Regulatory networks involved in Mycobacterium tuberculosis persistence

描述（由申请人提供）：结核病（TB）是一种主要的艾滋病相关感染。目前用于治疗结核病的冗长和繁琐的治疗方法导致了医疗不依从性和新出现的耐多药和广泛耐药结核病问题。这种长期治疗反映了结核分枝杆菌（Mtb）在受感染宿主体内以抗生素耐受性为特征的非复制状态持续存在的能力。抑制结核分枝杆菌生长的分子机制尚不清楚。我们和其他人已经证明，在生长限制条件下，警世酮过度磷酸化的鸟苷（(p)ppGpp）和调节分子无机聚磷酸盐（poly p）在结核分枝杆菌存活中发挥作用。然而，(p)ppGpp和poly p之间的调控关系以及该网络在结核分枝杆菌生长限制和抗生素耐受性中的确切作用尚未阐明。本课题拟利用Mtb重组菌株条件过表达负责(p)ppGpp合成的严格反应酶RelMtb，验证(p)ppGpp是抑制Mtb生长和耐药的分子“刹车”假说。接下来，我们将使用聚磷缺乏和聚磷积累的Mtb重组菌株，验证聚磷调节Mtb生长限制和抗生素耐受性的假设。最后，我们将验证(p)ppGpp和poly p构成一个复杂的反馈调节回路的假设，该回路涉及poly p依赖于relMtb的表达和(p)ppGpp介导的poly p水解抑制。尽管聚磷存在于所有细胞中，但在哺乳动物细胞中尚未发现负责结核分枝杆菌中聚磷合成的高度保守的细菌酶，因此使其成为药物开发的潜在吸引力靶点。一个小分子的RelMtb抑制剂可以减少Mtb合成(p)ppGpp。抑制这一调控网络可能导致持久性杆菌的存活减少，并有可能缩短结核化疗的持续时间。除了提高医疗依从性和减少产生耐药性的可能性之外，治疗活动性结核病的简化药物方案可能对合并感染艾滋病毒的患者特别有用，因为药物-药物相互作用和免疫重建可能使同时治疗两种感染复杂化。公共卫生相关性：结核病治疗需要至少6个月的治疗，因为引起结核病的细菌在遇到压力时会进入“休眠”状态，用目前的抗生素杀死分裂细菌变得非常困难。在这个提议中，我们计划研究一些导致结核细菌停止分裂的重要机制。如果我们能弄清楚结核病菌是如何“休眠”的，我们也许就能开发出新的方法来攻击这些病菌，并缩短治愈这种与艾滋病有关的主要感染所需的时间。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a major AIDS-related infection. The lengthy and cumbersome therapy currently available to treat TB has contributed to medical nonadherence and the emerging problems of multi-drug resistant (MDR)- and extensive-drug resistant (XDR)-TB. This prolonged therapy reflects the ability of Mycobacterium tuberculosis (Mtb) to persist in the infected host in a nonreplicating state characterized by antibiotic tolerance. The molecular mechanisms underlying Mtb growth restriction are unknown. We and others have shown that the alarmone hyperphosphorylated guanosine ((p)ppGpp) and the regulatory molecule inorganic polyphosphate (poly P) play a role in Mtb survival under growth-limiting conditions. However, the regulatory relationship between (p)ppGpp and poly P and the precise role of this network on Mtb growth restriction and antibiotic tolerance have not been elucidated. In this proposal, we plan to use Mtb recombinant strains conditionally overexpressing RelMtb, the stringent response enzyme responsible for (p)ppGpp synthesis, in order to test the hypothesis that (p)ppGpp is a molecular "brake" responsible for Mtb growth restriction and antibiotic tolerance. Next, using both poly P-deficient and poly P-accumulating Mtb recombinant strains, we will test the hypothesis that poly P regulates Mtb growth restriction and antibiotic tolerance. Finally, we will test the hypothesis that (p)ppGpp and poly P constitute a complex, feedback regulatory loop involving poly P- dependent expression of relMtb and (p)ppGpp-mediated inhibition of poly P hydrolysis. Although poly P is present in all cells, the highly-conserved bacterial enzyme responsible for poly P synthesis in Mtb has not been identified in mammalian cells, thus making it a potentially attractive target for drug development. A small molecule inhibitor of RelMtb would be predicted to lead to reduced Mtb synthesis of (p)ppGpp. Inhibition of this regulatory network may lead to reduced survival of persistent bacilli, with the potential to shorten the duration of TB chemotherapy. In addition to improving medical adherence and reducing the potential for the development of drug resistance, an abbreviated drug regimen to treat active TB could be especially useful in HIV co-infected patients, since drug-drug interactions and immune reconstitution may complicate the concurrent management of both infections. PUBLIC HEALTH RELEVANCE: TB treatment requires at least 6 months of therapy because the germs that cause TB can go "dormant" when they encounter stress, becoming very difficult to kill with current antibiotics, which kill dividing bacteria. In this proposal, we plan to study some of the important mechanisms that lead TB germs to stop dividing. If we can figure out how TB germs go "dormant", we may be able to develop new ways to attack these germs and shorten the time it takes to cure this major AIDS-related infection.