculosis (Mtb) are not well characterized. To understand the mechanisms, 16 Cfz-resistant and 16 Cfz-susceptible Mtb were subjected to whole genome sequencing to identify Cfz resistance associated genes, which were then experimentally confirmed using further clinical isolates and site-directed mutagenesis. The results indicated the association between ndh gene mutation and Cfz resistance. Type II NADH: menaquinone oxidoreductase (NDH-2), which coded by ndh gene, could reduce Cfz and nonenzymatic oxidation of reduced Cfz by O2 yielding CFZ and reactive oxygen species (ROS). High levels of intracellular ROS could be bactericidal. The hypothesis is that resistance to Cfz can be mediated by mutations in ndh gene, which could alter the activity of NDH-2 and would affect the reduction of Cfz and yields of ROS. To address the hypothesis, the association between ndh mutation and Cfz resistance will be confirmed using lots of clinical isolates and site-directed mutagenesis and complementary assay. The activity of the mutant variants of NDH-2 and the yields of ROS will be measured. The effects of ndh mutation on the bacterium fitness will be assessed, including growh temperature, growth rates, competing for growth, and drug susceptibility. Our work will identify the molecular mechanism associated with clofazimine resistance of mycobacterium tuberculosis caused by mutation of ndh gene. Our finding will provide useful information for rapid detection of Cfz resistance.

氯法齐明（Cfz）在耐多药结核病短程化疗中发挥重要作用，但结核分枝杆菌（Mtb）对Cfz的耐药机制仍不清楚。前期研究对比分析了16株Cfz耐药株和16株Cfz敏感株的全基因组序列，发现ndh突变可能与Cfz耐药相关，之后的临床菌株验证和ndh G301D定点诱变结果也支持该推论。Cfz可被ndh编码的Ⅱ型 NADH：甲基萘醌氧化还原酶（NDH-2）还原，之后自发氧化过程中产生的活性氧类（ROS）可杀灭Mtb。研究假设为ndh突变导致NDH-2酶活性改变影响Cfz还原，改变ROS产量，从而致Mtb对Cfz耐药。本研究拟通过大样本验证耐药基因的临床相关性、应用定点诱变和回复突变试验研究ndh突变对Cfz耐药的影响、通过测定突变NDH-2酶活性变化确定ndh突变对ROS产量的影响、评估ndh突变对Mtb适应度代价的影响，从而阐明ndh突变致Mtb对Cfz耐药的分子机制，便于及时诊断Cfz耐药。

氯法齐明（Cfz）在耐多药结核病短程化疗中发挥重要作用，但结核分枝杆菌（Mtb）对Cfz的耐药机制仍不清楚。本研究测定了391株耐多药结核分枝杆菌（MDR-TB）对莫西沙星、贝达喹啉、利奈唑胺、Cfz、环丝氨酸、德拉马尼和吡嗪酰胺的药物敏感性。德拉马尼、利奈唑胺、Cfz和贝达喹啉的耐药率最低，分别为3.3%(13/391)、3.8%(15/391)、6.6%(26/391)和7.2%(28/391)。莫西沙星(12.0%,47/391)和环丝氨酸(13.6%,53/391)对MDR-TB在体外也具有较好的抗菌效果。然而，吡嗪酰胺(38.4%,150/391)显示出较高的耐药率。贝达喹啉、利奈唑胺、Cfz、环丝氨酸和德拉马尼在单纯MDR、Pre-XDR和XDR中的耐药率比较相似，都在非常低的水平。然后对357株MDR-TB和67株敏感菌株进行了全基因组测序。共筛选到42个耐药相关的SNPs和44个耐药相关基因，其中31个耐药相关基因为本次研究首次发现，为耐药结核的诊治提供了新靶标。另外还绘制了357株MDR-TB的进化树，357株MDR-TB中293株（82.1%）为L2系，3株（0.8%）为L3系，61株（17.1%）为L4系。贝叶斯skyline分析结果表明我国MDR-TB种群大小的改变与我国经济社会环境和国家结核防控体系建设紧密相关。该结果全面解析了我国MDR-TB的流行病学/耐药特征，提供了新的潜在的耐药结核病诊治靶点，并揭示了影响我国MDR-TB种群变化的各种因素，为我国耐药结核的精准诊治提供了坚实的数据及理论基础，并对全球耐药结核的防控提供了有益的借鉴及参考。本研究还确定了26株Cfz耐药菌株的耐药相关基因突变，结果显示在结核分枝杆菌中Rv0678突变与Cfz低水平耐药相关，在鸟胞内分枝杆菌复合群中Rv0678同源基因及mmpT5突变与Cfz耐药相关。有利于Cfz耐药分子诊断技术研发。.研究成果共发表SCI论文1篇，中文核心期刊论文3篇，参编论著5部，申请发明专利4项。.项目实施期间指导了2名硕士、2名博士研究生毕业，3人晋升正高级职称，1人晋升副高级职称，2人晋升中级职称。课题负责人入选北京市优秀人才培养青年拔尖个人项目和通州区“两高”人才工程“领军人才”。