DosR乙酰化修饰调控结核菌潜伏感染的机制研究

Mycobacterium tuberculosis (MTB) can induce the latent infection and tolerate the drug effect via adaption to the hypoxic environment in granuloma and transition to dormancy. The transcriptional regulator DosR plays a key role in the response to hypoxia and latent infection of MTB by initiating the transcription and expression of hypoxia genes downstream, but the regulation mechanism is still not very clear. Based on the quantitative acetylation proteomic profiling of MTB cultured under aeration & hypoxia, we found that K182 in DosR could be acetylated, and the acetylation level of DosR was significantly downregulated under hypoxia. From the function detection results of gene knockout and complement strains of MTB, the transcriptions of hypoxia related genes were upregulated and the hypoxia response was promoted for the DosR deacetylation mutant; furthermore, the intracellular survival and the acute virulence to mice of MTB were influenced, too, which all suggested that the acetylation of DosR may play a key regulation role in the adaption to the adverse environment of host and induction of latent tuberculosis infection. In this project, based on the human PBMC cell granuloma model and Cornell latent tuberculosis infection model of C3HeB/FeJ mice, the regulatory role and mechanism of DosR acetylation in the latent tuberculosis infection will be analyzed, which will deepen the comprehension to the pathogenesis mechanism of MTB latent infection and further provide new target and theoretical basis for developing new drugs to contradict the latent infection of MTB.

结核菌(MTB)通过适应肉芽肿厌氧环境并进入休眠状态使其能够潜伏感染和耐受药物作用。DosR通过启动下游基因的转录表达从而在MTB厌氧应答及潜伏感染中发挥关键作用，但其调控机制尚不完全清楚。我们前期通过MTB有氧/厌氧定量乙酰化组学发现DosR的K182位可发生乙酰化修饰，并且厌氧导致DosR乙酰化水平降低；MTB基因敲除及回转菌株功能检测发现，DosR去乙酰化突变可上调厌氧基因的转录，促进MTB的厌氧应答，进而影响其胞内存活和小鼠急性感染致病力。这些前期结果提示DosR的乙酰化修饰可能在MTB适应宿主不利环境并引起潜伏感染中发挥重要调控作用。本项目进一步利用PBMC细胞肉芽肿模型和C3HeB/FeJ小鼠Cornell结核病潜伏感染模型，探索分析DosR乙酰化修饰调控MTB潜伏感染的功能及机制。研究结果将加深对结核菌潜伏感染发病机制的了解，并为开发抗MTB潜伏感染药物提供新的靶点和依据。

由结核分枝杆菌(Mycobacterium tuberculosis, Mtb)感染引起的结核病仍然是严重的全球公共卫生问题。Mtb通过适应肉芽肿厌氧环境进入休眠状态使其能够潜伏感染，伺机增殖促进肉芽肿恶化，其关键调控分子及机制尚不清楚。前期通过Mtb有氧/厌氧定量乙酰化组学发现Mtb重要的休眠潜伏调控分子DosR的K182位可发生乙酰化修饰，并且厌氧导致DosR乙酰化水平降低。本项目通过制备DosR及其点突变重组蛋白，发现K182位点发生模拟去乙酰化突变后，DNA 结合活性明显增强，从而显著上调下游厌氧基因的转录，促进Mtb的厌氧应答，有利于其胞内存活和小鼠感染致病力；进一步成功建立PBMC细胞肉芽肿模型，Mtb感染C3HeB/FeJ小鼠肉芽肿模型，海分枝杆菌感染斑马鱼肉芽肿模型以及DosR乙酰化修饰的乙酰转移酶Rv0998、去乙酰化酶Rv1151c基因敲除菌，明确Rv0998促进DosR的K182乙酰化修饰，导致其无法与下游靶基因的转录启动子区域结合，抑制下游基因转录，进而降低Mtb的体外生长，胞内存活以及结核性肉芽肿病变，而Rv1151c则发挥相反作用。同时，为了分析Mtb响应厌氧调控宿主免疫应答的关键分泌蛋白，本项目进一步开展了Mtb有氧厌氧培养上清的定量蛋白组学分析，发现乙酰转移酶FadA是一种厌氧诱导的Mtb关键分泌蛋白，作为一种乙酰辅酶A乙酰转移酶，通过催化两个乙酰辅酶A分子合成乙酰乙酰辅酶A，下调感染宿主巨噬细胞胞浆乙酰辅酶A水平，进一步降低组蛋白H3K9的乙酰化修饰水平，抑制保护性炎症因子Il6的表达，从而促进分枝杆菌体内存活和结核性肉芽肿恶化。补充乙酸可显著增加乙酰辅酶A水平，阻断FadA对Il6表达的抑制作用，降低感染动物的细菌负荷和肉芽肿恶化，发挥治疗作用。研究结果首次证明乙酰转移酶Rv0998去乙酰化酶Rv1151c催化的乙酰化修饰调控DosR的DNA结合活性以及厌氧基因转录，促进Mtb厌氧应答以及宿主肉芽肿病变的机制。首次证明乙酰转移酶FadA，下调依赖于乙酰辅酶A的组蛋白H3K9乙酰化修饰及Il6转录翻译，促进结核性肉芽肿恶化的机制，加深了对结核菌潜伏感染发病及乙酰化修饰调控机制的了解，为认识结核病的发病机制提供了全新的研究思路和重要理论基础，也为通过靶向分枝杆菌和宿主脂肪酸代谢之间的界面，来开发抗Mtb潜伏感染药物提供新的靶点和依据。