Mechanism of action of Dapsone in Mycobacterium leprae

项目摘要 目前对麻风的耐药性监测完全基于对临床症状和 对已知耐药基因的特定突变的分子鉴定，这些突变很少，而且无法 涵盖了导致治疗失败的各种分子机制。我们使用比较级 药物敏感和耐药麻风分枝杆菌的基因组学鉴定新分子 麻风患者抗生素耐药性的标记物。可能与多态相关的首选候选基因 用一种替代分枝杆菌(结核分枝杆菌)来表征具有耐药性的 因为麻风杆菌不能在体外培养。我们的初步结果显示，删除一名候选人 结核分枝杆菌中的fadD9基因显著增强了氨苯砜的耐药性。三、分析了 该基因的潜在功能与早期代谢组学研究的结果相结合 抗结核分枝杆菌代谢中的抗叶酸表明氨苯砜存在一个以前未知的靶点， 不依赖于叶酸途径的FolP1酶，其有害的药理抑制 通过突变降低或抑制FadD9的活性来缓解。这一探索性项目旨在 描述这些分枝杆菌对氨苯砜的敏感性和耐药性的新机制。 具体地说，我们假设氨苯松抑制g-氨基丁酸(GABA)氨基转移酶GabT， 负责从GABA中生产琥珀酸半醛(SSA)，从而限制 琥珀酸通过GABA分流进入TCA循环。我们进一步假设FadD9将 从而防止了FadD9功能突变丢失/减少 氨苯砜抑制的GabT产生的有限数量的SSA不会从TCA转移 周而复始。 目的1将使用遗传学和酶学方法来验证氨苯砜抑制GabT的假说。目标2 类似地，我将使用无细胞方法和基于全细胞的方法的组合来检验假设：(I) FadD9将SSA转化为琥珀醛，以及(Ii)临床相关突变导致SSA减少或丢失 FadD9活动。目标3最终将尝试将不同水平的氨苯砜耐药性关联在一组 明确定义的麻风杆菌分离株存在folP1、fadD9和/或潜在的gabT突变，在 同样的菌株。

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.