Cholesterol oxidation products in TB pathogenesis and as biomarkers of disease

结核病发病机制中的胆固醇氧化产物及其作为疾病的生物标志物

• 批准号: 10343850
• 负责人: JENNIFER A PHILIPS
• 金额: $ 19.69万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-05 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10343850
• 关键词: Alkynes; Bacillus; Bacteria; Biological Markers; Carbon; Cells; Cellular Membrane; Cellular biology; Cholesterol; Cholesterol Esters; Cholesterol Homeostasis; Cholesterol Oxidase; Clinical; Conflict (Psychology); Consensus; Data; Degradation Pathway; Development; Diagnostic; Diagnostic tests; Diazomethane; Disease; Enzymes; Epidemic; Fatty Acids; Foamy Macrophage; Foundations; Fractionation; Genetic; Genetic Determinism; Genome; Goals; Growth; Human; Hydroxysteroid Dehydrogenases; Immune Evasion; Immunity; Impairment; In Vitro; Individual; Infection; Infectious Agent; Lead; Lipids; Literature; Mass Spectrum Analysis; Measures; Mediating; Membrane; Membrane Lipids; Membrane Microdomains; Metabolic; Metabolic Pathway; Metabolism; Microscopy; Monitor; Mycobacterium leprae; Mycobacterium tuberculosis; NADPH Oxidase; Natural Immunity; Nutrient; Oxides; Pathogenesis; Patient Care; Patients; Persons; Plasma; Play; Production; Protocols documentation; Role; Sampling; Signal Transduction; Source; Sphingolipids; Sputum; System; Testing; Tuberculosis; Vaccines; Virulence; antimicrobial; base; burden of illness; cell motility; diagnostic tool; differential expression; innovation; insight; macrophage; metabolomics; novel; novel diagnostics; novel therapeutics; oxidation; pathogen; predictive marker; prevent; response; success; trafficking; treatment response; tuberculosis diagnostics; tuberculosis treatment; uptake

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.

总结 结核分枝杆菌（Mtb）导致结核病（TB），比任何其他病原体杀死更多的人。MTB 能够破坏先天免疫，损害巨噬细胞的抗菌功能。结核病诱导 形成充满脂滴或“泡沫状”的巨噬细胞，并且可以在近距离内发现杆菌本身。 接近细胞内脂滴，被认为是作为营养物质的形式来源， 胆固醇酯和脂肪酸。当我们对结核分枝杆菌感染者进行整体代谢分析时， 在巨噬细胞中，我们发现胆固醇代谢产物，胆甾烯酮，是第二大差异， 在感染和未感染的细胞之间表达代谢产物。阿替烯酮的存在反映了 宿主细胞是胆固醇的来源，细菌胆固醇氧化酶也是。结核病是已知的 氧化胆固醇，这被认为是使结核分枝杆菌能够利用 胆固醇作为碳源。我们假设氧化胆固醇不仅仅是一种中间体， 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，乌藨子烯酮将是一个有用的生物标志物的结核病负担。在这里， 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， 它们可用于诊断，以评估疾病负担。已知胆甾烯酮会破坏细胞 膜，干扰脂筏。NADPH氧化酶的组装和活性对 膜脂组成和筏完整性。我们将确定乌藨子烯酮是否损害NADPH氧化酶 组装、溶酶体运输和细胞内生长，使用在实验室中良好建立的方案。我们 将使用质谱法监测受感染巨噬细胞中的乌替尼酮分布， 分馏策略和新的二氮杂环丙烯炔探针，以可视化其亚细胞定位。虽然 虽然Mtb可以利用胆固醇作为碳源是共识，但关于Mtb的身份存在分歧。 胆固醇氧化酶我们将确定是否3β-羟基类固醇脱氢酶和ChoD，假定的胆固醇 氧化酶和Mce 4，一种胆固醇摄取系统，是体外胆固醇氧化所必需的， 巨噬细胞感染最后，我们将确定乌藨子烯酮的丰度是否能区分痰液和 来自未感染对照的TB感染个体。目前，没有生物标志物可以预测疾病负担 或对治疗的反应。因此，反映细菌负荷和对治疗的反应的生物标志物将具有 对病人护理产生重大影响。总的来说，我们的研究有可能对其中一个领域产生重要影响。 最强大的病原体（1）阐明结核病发病机制，（2）阐明一个重要的 代谢途径，和（3）导致急需的，新的诊断的发展。