Cholesterol oxidation products in TB pathogenesis and as biomarkers of disease

摘要 结核分枝杆菌(Mtb)导致结核病(TB)，比任何其他病原体导致的死亡人数都多。山地车 能够颠覆先天免疫，损害巨噬细胞的抗菌功能。结核分枝杆菌引起 可近距离观察到充满脂滴或“泡沫”的巨噬细胞的形成以及细菌本身。 近似于细胞内的脂滴，被认为是营养物质的来源，其形式 细菌所需的胆固醇酯和脂肪酸。当我们对感染的结核分枝杆菌进行全球代谢分析时 巨噬细胞，我们发现胆固醇代谢物，胆固酮，是差异第二大的。 感染细胞和未感染细胞之间表达的代谢物。胆固酮的存在反映了这种活性 作为胆固醇来源的宿主细胞和细菌胆固醇氧化酶。众所周知，MTB 氧化胆固醇，这被认为是降解途径的第一步，使结核分枝杆菌能够利用 胆固醇作为碳源。我们假设氧化的胆固醇不只是一种中间产物 降解途径；我们认为它通过干扰细菌的抗菌功能在毒力中发挥作用 巨噬细胞。此外，我们建议胆固酮将是一个有用的结核病疾病负担的生物标志物。这里, 我们将确定细菌衍生的胆固醇代谢产物是否有助于免疫逃避，以及是否 它们可用于诊断，以评估疾病负担。已知Cholestenone可以破坏细胞 膜，干扰脂筏。NADPH氧化酶的组装和活性对 膜脂成分和浮筏完整性。我们将确定胆固酮是否会损害NADPH氧化酶 使用实验室中成熟的方案进行组装、溶酶体运输和细胞内生长。我们 将使用兼容质谱仪监测受感染巨噬细胞中胆固酮的分布 分级策略和新的二氮杂炔探针，以可视化其亚细胞定位。尽管在那里 人们一致认为结核分枝杆菌可以利用胆固醇作为碳源，但对于 胆固醇氧化酶。我们将确定3-β-羟基类固醇脱氢酶和CHOD，假定的胆固醇 氧化酶和胆固醇摄取系统Mce4是胆固醇在体外和体内氧化所必需的。 巨噬细胞感染。最后，我们将确定胆固酮的丰度是否能区分痰和痰 来自未感染对照的结核病感染者。目前，还没有预测疾病负担的生物标记物。 或对治疗的反应。因此，反映细菌负荷和治疗反应的生物标记物将具有 对病人护理的重大影响。总体而言，我们的研究有可能对 最可怕的病原体通过(1)阐明结核分枝杆菌的发病机制，(2)澄清一个重要的 代谢途径，以及(3)导致急需的新诊断方法的发展。

SUMMARY Mycobacterium tuberculosis (Mtb) causes tuberculosis (TB), killing more people than any other pathogen. Mtb is able to subvert innate immunity, impairing the antimicrobial function of macrophages. Mtb induces the formation of lipid-droplet-filled or “foamy” macrophages, and the bacilli themselves can be found in close approximation to intracellular lipid droplets, which are thought to serve as a source of nutrients in the form of cholesterol esters and fatty acids for the bacilli. When we performed global metabolic profiling of Mtb infected macrophages, we found that the cholesterol metabolite, cholestenone, was the second most differentially expressed metabolite between infected and uninfected cells. The presence of cholestenone reflects the activity of both the host cell, which is the source of cholesterol, and a bacterial cholesterol oxidase. Mtb is known to oxidize cholesterol, which is thought to be the first step in a degradative pathway that enables Mtb to utilize cholesterol as a carbon source. We hypothesize that oxidized cholesterol is more than an intermediate in a degradative pathway; we propose that it plays a role in virulence by interfering with antimicrobial function of macrophages. Further, we propose that cholestenone will be a useful biomarker of TB disease burden. Here, we will establish whether bacterial-derived cholesterol metabolites contribute to immune evasion and whether they can be used for diagnostics to evaluate disease burden. Cholestenone is known to disrupt cellular membranes, interfering with lipid rafts. The assembly and activity of the NADPH oxidase is sensitive to membrane lipid composition and raft integrity. We will establish whether cholestenone impairs NADPH oxidase assembly, lysosomal trafficking, and intracellular growth using protocols that are well-established in the lab. We will monitor the distribution of cholestenone in infected macrophages using mass-spectrometry compatible fractionation strategies and novel diazirine alkyne probes to visualize its subcellular localization. Although there is consensus that Mtb can utilize cholesterol as a carbon source, there is discrepancy as to the identity of the cholesterol oxidase. We will establish whether 3β-hydroxysteroid dehydrogenase and ChoD, putative cholesterol oxidases, and Mce4, a cholesterol uptake system, are required for cholesterol oxidation in vitro and during macrophage infection. Finally, we will determine whether cholestenone abundance distinguishes sputum from TB-infected individuals from uninfected controls. Currently, there are no biomarkers that predict disease burden or response to therapy. Thus, a biomarker that reflects bacterial burden and response to therapy would have a major impact on patient care. Overall, our studies have the potential to make an important impact on one of the most formidable pathogens by (1) elucidating mechanisms of Mtb pathogenesis, (2) clarifying an important metabolic pathway, and (3) leading to the development of much needed, novel diagnostics.