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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

基本信息

批准号：
10216045
负责人：
JENNIFER A PHILIPS
金额：
$ 23.63万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-05 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10216045
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 Plasma Play Production Protocols documentation Role Sampling Signal Transduction Source Sphingolipids Sputum System Testing Tuberculosis Vaccines Virulence antimicrobial base burden of illness cell motility differential expression innovation insight macrophage metabolomics novel novel diagnostics novel therapeutics oxidation pathogen predictive marker prevent response success tool trafficking tuberculosis diagnostics tuberculosis treatment uptake

项目摘要

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