DevR-DevS双组份系统在MDR-TB发生中的作用和分子机制

Muti-drug resistant Mycobacterium Tuberculosis (MDR-TB) is generated from accumulation of gene mutation under dynamic concentrate chemical pressure. The dormant state can protect the bacillus from being killed, but its role and the molecular mechanism in the development of MDR are not clear. It was noted that two component system (TCS) DevR-DevS controls the dormant process. And our studies showed a high expression of DevR in MDR-TB strains. Furthermore, we found that anti-tuberculosis drugs could induce the expression of DevR. So we hypothesize that the conversion cycle of dormant and resuscitation regulated by DevR might be the key model to generate MDR-TB. In this study, a series of DevR mutants with different expression level of DevR will be firstly constructed. Then the activity of anti-tuberculosis drugs like isoniazid and rifampicin to trigger DevS will be identified. Then the role of DevR in development of latent and dormant under drugs condition will be investigated. And the gene mutation rate will be confirmed by inducing the cycles of latent, dormant and resuscitation conversion in vitro and in vivo. It would demonstrate not only the role of DevR in monitoring the development of MDR-TB, but also the value of inhibiting DevR in decrease the happening of MDR-TB. That would be much helpful in making new strategy to defend MDR-TB.

耐多药结核分枝杆菌（MDR-TB）是结核菌(MTB)在长期动态浓度药物环境压力下耐药相关基因突变的积累的结果。休眠是MTB耐受环境压力的重要机制之一，但其在MDR发生中的作用及其分子机制尚不明确。鉴于DevR-DevS双组份系统在调控MTB休眠中的作用，和我们前期试验证明DevR在MDR-TB中的高表达，以及抗结核药物可以诱导DevR表达增强的结果，我们提出DevR-DevS调控的休眠与复苏过程再循环的MDR发生模式。本研究将以构建DevR表达水平不同的MTB突变株为基础，深入解析异烟肼、利福平对DevR-DevS表达和活化的调节作用；DevR介导的MTB潜伏、休眠状态下的基因突变机制；MTB在体内外潜伏、休眠、复苏及该过程的再循环对耐药基因突变率的影响；阐明检测DevR表达水平对MTB耐药状态监测的意义，和抑制DevR表达以减少MDR发生的作用，为MDR-TB的防治提供新的策略。

耐多药结核分枝杆菌（MDR-TB）是结核菌在长期动态药物作用下耐药相关基因突变的积累的结果。休眠是结核菌抵抗环境压力的重要机制之一，是结核菌与动态药物相互作用中的抗衡因素，但其分子机制，以及其与耐多药发生的相互关系尚不明确。鉴于DevR-DevS双组份系统在调控结核菌休眠中的作用，和我们前期试验证明DevR在MDR-TB中的高表达，以及抗结核药物可以诱导DevR表达增强的发现，我们假想认为，DevR调控的结核菌休眠于复苏的循环可能是产生MDR的重要模式。本研究建立了DevR敲除菌株，和休眠模型，进而完成了DevR/S与结核分枝杆菌耐药的关系鉴定，发现了DevR、HspX和HBHA随其休眠而高表达的情况；利用DevR敲除菌株和质谱分析，明确了DevR/S调控结核分枝杆菌耐多药基因的关键分子节点，即HBHA；采用蛋白质谱、定量PCR等方法分析了DevR/S及其下游调控分子HBHA在临床分离菌株中的存在和表达情况，证实了其在MDR-TB中的高表达；HBHA敲除H37Rv菌株已经建立成功，初步明确了其在结核耐药中的作用，阐明了DevR/S-HBHA信号通路抑制肺泡上皮细胞核巨噬细胞自噬、促进细胞凋亡的作用。该研究明确了DevR/S与结核分枝杆菌耐多药的相关性；初步揭示了DevR/S-HBHA引起结核分枝杆菌耐多药的分子机制，阐明了DevR/S-HBHA对宿主细胞抗感染作用的影响。为开发以HBHA为靶点的抗耐药研究和鉴别诊断活动/潜伏感染提供了新的理论依据。