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漫长而繁琐的疗法导致了医疗不依从性和出现的多药耐药（MDR）和广泛耐药（XDR）结核病问题。这种长期治疗反映了结核分枝杆菌（Mtb）在感染宿主中以非复制状态持续存在的能力，其特征在于抗生素耐受性。Mtb生长限制的分子机制尚不清楚。我们和其他人已经表明，alarmone过度磷酸化鸟苷（（p）ppGpp）和调节分子无机多磷酸盐（聚P）在生长限制条件下的结核分枝杆菌生存中发挥作用。然而，（p）ppGpp和聚P之间的调节关系和该网络对结核分枝杆菌生长限制和抗生素耐受性的确切作用尚未阐明。在该提案中，我们计划使用条件性过表达RelMtb（负责（p）ppGpp合成的严格反应酶）的Mtb重组菌株，以检验（p）ppGpp是负责Mtb生长限制和抗生素耐受性的分子“制动器”的假设。接下来，使用聚P缺陷型和聚P积累型Mtb重组菌株，我们将测试聚P调节Mtb生长限制和抗生素耐受性的假设。最后，我们将检验以下假设：（p）ppGpp和聚P构成复杂的反馈调节环，其涉及relMtb的聚P依赖性表达和（p）ppGpp介导的聚P水解抑制。虽然poly P存在于所有细胞中，但在哺乳动物细胞中尚未鉴定出负责Mtb中poly P合成的高度保守的细菌酶，因此使其成为药物开发的潜在有吸引力的靶标。预计RelMtb的小分子抑制剂会导致（p）ppGpp的Mtb合成减少。抑制这种调节网络可能导致持续性杆菌的存活率降低，并有可能缩短结核病化疗的持续时间。除了提高医疗依从性和减少耐药性的发展潜力外，治疗活动性结核病的简化药物方案对HIV合并感染患者特别有用，因为药物相互作用和免疫重建可能使两种感染的并发管理复杂化。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，因为抗生素会杀死分裂的细菌。在这项提案中，我们计划研究导致结核病菌停止分裂的一些重要机制。如果我们能弄清楚结核病菌是如何“休眠”的，我们就有可能开发出新的方法来攻击这些病菌，并缩短治愈这种主要的艾滋病相关感染的时间。

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.