结核分枝杆菌RbpA转录调控及在利福平耐药中的功能研究

Mycobacterium tuberculosis is one of the most successful human pathogens able to persist in drug-resistant state in the host for years. As reported by the World Health Organization that M. tuberculosis infects almost one third of the world population and continue to kill about 1.5 million people each year. Multiplication of the multi-drug resistant forms of the tuberculosis during the last decades raises a serious threat for public health worldwide. Thus, development of new anti-tuberculosis therapeutic strategies is essential task and necessitates the mechanistic studies of the resistance mechanisms and the new drug targets. Rifampicin, the antibiotic targeting bacterial RNA polymerase (RNAP), still remains the first-line drug used to cure tuberculosis infection. However, bacteria have developed multiple mechanisms to escape from the antibiotic treatment. Most of these mechanisms, such like mutations in the drug target, activation of the stress-response and switch to the persistent state are linked to the regulation of gene transcription. RbpA is a 14 kDa RNAP-binding protein that was reported to be involved in rifampicin tolerance, because overexpression of RbpA in Mycobacterium was shown to decrease its sensitivity to rifampicin, and expression of the rbpA gene was reported to be induced during the rifampicin treatment. Regarding these proofs, mechanism(s) about the role(s) of RbpA in rifampicin tolerance is not clear. Transcription of rbpA in presence of rifampicin is very important for its role in rifampicin tolerance, but its transcription regulation has never been studied yet. In this project, we suppose to identify the regulators that involved in rbpA regulation in M. tuberculosis and investigate the mechanism(s) for the rifampicin-induced rbpA transcription. Effects of RbpA to M. tuberculosis RNAP activity will also be studied to show the details for the role(s) of RbpA in RNAP activity regulation and rifampicn tolerance. The final goal of this project is to show a new mechanism for rifampicin tolerance in aspect of gene transcription regulation. RbpA is essential for M. tuberculosis and specific to Actinomycetes. For these, RbpA is a potential target for new anti-tuberculosis drug development. Our project will greatly promote this process.

结核分枝杆菌是引起结核病的重要病原细菌，随着现有抗结核药物的长期使用，耐药菌株的出现给结核的防治提出了新的挑战，耐药机理的研究一直是研究者热点关注的问题。利福平是重要的一线抗结核药物，其作用靶标为RNA聚合酶(RNAP)，利福平耐药的主要原因是RNAP的突变及活性调控。RbpA是一种新发现的RNAP活性调控蛋白，其表达受利福平诱导，且该蛋白的过表达能增强细菌对利福平的耐药性，但有关RbpA表达调控及在利福平耐药中的作用机理还未有清楚阐释。本项目将系统鉴定结核分枝杆菌RbpA的转录调控蛋白，探讨其调控细节，揭示RbpA受利福平诱导表达过程，并分析RbpA对RNAP活性调控的作用机制，最终从基因表达与RNAP调控的角度阐释结核分枝杆菌利福平耐药新机理。由于结核分枝杆菌RbpA是必须蛋白，且仅存在于放线菌，具备药物作用靶点的基本特征，本项目的开展将为RbpA作为新的抗结核药物靶点奠定基础。

RbpA 是一种新发现的RNAP 活性调控蛋白，其表达受利福平诱导，但有关其表达调控及在压力耐受过程中的作用机理还不清楚。本项目对结核分枝杆菌rbpA基因的转录调控过程及转录调控蛋白进行了研究，取得了以下结果：. 1. 首次对rbpA基因在利福平等压力条件下的转录调控进行系统分析，鉴定出两种压力耐受相关的sigma因子SigE和SigB参与rbpA基因的转录起始过程；. 2. 对结核分枝杆菌RbpA在利福平等压力激活表达后发挥怎样的功能进行了探讨，发现rbpA基因表达水平下调时分枝杆菌对利福平等压力条件的敏感性增强，说明RbpA参与了分枝杆菌压力耐受过程。通过体外转录实验，我们排除RbpA直接与利福平竞争性结合RNA聚合酶来影响利福平耐受的假说，证明RbpA是通过调控基因表达来间接影响利福平等压力的耐受过程。进一步的调控靶标研究发现RbpA对furA-katG操纵子的调控在其帮助应对压力过程中发挥重要作用，首次阐释了RbpA蛋白在分枝杆菌压力耐受过程中的具体功能；. 3. 证明转录调控蛋白PhoP能抑制rbpA的转录，并找到了其在rbpA启动子上的作用区域，发现PhoP识别的DNA序列与经典的结合序列不同，说明其以一种新的机制来调控rbpA的转录。. 本项目首次阐释了RbpA在分枝杆菌应对利福平等压力条件下的转录及调控机制，首次对该蛋白在压力应对过程中的功能进行了体内水平的探讨。为下一步深入探讨其调控机制等研究奠定了重要基础。