Specific target of NO-mediated damage in M tuberculosis

描述(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，对治疗结核分枝杆菌的新型化疗药物的需求从未像现在这样大。结核分枝杆菌作为病原体的持久成功在很大程度上要归功于它能够通过进化机制来化解或修复有害的侮辱，从而逃避来自先天免疫系统的绝育反应。因此，对先天免疫系统使用的抗微生物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反应性氮中间产物(NO/RNI)是这种先天免疫反应的重要组成部分。令人惊讶的是，还没有确定NO/RNI介导的损伤的特定蛋白质靶点。本工作的重点是确定NO/RNI介导的蛋白-S亚硝化的靶点以及NO/RNI对结核分枝杆菌生物素基化蛋白的新作用。越来越多的证据支持蛋白质-S-亚硝化作为NO/RNI抗菌作用的重要介体。生物素在脂质生物合成中的必需作用和脂类在结核分枝杆菌毒力中的重要性表明，对于我们初步研究中发现的NO/RNI依赖效应，生物素具有类似的重要抗微生物作用。我们建议评估这些NO/RNI依赖效应的重要性，使用‘生物素开关’方法在蛋白质组学水平上识别SITRIP化蛋白；基于生物素的亲和化学研究NO诱导的生物素化蛋白的变化；结合酶分析、化学抑制剂研究和巨噬细胞感染实验使用定点突变和靶向基因敲除来评估这些靶点的功能重要性。在结核分枝杆菌中识别NOIRNI介导的损伤的真正靶点，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结核分枝杆菌感染的自然免疫反应，同时绕过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.