Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA.
Department of Life Sciences, Faculty of Science and Technology, University of Westminstergrid.12896.34, London, United Kingdom.
mBio. 2022 Feb 22;13(1):e0355921. doi: 10.1128/mbio.03559-21. Epub 2022 Jan 11.
Mycobacterium tuberculosis can cocatabolize a range of carbon sources. Fatty acids are among the carbons available inside the host's macrophages. Here, we investigated the metabolic changes of the fatty acid-induced dormancy-like state of and its involvement in the acquisition of drug tolerance. We conducted metabolomics profiling using a phosphoenolpyruvate carboxykinase (PEPCK)-deficient strain in an acetate-induced dormancy-like state, highlighting an overaccumulation of methylcitrate cycle (MCC) intermediates that correlates with enhanced drug tolerance against isoniazid and bedaquiline. Further metabolomics analyses of two mutants, an ICL knockdown (KD) strain and PrpD knockout (KO) strain, each lacking an MCC enzyme-isocitrate lyase (ICL) and 2-methylcitrate dehydratase (PrpD), respectively-were conducted after treatment with antibiotics. The ICL KD strain, which lacks the last enzyme of the MCC, showed an overaccumulation of MCC intermediates and a high level of drug tolerance. The PrpD KO strain, however, failed to accumulate MCC intermediates as it lacks the second step of the MCC and showed only a minor level of drug tolerance compared to the ICL KD mutant and its parental strain (CDC1551). Notably, addition of authentic 2-methylisocitrate, an MCC intermediate, improved the drug tolerance against antibiotics even in glycerol medium. Furthermore, wild-type displayed levels of drug tolerance when cultured in acetate medium significantly greater than those in glycerol medium. Taken together, the fatty acid-induced dormancy-like state remodels the central carbon metabolism of that is functionally relevant to acquisition of drug tolerance. Understanding the mechanisms underlying adaptive strategies to achieve drug tolerance is crucial for the identification of new targets and the development of new drugs. Here, we show that acetate medium triggers a drug-tolerant state in when challenged with antituberculosis (anti-TB) drugs. This carbon-induced drug-tolerant state is linked to an accumulation of the methylcitrate cycle (MCC) intermediates, whose role was previously known as a detox pathway for propionate metabolism. Three mutant strains with mutations in gluconeogenesis and MCC were used to investigate the correlation between drug tolerance and the accumulation of MCC metabolites. We herein report a new role of the MCC used to provide a survival advantage to as a species against both anti-TB drugs upon specific carbon sources.
结核分枝杆菌可以共代谢一系列碳源。脂肪酸是宿主巨噬细胞内可用的碳源之一。在这里，我们研究了脂肪酸诱导的休眠样状态下结核分枝杆菌的代谢变化及其在获得药物耐受性中的作用。我们使用磷酸烯醇丙酮酸羧激酶 (PEPCK) 缺陷型结核分枝杆菌在乙酸盐诱导的休眠样状态下进行代谢组学分析，突出显示甲基柠檬酸循环 (MCC) 中间产物的过度积累，这与异烟肼和贝达喹啉的药物耐受性增强相关。进一步对两种结核分枝杆菌突变体，即 ICL 敲低 (KD) 菌株和 PrpD 敲除 (KO) 菌株进行代谢组学分析，这两种突变体分别缺乏 MCC 酶异柠檬酸裂解酶 (ICL) 和 2-甲基柠檬酸脱水酶 (PrpD)，在用抗生素处理后进行。缺乏 MCC 最后一个酶的 ICL KD 菌株表现出 MCC 中间产物的过度积累和高水平的药物耐受性。然而，PrpD KO 菌株由于缺乏 MCC 的第二步而未能积累 MCC 中间产物，与 ICL KD 突变体及其亲本菌株 (CDC1551) 相比，仅表现出较小程度的药物耐受性。值得注意的是，添加真实的 2-甲基异柠檬酸，一种 MCC 中间产物，即使在甘油培养基中也能提高结核分枝杆菌对抗生素的药物耐受性。此外，野生型结核分枝杆菌在乙酸盐培养基中培养时表现出的药物耐受性水平明显高于在甘油培养基中培养时的水平。总之，脂肪酸诱导的休眠样状态重塑了结核分枝杆菌的中心碳代谢，这与获得结核分枝杆菌药物耐受性的功能相关。了解结核分枝杆菌获得药物耐受性的适应策略的机制对于确定新的靶点和开发新的药物至关重要。在这里，我们表明，当用抗结核 (anti-TB) 药物挑战时，乙酸盐培养基会引发结核分枝杆菌的药物耐受状态。这种碳诱导的药物耐受状态与甲基柠檬酸循环 (MCC) 中间产物的积累有关，其先前的作用是作为丙酸代谢的解毒途径。使用三种在糖异生和 MCC 中发生突变的突变菌株来研究药物耐受性与 MCC 代谢物积累之间的相关性。我们在此报告了 MCC 的一个新作用，即作为一种物种，当存在特定碳源时，MCC 用于为结核分枝杆菌提供对抗抗结核药物的生存优势。
