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.

概括 结核分枝杆菌（MTB）引起结核病（TB），杀死的人比任何其他病原体都多。 MTB 能够颠覆先天的免疫力，从而损害巨噬细胞的抗菌功能。 MTB诱导 可以在近距离近距离发现脂质的脂肪填充或“泡沫”巨噬细胞的形成 细胞内脂质液滴的近似值，被认为是以营养的形式来源的来源 芽孢杆菌的胆固醇酯和脂肪酸。当我们对MTB感染的全球代谢分析进行 巨噬细胞，我们发现胆固醇代谢产物胆固醇是第二个最差异的 在感染和未感染细胞之间表达代谢产物。胆固醇的存在反映了活性 宿主细胞的胆固醇和细菌胆固醇的来源。 MTB已知 氧化胆固醇，这被认为是降解途径的第一步，使MTB能够使用 胆固醇作为碳源。我们假设氧化的胆固醇不仅仅是一个中间体 降解途径；我们建议它通过干扰抗菌功能在病毒中起作用 巨噬细胞。此外，我们建议胆固醇将是结核病疾病负担的有用生物标志物。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进化以及是否有助于 它们可用于诊断来评估伯恩疾病。已知胆碱会破坏细胞 膜，干扰脂质筏。 NADPH氧化物的组装和活性对 膜脂质组成和筏完整性。我们将确定胆固醇是否会损害NADPH氧化物 使用实验室中建立的方案的方案，组装，溶酶体运输和细胞内生长。我们 将使用质量光谱法兼容，监测感染巨噬细胞中胆固醇的分布 分馏策略和新型的重氮嗪炔烃问题，可视化其亚细胞定位。虽然那里 达成共识，即MTB可以利用胆固醇作为碳源，关于该身份存在差异 胆固醇氧化物。我们将确定3β-羟基固醇脱氢酶和CHOD是否是否 氧化物和MCE4是一种胆固醇摄取系统，在体外和期间需要胆固醇氧化系统。 巨噬细胞感染。最后，我们将确定胆固醇的抽象是否将痰与 来自未感染的对照的结核病感染者。目前，没有生物标志物可以预测疾病伯恩 或对治疗的反应。那是一种反映细菌燃烧和对治疗反应的生物标志物将具有 对患者护理的重大影响。总体而言，我们的研究有可能对其中一个产生重要影响 通过（1）阐明MTB发病机理的最强大的病原体，（2） 代谢途径，以及（3）导致急需的新型诊断。