Mechanism of action of Dapsone in Mycobacterium leprae

氨苯砜对麻风分枝杆菌的作用机制

• 批准号: 10643361
• 负责人: Charlotte Avanzi
• 金额: $ 19.19万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-25 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10643361
• 关键词: 4-Aminobenzoic Acid; 4-Aminobutyrate aminotransferase; Aldehydes; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biological Assay; Candidate Disease Gene; Carbon; Carboxylic Acids; Cells; Citric Acid Cycle; Clinic; Clinical; Collection; Complement component C4a; Development; Dihydropteroate Synthase; Drug Monitoring; Drug resistance; Energy Metabolism; Enzymes; Exposure to; Fatty Acids; Folate Biosynthesis Pathway; Folic Acid; Folic Acid Antagonists; Gene Cluster; Gene Combinations; Genes; Genetic; Genetic Polymorphism; Genome; Genus Mycobacterium; In Vitro; Infection; Knowledge; Lead; Leprosy; Measures; Metabolism; Molecular; Monitor; Mus; Mutation; Mycobacterium leprae; Mycobacterium marinum; Mycobacterium tuberculosis; Orthologous Gene; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Pharmacotherapy; Predisposition; Production; Recombinants; Recommendation; Regimen; Resistance; Resistance development; Rifampin; Role; Shunt Device; Succinates; Sulfonamides; Symptoms; Testing; Treatment Failure; Vaccines; World Health Organization; aminobutyrate; clinically relevant; comparative genomics; enzyme activity; fighting; gene function; metabolomics; molecular marker; mycobacterial; nonsynonymous mutation; novel; overexpression; pharmacologic; prevent; resistance gene; resistance mechanism; success; weapons; whole genome

Project Summary Current drug resistance surveillance for leprosy is solely based on the monitoring of clinical symptoms and the molecular identification of specific mutations in known drug resistance genes, which are few and fail to encompass the range of molecular mechanisms responsible for treatment failure. We used comparative genomics of drug-susceptible and drug-resistant Mycobacterium leprae strains to identify novel molecular markers of antibiotic resistance in leprosy. Top candidate genes whose polymorphism potentially associated with drug resistance were characterized using a surrogate Mycobacterium (Mycobacterium tuberculosis) since M. leprae cannot be cultured in vitro. Our preliminary results show that the deletion of one candidate gene in particular, fadD9, in M. tuberculosis significantly enhances dapsone resistance. Analysis of the potential function of this gene combined with the results of an earlier metabolomics study on the effects of antifolates on M. tuberculosis metabolism point to the existence of a previously unknown target of dapsone, independent of the FolP1 enzyme from the folate pathway, the deleterious pharmacological inhibition of which is mitigated by mutations reducing or inhibiting the activity of FadD9. This exploratory project aims to characterize these new mechanisms of susceptibility and resistance to dapsone in mycobacteria. Specifically, we hypothesize that dapsone inhibits the g-aminobutyrate (GABA) aminotransferase, GabT, responsible for the production of succinate semialdehyde (SSA) from GABA, thereby limiting the amount of succinate entering the TCA cycle through the GABA shunt. We further hypothesize that FadD9 converts the product of GabT, SSA, to succinaldehyde and that loss of/reduced function mutations in FadD9 thus prevent the limited amounts of SSA produced by the dapsone-inhibited GabT from being diverted away from the TCA cycle. Aim 1 will use genetic and enzymatic approaches to test the hypothesis that dapsone inhibits GabT. Aim 2 will similarly use a combination of cell-free and whole cell-based approaches to test the hypotheses that (i) FadD9 converts SSA to succinaldehyde and that (ii) clinically-relevant mutations lead to reduced or loss of FadD9 activity. Aim 3 will finally attempt to correlate different levels of dapsone resistance in a collection of well-defined M. leprae isolates to the presence of mutations in folP1, fadD9, and/or potentially gabT, in the same isolates.

项目摘要 目前对麻风的耐药性监测仅基于对临床症状的监测， 已知耐药基因中特定突变的分子鉴定，这是很少的， 包括导致治疗失败的分子机制的范围。我们使用比较 麻风分枝杆菌药物敏感和耐药菌株的基因组学，以鉴定新分子 麻风病的抗生素耐药性标志。多态性可能与以下基因相关的热门候选基因 使用替代分枝杆菌（结核分枝杆菌） 自从M.麻风不能在体外培养。我们的初步结果表明，删除一个候选人， 特别是fadD 9基因。结核病显著增强了对氨苯砜的抗性。分析 该基因的潜在功能结合早期代谢组学研究的结果， 叶酸拮抗剂对M.结核病代谢指出存在以前未知的氨苯砜靶标， 不依赖于叶酸途径的FolP 1酶，其有害的药理学抑制 通过降低或抑制FadD 9活性的突变来减轻。该探索性项目旨在 描述了分枝杆菌对氨苯砜敏感和耐药的新机制。 具体来说，我们假设氨苯砜抑制γ-氨基丁酸（GABA）转氨酶，GabT， 负责从GABA产生琥珀酸半醛（SSA），从而限制了GABA的量。 琥珀酸通过GABA分流进入TCA循环。我们进一步假设，FadD 9将 FadD 9中的功能丧失/降低突变因此阻止了GABT、SSA转化为琥珀醛， 由氨苯砜抑制的GabT产生的有限量的SSA从TCA转移开 周期 目的1将使用遗传学和酶的方法来验证氨苯砜抑制GabT的假设。目的2 将类似地使用无细胞和基于全细胞的方法的组合来测试以下假设： FadD 9将SSA转化为琥珀醛，并且（ii）临床相关突变导致SSA减少或丧失。 FadD 9活性。Aim 3将最终尝试在一组受试者中将不同水平的氨苯砜耐药性联系起来。 定义明确的M。麻风分离株与folP 1、fadD 9和/或潜在gabT突变的存在有关， 同样的隔离。