Regulatory networks involved in Mycobacterium tuberculosis persistence

简介(申请人提供)：结核病(TB)是一种与艾滋病相关的主要传染病。目前可用于治疗结核病的漫长而繁琐的治疗方法导致了医学上的不依从性，以及出现了多药耐药(MDR)和广泛耐药(XDR)-结核病的问题。这一长期治疗反映了结核分枝杆菌(Mtb)以抗生素耐受为特征的非复制状态在受感染宿主中持续存在的能力。结核分枝杆菌生长受限的分子机制尚不清楚。我们和其他人已经证明，警报蛋白过度磷酸化的鸟苷((P)ppGpp)和调节分子无机聚磷酸(PolyP)在限制生长的条件下对结核分枝杆菌的存活起到了作用。然而，(P)ppGpp和PolyP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药性中的确切作用尚未阐明。在这个建议中，我们计划使用Mtb重组菌株有条件地过度表达RelMtb，RelMtb是负责合成(P)ppGpp的严格反应酶，以检验(P)ppGpp是导致Mtb生长限制和抗生素耐受的分子“刹车”的假设。接下来，使用聚P缺乏和聚P积累Mtb重组菌株，我们将检验PolyP调节Mtb生长限制和抗生素耐受性的假说。最后，我们将检验这样的假设，即(P)ppGpp和聚P构成一个复杂的反馈调控环，涉及聚P依赖的relMtb的表达和(P)ppGpp介导的抑制聚P的水解。尽管Poly P存在于所有细胞中，但在哺乳动物细胞中尚未鉴定出高度保守的负责Mtb中PolyP合成的细菌酶，因此使其成为潜在的有吸引力的药物开发靶点。预测RelMtb的小分子抑制剂将导致(P)ppGpp的Mtb合成减少。抑制这一调控网络可能会导致持久性杆菌的存活率降低，从而有可能缩短结核病化疗的持续时间。除了改善服药依从性和减少产生耐药性的可能性外，治疗活动性结核病的简化药物方案对艾滋病毒合并感染的患者可能特别有用，因为药物-药物相互作用和免疫重建可能会使两种感染的同时管理复杂化。与公共卫生相关：结核病的治疗需要至少6个月的治疗，因为导致结核病的细菌在遇到压力时会进入“休眠”状态，用目前的抗生素很难杀死正在分裂的细菌。在这项提案中，我们计划研究导致结核病细菌停止分裂的一些重要机制。如果我们能弄清楚结核细菌是如何“休眠”的，我们或许能开发出攻击这些细菌的新方法，并缩短治愈这种与艾滋病相关的重大感染所需的时间。

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.