结核分枝杆菌广谱胁迫蛋白Rv1996介导的异烟肼耐药机制研究

Tuberculosis (TB), caused by the pathogen Mycobacterium tuberculosis, is one of the most serious infectious diseases in the world and caused millions of people deaths per year. M. tuberculosis, as an intracellular pathogen, has evolved special mechanisms of latency in host cells protecting itself from clearance. The latent M. tuberculosis is a potential source of development of active TB patients. But the related mechanisms such as the bacilli how to switch to latent status and how to resuscitate have not been fully explored. Moreover, the prevalence drug-resistant M. tuberculosis worsens the situation. Fast, accuracy of clinical diagnosis of the physiological status in patients and the antibiotic resistance of M. tuberculosis is urgently required for selection of therapy regiment, but the development of it is arrested due to being lack of scientific basis. Isoniazid (INH) is an important first-line drug for the treatment of active and latent TB. Recently studies showed that INH resistance is growth phase-dependent. But phenotypic resistance to INH is poorly understood. Our previous study showed that overexpresses the latent protein universal stress protein (USP) Rv1996 increased susceptibility to INH compared with the wild-type strain. Following our previous studies, we are going to 1) define how Rv1996 regulate KatG by using genetics and biochemical approaches; 2) explore the pathway of Rv1996 mediated INH resistance through KatG. Our studies will give an insight in the development of new diagnosis methods and providing the potential drug selection targets.

结核病是由结核分枝杆菌引起的、导致人类死亡最多的传染病之一。结核菌重要特征是感染人体可以潜伏存在，细菌处于不分裂的状态，在此状态下为生理性耐药，临床表现对抗生素不敏感。结核菌的潜伏感染和耐药是结核防治的难题，对结核菌耐药机制和潜伏感染深入研究为获得药物筛选的靶标及筛选模型的建立提供科学理论依据。异烟肼是治疗活动性结核和干扰潜伏感染的重要药物，我们前期研究显示潜伏相关蛋白结核广谱胁迫蛋白Rv1996与异烟肼的耐药性相关，我们将以此为基础1）利用遗传学、生物化学等技术阐释Rv1996与KatG的作用方式及对异烟肼抗性的影响；2）通过体内垂钓、定量质谱组学及生化分析，鉴定以Rv1996为核心的调控异烟肼抗性的关键蛋白。我们希望本项目能够揭示以Rv1996为核心的影响KatG及异烟肼抗性的关键蛋白及相关通路，获得结核菌耐异烟肼的新机制，为开发准确的分子诊断技术和研发高效的抗结核药物提供理论基础。

结核病是由病原菌结核分枝杆菌引起的威胁全人类公共卫生的传染性疾病。全世界有1/4的人口感染结核菌，潜伏感染是结核防治的难题。广谱胁迫反应蛋白（Universal Stress Protein, USP）是结核菌进入潜伏状态的重要调控蛋白。我们研究显示USP-Rv1996调控结核菌异烟肼的抗性，利用Rv1996进行体内垂钓和生化验证，在检测的条件下未能检测到Rv1996与KatG的直接相互作用，Rv1996通过调控异烟肼胁迫的氧化还原调控网络中的关键蛋白，间接影响了细菌的耐药性。结核菌中有10个USP，USP蛋白家族存在功能冗余，但是不同USP调控不同的途径。利用低氧模型和细胞杀伤实验，我们发现USP-Rv2624c通过调控细菌中精氨酸代谢通路基因的表达，提高了细菌在低氧胁迫环境如宿主细胞中的存活率，暗示Rv2624c在结核菌建立潜伏感染过程中起着重要的作用。综上，我们发现USP家族的Rv1996、Rv2624c都参与调控细菌氧化还原网络，但是它们由于调控不同的通路产生了不同的效应，Rv1996调节异烟肼耐药性而Rv2624c调节细菌的致病力。我们的工作为精细研究结核菌的氧化还原调控网络提供了研究思路，同时为进一步鉴定分枝杆菌蛋白家族同源蛋白的功能提供了研究策略。