结核分枝杆菌重要毒力因子蛋白酪氨酸磷酸酶PtpA操纵子转录调控机制研究

The ptkA-ptpA-rv2235 operon, which encode a protein tyrosine phosphatase A (PtpA), in Mycobacterium tuberculosis(Mtb) plays critical role during its infection.Mtb secrete PtpA into human macrophage to inhibit the innate immunity, apoptosis，phagosome-lysosome fusion and modulate macrophage bioenergetics state by interfering multiple pathways. Till now, most of the researches about PtpA are mainly focus on its host targets identification and regulation, post-translational modification and inhibitor screening. However, little is known about the regulation of ptpA gene (encoding PtpA) transcription. Considering the importance of transcriptional regulation of ptpA gene in Mtb and our previous work that ptka-ptpa-rv2235 form an operon in which all tree genes were up-expressed after macrophage infection, we plan to carry out the following researches to decipher its transcriptional regulatory pathway: 1) Identify the sigma factor that responsible for initiating ptpA gene transcription by using in vitro transcription assay and locate the binding sites; 2) The upstream DNA region of the promoter will be used as bait to fishing the transcriptional regulator from Mtb lysate by DNA affinity chromatography and the mechanism of regulation will also be investigated; 3) A mycobacterium protein two hybrid system will be used to screen against a Mtb whole genome expression library to identifying the protein that command the sigma factor and transcriptional regulator. Base on these, the transcriptional regulatory pathway of ptpA gene will be delineated, and the critical gene in this pathway will be deleted in Mtb to confirm their regulation both in culture medium and infected macrophage. These will help us to better understanding of pathogenic mechanism mediated by PtpA. More importantly, this makes it possible to block PtpA function by targeting its transcriptional regulatory pathway instead of targeting the protein itself, which faces a strait due to the high identity with human protein tyrosine phosphatase. It will also provide reference for the transcriptional regulation mechanism of this operon in other bacteria.

操纵子ptkA-ptpA-rv2235在结核菌感染中发挥重要作用，特别是分泌型蛋白酪氨酸磷酸酶A（PtpA），经多条途径抑制宿主天然免疫、细胞凋亡及吞噬体-溶酶体融合等，逃逸免疫杀伤，还能调控宿主能量代谢。对PtpA的研究主要集中在宿主调控机制、翻译后修饰调控及抑制剂研发等方面，其转录调控至关重要但却缺乏研究。本项目拟研究：1)鉴定识别其启动子的σ因子并揭示识别位点；2)钓取涉及调控的转录调控因子并揭示调控机制；3)筛选和鉴定作用于转录调控因子和σ因子的上游调控蛋白并阐明调控机制；4)在结核菌中敲除或RNAi干扰关键性上游调控蛋白编码基因，在巨噬细胞感染模型中验证其调控作用。揭示结核菌感染宿主后从信号感应到介导该操纵子表达调控的分子机制，为绕开直接靶向PtpA药物筛选面临的各种困难，从转录调控通路干扰其PtpA的功能奠定基础，也为揭示其它细菌中该操纵子的转录调控提供参考。

结核分枝杆菌中，蛋白酪氨酸激酶PtkA及蛋白酪氨酸磷酸酶PtpA发挥了重要的生理功能，但其转录调控机制尚不清楚。本项目在执行期间，首先验证了结核菌ptkA-ptpA-rv2235组成同一操纵子。在此的基础上，通过克隆、表达和纯化结核菌的11个σ因子，以ptkA-ptpA-rv2235操纵子启动子片段为模板，结合EMSA验证及体外转录实验，发现负责该操纵子转录的σ因子。另一方面，结合生物信息学及DNA亲和层析实验，初步发现了转录调控因子Rv1353c和IdeR，类核相关蛋白EspR和mIHF涉及该操纵子的转录调控。基于此，初步提出ptkA-ptpA-rv2235操纵子转录调控模型：该操纵子转录起始位点位于ptkA基因起始密码子上游约40bp左右。通常情况下，由主要功能σ因子SigA或主要功能样σ因子SigB介导该操纵子进行组成性表达，而在面对氧化应激的条件下，胞外功能σ因子SigJ介导和增强其转录。此外，该操纵子在宏观上可能受到类核相关蛋白mIHF和EspR的全局性调控；同时，可能在环境Fe2+充足的情况下，转录调控因子IdeR结合Fe2+被激活，抑制该操纵子的转录，而当Fe2+不足时，该抑制作用被解除，转录被激活或增强。为后期进一步深入揭示及精确信号转导及调控机制鉴定了基础。项目申请书中研究目标基本完成，成果指标按量完成，相关研究成果也在持续整理完善中。