Role of FHL-mediated formate metabolism in Mycobacterium tuberculosis persistence

描述(申请人提供)：结核分枝杆菌是一种杆状、抗酸、人类特有的病原体，是呼吸道疾病结核病(TB)的病原体。这种细菌是世界范围内发病率和死亡率的重要来源，目前预计将感染约20亿人。结核分枝杆菌作为病原体的成功与其在易感个体中建立潜伏感染并在稍后免疫抑制期间重新激活的能力密切相关。对结核分枝杆菌在宿主内长期、持续感染后的建立、维持和重新激活具有重要作用的决定因素知之甚少。据认为，潜伏期始于结核分枝杆菌包裹在肉芽肿性病变内，以及对这种环境中存在的抑制有氧呼吸和促进过渡的特定信号的识别。 结核分枝杆菌进入一种改变的非复制持久性(NRP)的生理状态。最近发现，结核分枝杆菌在体外暴露于促进NRP的条件(低氧和一氧化氮)后，一个被预测为编码包含甲酸氢解酶(FHL)的决定因素的基因(Rv0081-Rv0088)上调。此外，该基因还被发现由两个反应调节因子(DosR/DevR和MprA)直接调节，这两个反应调节因子在体外和体内都有助于结核分枝杆菌的持久性。在微需氧/厌氧生长期间，FHL通过在没有外部电子受体的情况下介导甲酸盐氧化为CO2和H2，在肠道细菌内的能量代谢中发挥重要作用。这项建议试图通过研究在生理相关条件下结核分枝杆菌新陈代谢的各个方面来填补我们目前知识上的空白。我们假设，结核分枝杆菌合成了一种功能性的FHL酶复合体，这是能量代谢和NRP期间的生存所必需的。为了解决这一假设，提出了两个具体目标。首先，将使用分光光度和基于气相色谱的方法来检测结核分枝杆菌FHL将甲酸盐氧化为CO2和H2的能力。其次，将检查构成预测的FHL的决定因素，以确定它们是否相互作用形成FHL，并在NRP期间是结核分枝杆菌生存所必需的。总的来说，这些研究有望为结核分枝杆菌在与NRP和潜伏期相关的条件下的生理学提供新的见解。甲酸盐代谢基础的描述还可以确定新的酶，这些酶可以作为结核分枝杆菌在NRP期间的治疗干预的靶点，而此时细菌对现有的抗生素是顽固的。 公共卫生相关性：本提案中描述的实验将调查在非复制持久性期间FHL中介甲酸盐代谢在结核分枝杆菌生理学中的重要性。对构成这种酶复合体的决定因素的检查有望通过加深我们对与持续感染相关的条件下的结核分枝杆菌代谢的了解，填补我们对结核分枝杆菌生理学知识的关键空白。这项建议的完成可能导致鉴定新的酶，这些酶可以在结核分枝杆菌对目前可用的抗生素不敏感的潜伏期内作为治疗干预的靶点。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is a rod-shaped, acid-fast, human-specific pathogen and is the causative agent of the respiratory disease tuberculosis (TB). The bacterium is a significant source of morbidity and mortality worldwide, and is currently predicted to infect approximately 2 billion individuals. The success of M. tuberculosis as a pathogen is closely linked with its ability to establish latent infections in susceptible individuas and reactivate at later timed during periods of immunosuppression. Determinants important for the establishment, maintenance, and reactivation of M. tuberculosis from long-term, persistent infection within the host are poorly understood. It is thought that latency initiates following encasement of M. tuberculosis within granulomatous lesions, and the recognition of specific signals present within this environment that inhibit aerobic respiration and promote the transition of M. tuberculosis into an altered physiological state of nonreplicating persistence (NRP). Recently, a locus (Rv0081-Rv0088) predicted to encode determinants comprising a formate hydrogenlyase (FHL) were found to be upregulated following exposure of M. tuberculosis to conditions (hypoxia and nitric oxide) promoting NRP in vitro. In addition, this locus was found to be directly regulated by two response regulators (DosR/DevR and MprA) known to contribute to persistence by M. tuberculosis in vitro and in vivo. FHL plays an important role in energy metabolism within enteric bacteria during periods of microaerophilic/anaerobic growth by mediating the oxidation of formate to CO2 and H2 in the absence of an external electron acceptor. This proposal seeks to fill a current gap in our knowledge by investigating aspects of M. tuberculosis metabolism under physiologically relevant conditions. We hypothesize that M. tuberculosis synthesizes a functional FHL enzyme complex that is required for energy metabolism and survival during periods of NRP. To address this hypothesis, two specific aims have been proposed. First, the ability of M. tuberculosis FHL to mediate the oxidation of formate to CO2 and H2 will be examined using spectrophotometric- and gas chromatographic-based approaches. Second, determinants comprising the predicted FHL will be examined to determine if they interact to form FHL and are required for survival of M. tuberculosis during NRP. Collectively, these studies are expected to provide novel insights into M. tuberculosis physiology under conditions associated with NRP and latency. Delineation of the basis for formate metabolism may also identify new enzymes that can be targeted for therapeutic intervention of M. tuberculosis during periods of NRP when the bacterium is otherwise recalcitrant to currently available antibiotics. PUBLIC HEALTH RELEVANCE: Experiments described in this proposal will investigate the importance of FHL-mediate formate metabolism in the physiology of Mycobacterium tuberculosis during non-replicating persistence. Examination of determinants comprising this enzyme complex is expected to fill a critical gap in our knowledge of M. tuberculosis physiology by furthering our understanding of M. tuberculosis metabolism under conditions associated with persistent infection. Completion of this proposal may lead to identification of new enzymes that can be targeted for therapeutic intervention of M. tuberculosis during latency when the bacterium is otherwise recalcitrant to currently available antibiotics.