Lipid storage and mobilization in M. tuberculosis as antilatency drugs targets

描述（由申请人提供）：结核病（TB）是可预防死亡的主要原因。结核分枝杆菌（Mtb）在宿主体内以非复制耐药状态潜伏数十年的能力，在人的免疫力减弱时重新激活，是治愈和根除这种每年导致近200万人死亡的疾病的主要障碍。已知脂肪酸是Mtb在宿主中持续存在所需的主要能量来源。然而，脂肪酸的来源仍然未知。我们假设结核分枝杆菌将能量储存为三酰甘油（TG）和蜡酯（WE），以成功地通过潜伏期。结核分枝杆菌用来储存和调动这些能量来源的步骤可能是消除潜伏性结核分枝杆菌的新型抗潜伏药物的理想目标。为了阐明这些步骤，我们建议：（1）产生tgs，fcr，fabp，fatp，cut和lip基因的基因破坏突变体，这些基因可能编码可能参与脂质储存和动员的酶，以成功地度过潜伏期。(2)检查Mt B中tgs、lip、cut和fcr基因的生物化学功能a）确定在诱导休眠样状态B的胁迫条件下，tgs、lip、cut和fcr基因的破坏对Mt B中脂质积累的影响b）阐明lip和cut突变体对储存脂质的动员的可能作用c）检查tgs、lip、cut和fcr基因的可能作用，在结核分枝杆菌脂质代谢的酰基转移反应中的lip和cut基因。(3)a）利用新的休眠的体外多重胁迫Mtb培养模型来鉴定在休眠发展期间参与TG和WE积累的tgs和fcr基因。B）开发一种使用多重应激体外模型来测试抗潜伏药物候选物的方法。(4)a）测试Mt B是否在缺氧条件下在THP-1衍生的巨噬细胞（TDM）中进入休眠状态B）确定在负载脂质的TDM内Mt B中积累的脂质体是否来源于宿主储存脂质，并鉴定涉及脂质积累的Mt B基因c）通过研究脂质积累缺陷的突变体是否与结核分枝杆菌在TDM中的脂质体积累有关，不能产生利福平耐药性。在两种体外休眠模型中均未能表现出休眠性状的基因破坏突变体将在TB潜伏期的兔遏制/地塞米松再活化模型中测试其成功经历休眠和再活化的能力。它们还将被提交给TARGET，用于在小鼠休眠的中空纤维人工肉芽肿模型中进行测试，并确定储存脂质积累是否参与该体内休眠模型。(5)在两种体外休眠模型中均未能表现出休眠性状的基因破坏突变体将在TB潜伏期的兔遏制/地塞米松再活化模型中测试其成功经历休眠和再活化的能力。它们还将被提交给TARGET，用于在小鼠休眠的中空纤维人工肉芽肿模型中进行测试，并确定储存脂质积累是否参与该体内休眠模型。预期的结果可能有助于确定可以消除潜伏性Mtb的新型药物的靶点。公共卫生相关性：结核病（TB）是可预防死亡的主要原因，每年造成200万人死亡。致病病原体可以停留在一个潜伏状态，并得到重新激活时，人的免疫系统被削弱了一些时间在生活中。世界人口的三分之一患有潜伏性结核病，目前没有可用的药物对潜伏性结核病有效。这项提议是为了发现有效对抗潜伏性结核病的新型药物的靶点。这些药物是完全治愈和全球根除结核病所必需的。

DESCRIPTION (provided by applicant): Tuberculosis (TB) is a leading cause of preventable death. The ability of Mycobacterium tuberculosis (Mtb) to remain latent in a nonreplicating drug resistant state in the host for decades, to be reactivated when the immunity of the person weakens, is a major impediment to curing and eradicating this disease that causes nearly two million deaths annually. Fatty acids are known to be the major energy source required for the persistence of Mtb in the host. However, the source of the fatty acids remains unknown. We postulate that Mtb stores energy as triacylglycerol (TG) and wax esters (WE) to successfully go through latency. The steps Mtb uses to store and mobilize these energy sources could be ideal targets for novel antilatency drugs that will eliminate latent Mtb. To elucidate these steps we propose to: (1) Generate gene-disrupted mutants for tgs, fcr, fabp, fatp, cut and lip genes that may encode enzymes that could be involved in the storage and mobilization of lipids for successfully going through latency. (2) Examine the biochemical functions of tgs, lip, cut, and fcr genes in Mtb a) Determine the effect of disruption of tgs, lip, cut and fcr genes in the accumulation of lipids in Mtb under stress conditions that induce a dormancy-like state b) Elucidate the possible role of the lip and cut mutants on the mobilization of the stored lipids c) Examine the possible role of tgs, lip and cut genes in acyl transfer reactions in Mtb lipid metabolism. (3) a) Utilize a novel in vitro multiple stress Mtb culture model of dormancy to identify the tgs and fcr genes involved in the accumulation of TG and WE during development of dormancy. b) Develop a method to use the multiple stress in vitro model to test antilatency drug candidates. (4) a) Test whether Mtb goes into a dormant state in THP-1 derived macrophages (TDM) under hypoxia b) Determine whether the lipid bodies that accumulate in Mtb within lipid-loaded TDM originate from host storage lipids and identify Mtb genes involved in lipid accumulation c) Determine whether the lipid body accumulation by Mtb in TDM is associated with development of Rif resistance by investigating whether mutants defective in lipid accumulation fail to develop Rif resistance. Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. (5) Gene-disrupted mutants that fail to manifest dormancy traits in both in vitro models of dormancy will be tested for their ability to successfully go through dormancy and reactivation in a rabbit containment/dexamethasone reactivation model of TB latency. They will also be submitted to TARGET for testing in a hollow fiber artificial granuloma model of dormancy in mice and determine whether storage lipid accumulation is involved in this in vivo dormancy model. The expected results are likely to help identify targets for novel drugs that can eliminate latent Mtb. PUBLIC HEALTH RELEVANCE: Tuberculosis (TB) is a leading cause of preventable death causing two million deaths annually. The causative pathogen can stay in a latent state and get reactivated when the person's immune system is weakened some time during life. One third of the world population has latent TB and no currently available drug is effective against latent TB. This proposal is to discover targets for novel drugs that will be effective against latent TB. Such drugs are required to get complete cure and global eradication of TB.