A.N. Bach Institute of Biochemistry, Federal Research Centre "Fundamentals of Biotechnology" of the Russian Academy of Sciences, Leninsky Prospect 33, Build. 2, Moscow, Russia, 119071.
Metabolomic Discoveries GmbH, Am Mühlenberg 11, 14476, Potsdam, Germany.
Metabolomics. 2020 Feb 6;16(2):24. doi: 10.1007/s11306-020-1645-8.
Under gradual acidification of the culture medium mycobacterial cells transit into a specific state characterized by low level of metabolic activity and morphological alterations. This state of non-replicative persistence (dormancy) is directly linked to physiological drug resistance, which complicates the efforts to eradicate the latent forms of TB. In order to find new anti-latent TB compounds, the metabolic processes which may occur in the state of dormancy and during the transition into the active state (reactivation) should be characterized.
In the current study we analyzed the untargeted metabolomic profiles of dormant and reactivating Mycolicibacterium smegmatis cells (a model microorganism, bearing many common physiological traits of MTB), on the global scale level, since the characterization and analysis of the metabolites' dynamics would provide a comprehensive overview on global biochemical responses of the bacteria to stress conditions.
The reactivation process was tracked by measuring the value of membrane potential, applying a ratio-metric approach, by the method of flow-cytometry. The crucial timepoints were selected and the bacteria were sampled to LC-MS metabolic profiling.
Reactivation of these cells after 60 days of storage revealed that this process proceeds in two stages: (I) a period, which lasts for 10 h and is characterized by a constant CFU number, unchangeable cell size, a minuscule increase of respiratory activity and a noticeable increase in membrane potential value, indicating the onset of the first metabolic processes during this time interval; the second phase (10-26 h) is characterized by acceleration of endogenous respiration, changes in the size of the cells and it finishes with the beginning of cells division. Analysis of the changes in the relative abundances of KEGG-annotated metabolites revealed that a significant number of metabolites, such as stearic acid, glycerol, D-glucose, trehalose-6-phosphate decrease their concentrations over the reactivation time, whereas in contrast, such metabolites as dodecanoic acid, mycobactin S, and other compounds of PG/AG biosynthesis are synthesized during reactivation. Differential analysis of metabolic profiles disclosed the activation of a number of metabolic pathways at the early reactivation stage: biosynthesis of secondary metabolites, purine and pyrimidine metabolism, glycerophospholipid and fatty acids metabolism etc. CONCLUSION: The data obtained indicate, despite the long-term storage of dormant cells in a state of minimal metabolic activity, according to metabolic profiling, they still retained a large number of metabolites. In the process of reactivation, the incremental stochastic assembly of the complete metabolic pathways occurs.
在培养基逐渐酸化的过程中，分枝杆菌细胞会进入一种特定的状态，其特征是代谢活性低和形态改变。这种非复制性持续（休眠）状态与生理耐药性直接相关，这使得根除潜伏性结核病的工作变得复杂。为了寻找新的抗潜伏性结核病化合物，应该描述休眠状态和进入活跃状态（再激活）时可能发生的代谢过程。
在当前的研究中，我们在全局水平上分析了休眠和再激活分枝杆菌 smegmatis 细胞（一种模式微生物，具有许多与 MTB 共同的生理特征）的非靶向代谢组学图谱，因为代谢物动态的特征和分析将提供对细菌应激条件下整体生化反应的综合概述。
通过流式细胞术的比率方法，测量膜电位值来跟踪再激活过程。选择关键时间点并取样进行 LC-MS 代谢组学分析。
储存 60 天后，这些细胞的再激活表明该过程分两个阶段进行：（I）持续 10 小时的时间段，其特征是 CFU 数量不变，细胞大小不变，呼吸活性稍有增加，膜电位值显著增加，表明在此时间间隔内开始了第一代谢过程；第二阶段（10-26 小时）的特征是内源性呼吸加速，细胞大小变化，并以细胞分裂开始结束。KEGG 注释代谢物相对丰度的变化分析表明，大量代谢物，如硬脂酸、甘油、D-葡萄糖、海藻糖-6-磷酸的浓度在再激活过程中降低，而相反，十二烷酸、分枝菌 S 和 PG/AG 生物合成的其他化合物等代谢物在再激活过程中被合成。代谢谱的差异分析揭示了许多代谢途径在再激活早期阶段的激活：次生代谢物合成、嘌呤和嘧啶代谢、甘油磷脂和脂肪酸代谢等。
尽管休眠细胞在最低代谢活性状态下长期储存，但根据代谢组学分析，它们仍保留了大量代谢物。在再激活过程中，完整代谢途径的增量随机组装发生。
