结核分枝杆菌基因组甲基化修饰与其耐药和致病机制研究

Mycobacterium tuberculosis(MTB) is one of serious infectious diseases worldwide. The emerging of multidrug resistance (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (TB) make its prevention and treatment tremendous difficulties. Present study showed that point mutation of the drug target genes responsible for most drug-resistant phenotype in MTB. But some drug-resistant phenotype can not be reasonably explained. Meanwhile pathogenesis of TB is not completely clear. Therefore, to reveal the pathogenesis of drug-resistant Mycobacterium tuberculosis is particularly urgent. Over the past two years, the third generation sequencing technology proveded its validity in the detection of methylation in genomic level in bacteria. And the epigenetic modification is potentially associated with drug resistance and virulence of bacteria. In this study, we obtained methylation information of the clinical multidrug-resistant and multi- sensitive Mycobacterium tuberculosis using the third generation genome sequencing technology, combined with bioinformatics analysis and functional verification of the methyltransferase, we are trying to reveal the association between the phenotype of multi- drug-resistant TB and its pathogenic mechanism. The results of this study will provide a potential drug target sites for MDR and XDR TB treatment.

结核分枝杆菌（TB）是肺结核的病原菌，其多耐药（MDR）和广泛耐药（XDR）菌株的出现给结核病的防治带来极大困难。TB的耐药性与致病性是TB研究中的难题，虽然多数情况下是由基因突变导致，但也有许多未知因素参与，只是目前还无法确认。对其他类病原菌的研究表明，细菌基因组表观修饰不但与细菌的致病性相关，也与细菌的耐药性相关。基于此，我们推测TB遗传表观修饰与致病性和耐药性有关联，因此拟以临床多耐药和毒力不同的TB菌株为研究对象，利用三代单分子DNA测序方法在基因组水平分析其甲基化修饰信息，并通过基因敲除和转录组分析等方法进行功能验证，明确甲基化修饰与TB多耐药表型和致病性的关联性。课题的前期实验在临床株中发现了两个全新的甲基转移酶，初步结果表明其可能参与了耐药和毒力相关基因的表达调控。课题研究将为TB多耐药表型的出现及其致病机理提供理论依据，为多耐药TB的治疗提供潜在的药物作用靶位点。

结核分枝杆菌（Mycobacterium tuberculosis）是结核病(Tuberculosis, TB)的病原菌，其多耐药（MDR）和广泛耐药（XDR）菌株的出现给结核病的防治带来极大困难。M. tuberculosis耐药性与致病性的具体机制是TB 研究的难题。. 我们对8株耐药水平不同的M. tuberculosis 进行了第三代单分子实时全基因组测序，并结合NCBI获得的136个完整基因组序列，完成了遗传学和生物信息学分析，鉴定得到一个广泛存在于结核分枝杆菌北京株的甲基化酶-HsdM。HsdM是结核分枝杆菌北京谱系中的优势甲基化酶。与亲本株相比，hsdM基因敲除株11826ΔhsdM中编码INH活化酶的katG基因的mRNA表达水平显著提高，与此一致，药敏实验结果也显示敲除株11826ΔhsdM对INH显著耐受；而11826ΔhsdM菌株中编码致病相关的蛋白基因mRNA表达水平显著下调，小鼠实验也显示，感染M. tuberculosis一个月后，小鼠肺部、脾脏中的11826ΔhsdM细菌负载量均比亲本株11826显著降低，说明HsdM的甲基化修饰显著影响了M. tuberculosis的毒力；hsdM基因过表达的耻垢分枝杆菌（Mycobacterium smegmatis）比对照菌株突变率高出约10倍，表明HsdM的甲基化修饰明显增加了菌株的突变率，从而解释了为什么结核分枝杆菌北京株的突变率高于H37Rv等L4株系菌株4倍；. 综上所述，我们的研究结果明确了HsdM甲基化修饰与M. tuberculosis多耐药表型和致病性的关联性，为M. tuberculosis多耐药表型的出现及其致病机理提供了理论依据，为多耐药TB 的治疗提供了潜在的新药作用靶点。