Metabolomic Analysis of Redox control in Mycobacterium tuberculosis persisters

描述(由申请人提供)：迫切需要新的化疗药物来控制结核病大流行，这是由于出现耐多药和广泛耐药的结核分枝杆菌菌株以及该细菌与艾滋病毒的灾难性联盟而加剧的。除了对药物的遗传耐药性外，结核分枝杆菌细胞还可以成为持久性细胞，即对药物杀灭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，这是改进结核病化疗的最大挑战。不幸的是，人们对持久者的新陈代谢状态知之甚少。为了阐明持久性结核分枝杆菌细胞的生理状态，我们利用质谱仪建立了结核分枝杆菌在有氧和低氧生长条件下的代谢谱(持续性模型)，并发现了几个调节NADH/NAD(氧化还原)代谢的关键控制点。为了进一步描述代谢特征，我们研究了结核分枝杆菌的三羧酸循环(TCA)。结合遗传和生化方法，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TCA的关键酶，通常在厌氧细菌中观察到。此外，我们还发现编码NADH脱氢酶II(NDH)的基因突变会导致对异烟肼和乙硫酰胺的耐药性。通过结合代谢物分析和稳定同位素通量分析，结合突变研究，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有效杀死持久性结核分枝杆菌细胞的新策略。 公共卫生相关性：迫切需要新的化疗药物来控制结核病大流行，结核病大流行是由于出现了对多种药物和广泛耐药的结核分枝杆菌菌株以及该细菌与艾滋病毒灾难性的联盟而加剧的。除了对药物的遗传耐药性外，结核分枝杆菌细胞还可以成为持久性细胞，即对药物杀灭具有表型抗性的细胞。这一建议旨在发展对持续存在的结核分枝杆菌细胞的新理解，为新的和缩短的化疗提供知识。

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.