Metabolome of non-replicating M. tuberculosis

描述（由申请人提供）：现在人们普遍认为需要新药来实现全球结核病控制。结核分枝杆菌基因组序列的完成为大大加快新药的发现奠定了基础。虽然已经报道了数量有限的结核病转录组和蛋白质组研究，但这些全局大分子分析仍然不能直接促进药物发现，除非已知基因/蛋白质功能或蛋白质晶体结构可用。阐明生物体中低分子量化合物的全部补体-代谢组-正被认为是对任何病原体的全面理解的补充，也许是最容易处理的方法，包括合理药物发现所必需的洞察力。这项R-21提案旨在确定结核杆菌在非复制持久性（NRP）阶段的代谢组，这一生理状态被认为是导致结核病所需的较长治疗时间的原因。代谢组将通过高分辨率色谱（CCC， LC）和光谱（MS， NMR）来阐明。最初的研究将利用高生物量的卡介苗来评估几种分离方案，以优化（次生）代谢物的分解。该方案将用于确定NRP中结核分枝杆菌的代谢组。后续的实验将集中于了解NRP下产生的独特代谢物的生长阶段特异性。所获得的信息不仅将补充细胞壁化学的广泛知识，而且还有望：1)识别可能与最近描述的其他分枝杆菌相关的新的次级代谢物；2)帮助澄清事件并识别代谢向低氧环境转变过程中的低MW标记，特别是考虑到在这种适应过程中上调的bbb70 %的基因功能未知。3)通过模拟在哺乳动物细胞中尚未发现的结核病特异性化合物，为药物开发提供线索；4)有助于了解新发现的抗结核病药物的作用机制。本研究的操作假设是，阐明结核分枝杆菌的代谢组将克服全球大分子分析固有的局限性，从而获得对休眠和药物发现的关键见解。

DESCRIPTION (provided by applicant): It is now generally accepted that new drugs are needed to make global tuberculosis control a reality. The completion of the sequence of the M. tuberculosis genome has laid the foundation for greatly accelerating new drug discovery. While a limited number of studies of the TB transcriptome and proteome have been reported, these global macromolecular analyses are still not able to contribute directly to drug discovery except where gene/protein function are known or protein crystal structures are available. Elucidating the full complement of low molecular weight compounds in an organism -the metabolome - is becoming recognized as a complementary and perhaps the most tractable approach to a comprehensive understanding of any pathogen including the insight necessary for rational drug discovery. This R-21 proposal seeks to define the metabolome of the tubercle bacillus in the stage of non-replicating persistence (NRP), the physiological state considered to be responsible for the required long treatment duration for TB. The metabolome will be elucidated by high-resolution chromatography (CCC, LC) and -spectroscopy (MS, NMR). Initial studies will utilize a high biomass of BCG to evaluate several separation schemes in optimizing the resolution of (secondary) metabolites. This protocol will then be used to define the metabolome of M. tuberculosis in NRP. Subsequent experiments will focus on understanding the growth-phase specificity of unique metabolites produced under NRP. The information obtained will not only complement the extensive knowledge of the chemistry of the cell wall but is also expected to I) identify novel secondary metabolites possibly related to those recently described for other mycobacteria, 2) help to clarify events and identify low MW markers during the metabolic shift to a low oxygen environment especially considering that >70% of genes up-regulated during this adaptation are of unknown function, 3) provide leads for drug development through analoging around TB-specific compounds not known to occur in mammalian cells and 4) assist in the understanding of the mechanism of action of newly discovered anti-TB agents. The operating hypothesis for this study is that elucidating the metabolome of Mycobacterium tuberculosis will overcome limitations inherent in global macromolecular analyses with respect to gaining key insights into dormancy and drug discovery.