Specific target of NO-mediated damage in M.tuberculosis

描述（由申请人提供）：在艾滋病毒、耐多药结核病和生物恐怖主义的时代，对新型结核分枝杆菌化疗药物的需求从未如此之大。M.结核病作为病原体主要归因于其通过缓解或修复有害损伤的机制的进化来逃避先天免疫系统的杀菌反应的能力。因此，对先天免疫系统所使用的抗菌机制的详细了解可能会揭示新的化疗靶点。巨噬细胞相关的一氧化氮和活性氮中间体（NO/RNI）是这种先天性免疫反应的关键组成部分。令人惊讶的是，没有特异性的蛋白质靶点的NO/RNI介导的损害已被确定。本研究的重点是确定NO/RNI介导的蛋白质S-亚硝基化的靶点，以及NO/RNI对M.结核越来越多的证据支持蛋白质-S-亚硝基化作为NO/RNI的抗菌作用的重要介质的新兴作用。生物素在脂类生物合成中的必然作用以及脂类在M.结核病表明，在我们的初步研究中发现的NO/RNI依赖性作用的类似重要的抗菌作用。我们建议使用“生物素开关”方法在蛋白质组水平鉴定亚硝基化蛋白质，基于生物素的亲和化学研究NO诱导的生物素化蛋白质改变，和酶测定的组合，化学抑制剂研究和巨噬细胞感染实验，定向诱变和靶向基因敲除，以评估这些靶标的功能重要性。NOIRNI介导的M.结核病的研究为开发新的抗结核药物提供了希望，这些药物可以增强或模拟对M.结核病，同时规避NO/RNI特异性防御。

DESCRIPTION (provided by applicant): In an era of HIV, multidrug resistant tuberculosis, and bioterrorism, the need for novel chemotherapeutic agents against Mycobactedum tuberculosis has never been greater. The durable success of M. tuberculosis as a pathogen is largely attributable to its ability to evade a sterilizing response from the innate immune system through the evolution of mechanisms that either defuse or repair noxious insults. A detailed understanding of the antimicrobial mechanisms used by the innate immune system, therefore, may reveal novel chemotherapeutic targets. Macrophage-associated nitric oxide and reactive nitrogen intermediates (NO/RNI) are a critical component of this innate immune response. Surprisingly, no specific protein targets of NO/RNI-mediated damage have been identified. The focus of the proposed work is to define target of NO/RNI-mediated protein-S-nitrosylation and novel effects of NO/RNI on biotinylated proteins of M. tuberculosis. Accumulating evidence supports an emerging role for protein-S-nitrosylation as an important mediator of the antimicrobial effects of NO/RNI. The obligatory role of biotin in lipid biosynthesis and the importance of lipids in the virulence of M. tuberculosis suggest a similarly important antimicrobial role for the NO/RNI-dependent effects discovered in our preliminary studies. We propose to evaluate the importance of these NO/RNI-dependent effects using the 'biotin-switch' method to identify, at the proteomic level, Snitrosylated proteins; biotin-based affinity chemistries to study NO-induced alterations in biotinylated proteins; and a combination of enzyme assays, chemical inhibitor studies and macrophage infection experiments using site-directed mutagenesis and targeted gene knockouts to assess the functional importance of these targets. The identification of bona fide targets of NOIRNI-mediated damage in M. tuberculosis offers the promise of developing novel antitubercular agents that augment or mimic the natural immune response to infection with M. tuberculosis while circumventing NO/RNI-specific defenses.