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.

项目概要 目前对麻风病的耐药性监测仅基于临床症状的监测和 对已知耐药基因中的特定突变进行分子鉴定，这些突变很少且无法检测到 涵盖了导致治疗失败的一系列分子机制。我们用比较法 对药物敏感和耐药麻风分枝杆菌菌株进行基因组学鉴定新分子 麻风病抗生素耐药性的标志物。多态性可能相关的顶级候选基因 使用替代分枝杆菌（结核分枝杆菌）对耐药性进行了表征 因为麻风分枝杆菌不能在体外培养。我们的初步结果显示，删除了一名候选人 结核分枝杆菌中的基因，特别是 fadD9，可显着增强氨苯砜耐药性。分析 该基因的潜在功能与早期代谢组学研究的结果相结合 抗叶酸剂对结核分枝杆菌代谢的影响表明存在先前未知的氨苯砜靶标， 独立于叶酸途径中的 FolP1 酶，其有害的药理抑制作用 通过减少或抑制 FadD9 活性的突变来缓解。这个探索性项目旨在 描述分枝杆菌中氨苯砜敏感性和耐药性的新机制。 具体来说，我们假设氨苯砜抑制 g-氨基丁酸 (GABA) 转氨酶 GabT， 负责从 GABA 生成琥珀酸半醛 (SSA)，从而限制了 琥珀酸通过GABA分流进入TCA循环。我们进一步假设 FadD9 将 GabT、SSA 到琥珀醛的产物，FadD9 中功能突变的丧失/减少从而阻止 氨苯砜抑制的 GabT 产生的 SSA 数量有限，以免从 TCA 中转移 循环。 目标 1 将使用遗传和酶学方法来检验氨苯砜抑制 GabT 的假设。目标2 同样将使用无细胞和全细胞方法的组合来检验以下假设：(i) FadD9 将 SSA 转化为琥珀醛，并且 (ii) 临床相关突变导致 FadD9 活动。目标 3 最终将尝试将一系列氨苯砜耐药性的不同水平关联起来。 明确定义的麻风分枝杆菌分离株中存在 folP1、fadD9 和/或潜在的 gabT 突变， 相同的分离株。