Metabolomic Analysis of Redox control in Mycobacterium tuberculosis persisters

描述（由申请人提供）：由于耐多药和广泛耐药结核分枝杆菌菌株的出现以及该细菌与艾滋病毒的灾难性结合，结核病的流行迫切需要新的化疗药物。除了对药物的遗传抗性外，结核分枝杆菌细胞还可以成为持久性细胞，即对药物杀伤具有表型抗性的细胞。这种进入持续状态的能力需要长时间的化疗，这是改善结核病化疗的最大挑战。不幸的是，我们对持久者的代谢状态知之甚少。为了阐明持久性结核分枝杆菌细胞的生理状态，我们使用质谱法生成了结核分枝杆菌在有氧和缺氧生长条件下（持久性模型）的代谢谱，并发现了调节NADH/NAD（氧化还原）代谢的几个关键控制点。为了进一步表征代谢谱，我们研究了结核分枝杆菌的三羧酸循环（TCA）。利用遗传和生化方法的结合，我们发现结核分枝杆菌具有一组编码酮戊二酸氧化还原酶的基因，酮戊二酸氧化还原酶是厌氧细菌中常见的TCA的关键酶。此外，我们发现编码NADH脱氢酶II （ndh）的基因突变赋予对异烟肼和乙硫酰胺的抗性。通过结合代谢物谱、稳定同位素通量谱和突变研究，我们打算描述结核分枝杆菌的持续状态。这一知识应该会导致新的策略来有效地杀死持久性结核分枝杆菌细胞。

DESCRIPTION (provided by applicant): New chemotherapeutics are urgently required to control the tuberculosis pandemic, which is fueled by the emergence of multi-drug- and extensively-drug-resistant Mycobacterium tuberculosis strains and the bacterium's catastrophic alliance with HIV. In addition to genetic resistance to drugs, M. tuberculosis cells can become persistors, cells that are phenotypically resistant to the killing by drugs. This ability to enter into a persistent state necessitates long periods of chemotherapy and represents the greatest challenge in improve TB chemotherapies. Unfortunately, little is known about the metabolic state of persistors. To elucidate the physiological state of persistent M. tuberculosis cells, we have used mass spectroscopy to generate metabolic profiles of M. tuberculosis when grown in aerobic and hypoxic growth conditions (a persistence model) and found several key control points modulating NADH/NAD (redox) metabolism. To further characterize the metabolic profiles, we have investigated the Tricarboxylic Acid Cycle (TCA) of M. tuberculosis. Using a combination of genetic and biochemical approaches, we have discovered that M. tuberculosis possesses a set of genes encoding ketoglutarate oxidoreductase, a key enzyme of the TCA that is generally observed in anaerobic bacteria. In addition, we have discovered that mutations in the gene encoding NADH dehydrogenase II (ndh) confer resistance to Isoniazid and Ethionamide. By combining metabolite profiling, and stable isotope flux profiling, with mutational studies we intend to characterize persistent states of M. tuberculosis. This knowledge should lead to novel strategies to effectively kill persistent M. tuberculosis cells. PUBLIC HEALTH RELEVANCE: New chemotherapeutics are urgently required to control the tuberculosis pandemic, which is fueled by the emergence of multi-drug- and extensively-drug-resistant Mycobacterium tuberculosis strains and the bacterium's catastrophic alliance with HIV. In addition to genetic resistance to drugs, M. tuberculosis cells can become persistors, cells that are phenotypically resistant to the killing by drugs. This proposal seeks to develop new understandings of persisting M. tuberculosis cells providing the knowledge for novel and shortened chemotherapies.