两株超级广泛耐药结核分枝杆菌全基因组比较分析

Comparative genome analysis of two super-extensive drug-resistant isolates of Mycobacterium tuberculosis

摘要:

【目的】结合现有数据，通过对两株临床超级广泛耐药的结核分枝杆菌全基因组的测序和分析，发现其型别相关的突变位点，解释发生广泛耐药的基因组突变机制。【方法】利用Solexa第二代测序技术对两株广泛耐药结核分枝杆菌(FJ05194和GuangZ0019)进行全基因组测序分析。以H37Rv为参考序列得到两株广泛耐药菌株的单核苷酸多态性(SNPs)，构建系统发育树鉴定菌株型别，判断突变位点中型别相关和非型别相关的SNPs。定位SNPs所在的基因组区域，对型别相关的突变基因进行KEGG通路的富集分析，对非型别相关的突变基因和间隔区判断是否与耐药相关。【结果】两株广泛耐药菌株分别属于Lineage2和Lineage4型别，两菌株在碱基替换方面存在差异性，Lineage2型别相关的基因功能富集于ABC转运蛋白和核苷酸切除修复的通路。耐药方面，发现了已知的耐药相关基因的突变(rpoB、katG、rpsl、gyrA、gyrB、embB和ethA等)，但卷曲霉素和卡那霉素相关的rrs、tlyA和eis启动子区域未发生突变，不足以解释其耐药性的产生。与最新报道的候选耐药基因比较，发现了卷曲霉素和卡那霉素相关的突变(Rv1393c、Rv0265c和narX等)和外排泵相关的pstB、Rv2333c和Rv2687c突变。【结论】结核分枝杆菌Lineage2型别相关的SNPs中含有影响结核分枝杆菌突变率和耐药性的突变。对于两株超级广泛耐药的结核菌，已知的激活药物或药靶相关的单耐药基因突变集合不能完全解释其广泛耐药性，还涉及新候选结核耐药基因、外排泵和补偿等其他潜在机制的相关基因突变。

Abstract:

[Objective] Based on the genome sequences of two super extensive drug resistant isolates of Mycobacterium tuberculosis, we attempted to identify the lineage-specific SNPs and found the drug resistant-associated mutations in them. [Methods] By using the next-generation sequencing technology on two super-extensive drug resistant isolates of mycobacterium tuberculosis (FJ05194 and GuangZ0019), we compared them with the reference sequence to identify the single nucleotide polymorphisms (SNP) and divided the SNPs into lineage-specific and isolate-specific SNPs. Check the coordinate of SNPs on the genome, then we did the enrichment analysis of the lineage-specific SNPs, and were able to found out the drug resistant mutations from the isolate-specific SNPs by overlapping with the candidate drug resistance associated regions. [Results] The Lineage2-specific SNPs was significantly enriched on ABC transpoters and nucleotide excision repair KEGG pathways, and the extensive drug resistance were mainly attributed to the mutation of the well-known genes (rpoB, katG, rpsl, gyrA, gyrB, embB, ethA and so on), but the capreomycin and kanamycin resistance was probably due to some candidate resistant-associated genes (Rv1393c, Rv0265c, narX) and the efflux pump genes (pstB, Rv2333c and Rv2687c). [Conclusion] The Lineage2-specific-SNPs possible result in the high mutation rate and drug resistance of the Lineage2. And the mechanism of super extensive drug resistance is more complex, it not only involve with the drug target and drug activation genes, but also some unclear candidate genes such as compensatory genes and efflux pump genes.