结核分枝杆菌优势亚型菌株三酰甘油酯过量累积的调控机制及其生理功能研究

Mycobacterium tuberculosis (Mtb) has genetically diverged into multiple lineages and sublineages during the microevolution and co-expansion with human populations. Tuberculosis burden in China was largely due to a recently evolved Mtb sublineage that is named predominant sublineage showing hyper-pathogenicity. However, the molecular mechanisms of its hyper-pathogenicity are still elusive. We previously discovered that triacylglycerol(TAG) was excessively accumulated in predominant sublineage strains and found tgs2 (encodes TAG synthase) was significantly upregulated. TAG functions as the major carbon/energy source during Mtb latent stage and is essential for the regrowth and exit from the hypoxic non-replicating state. Excessive accumulation of TAG might alter Mtb pathogenicity through impacting multiple biological processes. Thus, we hypothesize that upregulation of tgs2 overproduced TAG and contributes to the pathogenic evolution. In this project, we plan to validate the correlation between tgs2 upregulation and TAG accumulation, and study the regulatory mechanisms for the tgs2 upregulation; we also aim to investigate the changes in biological functions including growth rate, cell wall lipid synthesis and the capability of drug tolerance causing by excessive accumulation of TAG; we shall further verify the pathogenic phenotype evolution through cell and mice infection models. The expected findings of this research would elucidate or partially explain the molecular mechanisms for the hyper-pathogenicity of dominant sublineage and provide potential targets for new anti-tuberculosis drugs development.

结核分枝杆菌（简称结核菌）在进化过程中分化形成多个亚型。“优势亚型”在我国流行比例最高且具有高致病性特征，但其高致病机制尚不清楚。我们前期工作发现，优势亚型菌株的三酰甘油酯(TAG) 过量累积，且TAG合酶基因tgs2的表达上调。TAG是结核菌从“潜伏状态”发展成“活动结核”过程的重要碳源与能量供给，并且TAG过量累积可能从多方面影响其致病性。因此，我们提出“优势亚型菌株tgs2表达上调引起TAG过量累积，通过影响多种生理功能从而增强其致病性”的科学假设。本项目将首先验证tgs2表达上调对三酰甘油酯累积的影响，并从转录因子突变的角度探索tgs2表达上调的调控机制；通过细菌增殖、细胞壁毒力相关脂质组成、抗生素耐受能力等方面研究TAG过量累积引起的生理功能改变，并在细胞、小鼠感染模型验证其导致的致病性变化。预期成果有望解释或部分解释优势亚型的高致病机制，并为抗结核新药研发提供可能靶点。

结核分枝杆菌(简称结核菌)起源于非洲，随着人类迁徙扩散到世界各地。结核菌在与人类共进化过程中分化形成多个亚型，不同亚型在地域分布以及传播能力上存在差异。“优势亚型”(又名现代型北京菌株)是在我国本土分化形成且流行比例最高的亚型，流行病学研究和动物感染实验刻画了其高致病性特征，但其高致病机制尚不清楚。结核菌的细胞壁脂质在与宿主相互作用中扮演重要角色，本项目旨在刻画优势亚型菌株的脂质组成特征及其分子机理。本研究通过对结核菌临床菌株全基因组测序，挑选了优势亚型菌株(5株)与非优势亚型菌株(3株)进行脂质组学分析，发现优势亚型的三酰甘油酯(TAG)累积增加2.61倍(P<0.0001)；通过扩大临床菌株样本量，我们进一步挑选7株优势亚型菌株和5株非优势亚型菌株，验证了优势亚型菌株TAG累积增加这一表型特征。TAG是结核菌从“潜伏状态” 发展成“活动结核”过程的重要碳源与能量供给，并且TAG过量累积可能从多方面影响其致病性。我们通过转录组分析发现，优势亚型菌株的tgs2基因表达上调近3倍，该基因功能预测为TAG合成酶。为了验证tgs2的表达上调对TAG累积的影响，我们在模式菌海分枝杆菌中通过基因插入突变、回补以及过表达等实验确认Tgs2的TAG合成酶功能，验证了tgs2的表达上调可以导致海分枝杆菌TAG累积增加。我们进一步通过药物敏感实验研究TAG累积增加或者减少对抗结核一线药物的敏感性改变，但结果显示TAG的累积增加/减少均未改变海分枝杆菌对利福平/异烟肼的药物敏感性。本研究从结核菌脂质组成的角度刻画了优势亚型菌株与非优势亚型菌株的差异特征，发现优势亚型菌株的TAG的累积增加，并且这一表型改变可以通过tgs2基因表达上调得到解释。TAG的累积增加可作为一个潜在的关键分子表型，帮助解释优势亚型菌株的高致病性特征，并为后续研究阐明优势亚型菌株高致病性的分子机制奠定基础。