耐药结核分枝杆菌补偿性进化新机制的研究

Drug-resistant TB is the major threat to the TB control worldwide. M. tuberculosis acquires antibiotic resistance through chromosomal mutations, which usually coffer fitness-costs. Drug-resistant strains could recover their fitness through compensatory evolutions. However, due to limitation of technique, the mechanisms of compensatory evolution are poorly understood. By this project, we will take the high-throughput advantage of next-generation sequencing to study the potential compensatory mechanisms of M. tuberculosis at the whole-genome scale. We plan to sequence the whole genomes of serial resistant strains that isolated from the same patient during different stages of treatment. By comparative genomic and other bioinformatics analysis, we are expected to identify new compensatory mutations (CMs). Competitive growth experiment and complementary experiment will be applied to explore the relationship between newly identified CMs and the fitness recovery of resistant strains. The clinical importance of CMs will be evaluated by the frequencies of CMs in clinical resistant isolates. The identification of new compensatory mechanisms is important to prevent the transmission of highly fitted resistant strains. At the same time, the new compensatory mechanisms will be helpful to develop proper antibiotic treatment regimen and evaluate the effectiveness of drug target.

耐药结核是全球结核病防控的难题。结核分枝杆菌对抗生素的耐受性主要由其基因组上耐药相关基因的突变所致。耐药突变通常会导致细菌适应度下降，而耐药菌株可通过补偿性进化使其在体内的适应度得到恢复。由于技术限制及缺乏重视，前期对耐药结核分枝杆菌的补偿性进化机制了解较少。本项目将利用新一代测序技术的高通量优势，从全基因组范围研究耐药结核分枝杆菌的补偿性进化机制。项目拟对从同一病人不同时期分离的耐药菌株进行深度测序，运用比较基因组学及其他生物信息学方法鉴定可能的补偿性突变，并通过竞争性生长实验及互补实验衡量补偿性突变对耐药菌株适应度的影响。最后通过研究补偿性突变在临床耐药菌株中的频率，评估其对临床菌株适应度恢复的重要性。鉴定新的补偿性进化机制对预防高度适应性耐药菌株的传播具有重要意义，也可为制定合理化疗方案及评估药物靶标的有效性提供重要参考。

耐药结核是全球结核病防控的难题。结核分枝杆菌对抗生素的耐药性主要由其基因组上耐药相关基因突变所致。耐药突变通常会导致细菌适应度下降，而耐药菌可通过补偿性进化使其在体内的适应度得到恢复。由于技术限制，前期对耐药结核分枝杆菌的补偿性进化机制了解较少。本项目利用新一代测序技术的高通量优势，以及目前公共数据库中收录的大量基因组信息，从全基因组范围研究利福平和喹诺酮耐受结核分枝杆菌的补偿性进化机制。利福平方面，通过对38对（103株）耐多药结核分枝杆菌进行基因组测序和生物信息学分析，我们鉴定了21个分布于rpoB耐药决定区外的补偿性突变；进一步在临床菌株中分析其流行度发现，该区域突变在全球耐多药菌株中的频率约为20%。喹诺酮方面，通过对2715个全球范围的耐多药菌株基因组进行分析，我们鉴定了37个在gyrA/gyrB上携带多个突变的耐药菌株；通过在耻垢分枝杆菌上构建双突变并进行体外竞争性生长实验发现，gyrA/gyrB双突变之间仅少数存在补偿性效应；药物敏感性实验显示，双突变菌株可耐受高浓度的氧氟沙星和莫西沙星，提示临床结核分枝杆菌gyrA/gyrB双突变很可能是药物选择的结果，而非补偿性进化所致。本研究鉴定的rpoB基因补偿性突变可作为鉴定临床高适应度耐药菌株的重要分子标识；而鉴定gyrA/gyrB双突变菌株对评估临床病例是否对莫西沙星耐受具有重要意义。