The Mycobacterium Tuberculosis Dormancy Program

描述(申请人提供)：世界上三分之一的人口感染结核分枝杆菌(Mtb)，这些感染大多是潜伏性的。结核杆菌可以在肺部病变中保持不活跃，但几十年后才会出现，从而滋生新的结核病爆发。此外，结核病是最难治疗的细菌感染之一，并且继续导致比任何其他细菌感染更多的死亡。细菌以复制和非复制状态存在于一系列微环境中，这些微环境的氧气浓度和养分利用率各不相同。在潜伏感染期间存活的杆菌可能以非复制状态存在，而抗菌剂对活跃生长的细菌有效，但对非复制细菌往往无效。关于结核分枝杆菌在潜伏感染或坏死性干酪结节病变的恶劣微环境中存活所使用的代谢机制知之甚少，在这些微环境中，氧气有限，营养来源不佳。结核分枝杆菌不能生长，但在没有好氧呼吸的情况下可以生存。因此，无复制厌氧状态被认为是体内持久性杆菌的主要模型。在没有好氧呼吸的情况下，Mtb需要一个功能正常的电子传递系统来驱动ATP的合成。然而，细菌维持氧化还原平衡的核心代谢机制尚不清楚。因此，我们的中心研究问题是：当有氧呼吸被抑制时，结核分枝杆菌采用了什么代谢机制？至少有三个因素限制了结核分枝杆菌的有氧呼吸：巨噬细胞产生的一氧化氮和一氧化碳的抑制作用，以及成熟肉芽肿的结构。所有这三种情况都强烈地诱导了DosR调节子，这是厌氧生存所必需的调节子。然而，该调节子并不编码一个完整的可识别的中间代谢途径。我们的表达分析和生化数据有力地表明，Mtb是一种专性需氧微生物，在没有有氧呼吸的情况下，维持着一种独特的多方面的代谢中间途径。因此，在结核分枝杆菌作为一种坦率的人类病原体的整个进化过程中，它一直保持着一系列广泛的酶，这些酶似乎是专门针对厌氧代谢功能的。该途径预测Mtb具有以厌氧方式代谢脂类和所有其他主要碳源的潜力。设计用来杀死非复制型厌氧杆菌的抗菌剂是迫切需要的。我们的研究直接支持这一目标。新的抗分枝杆菌药物和药物组合经常被用于抗缺氧/厌氧杆菌的测试，但关于适当方案的问题以及我们对相关中间代谢途径的有限了解限制了药物设计的合理方法。我们的初步数据表明，我们正处于对结核分枝杆菌厌氧代谢的基本了解的边缘。我们的工作假设是：结核分枝杆菌利用一种新的无氧代谢周期与DosR调节子相结合，在结核病变内的非呼吸条件下提供生存。为了验证这一假设，我们将调查所提出的厌氧代谢途径的关键方面。

DESCRIPTION (provided by applicant): A third of the world's population is infected with Mycobacterium tuberculosis (Mtb), and most of these infections are latent. Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effective against actively growing bacteria, are often not effective against non-replicating bacteria. Little is known about the metabolic mechanisms employed by Mtb to survive latent infection or the hostile microenvironment of necrotic caseous tubercle lesions where oxygen is limited and nutrient sources suboptimal. Mtb cannot grow but endures in the absence of aerobic respiration. Therefore, the non-replicating anaerobic state is considered a prime model for persistent bacilli in vivo. In the absence of aerobic respiration Mtb requires a functional electron transport system to drive ATP synthesis. However, the core metabolic mechanisms by which the bacilli maintain redox balance are unknown. Therefore, our central research question is "What metabolic mechanisms are employed by Mtb when aerobic respiration is inhibited?" At least three factors limit Mtb aerobic respiration: the inhibitory effect of macrophage-produced nitric oxide and carbon monoxide, and the structure of mature granulomas. All three of these conditions strongly induce the DosR regulon, a regulon essential for anaerobic survival. However, the regulon does not encode a complete recognizable intermediary metabolic pathway. Our expression analysis and biochemical data strongly indicate that Mtb - an obligate aerobe - maintains a unique multifaceted intermediary pathway for metabolism in the absence of aerobic respiration. Thus, throughout the evolution of Mtb as a frank human pathogen, it has maintained an extensive array of enzymes which appear to be geared specifically for anaerobic metabolic functions. The pathway predicts that Mtb has the potential to metabolize lipids and all other major carbon sources anaerobically. Antimicrobials designed to kill non-replicating anaerobic bacilli are sorely needed. Our research directly supports this goal. New antimycobacterial drugs and drug combinations are routinely tested against hypoxic/anaerobic bacilli, but questions about the proper protocol and our limited understanding of relevant intermediary metabolic pathways limit a rational approach to drug design. Our preliminary data suggests we are at the brink of a fundamental understanding of Mtb anaerobic metabolism. Our working hypothesis is: Mtb employs a novel anaerobic metabolic cycle in conjunction with the DosR regulon to confer survival during non- respiring conditions within TB lesions. To test this hypothesis we will investigate key aspects of the proposed anaerobic metabolic pathway.