INTRODUCTION 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. OBJECTIVES 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. METHODS 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. RESULTS 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.

中文翻译：

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休眠性耻垢分枝杆菌细胞重新激活的代谢谱分析揭示了代谢过程的逐步组装。

引言随着培养基的逐渐酸化，分枝杆菌细胞进入特定状态，该状态的代谢活性和形态变化水平较低。这种非复制性持久性（休眠）状态与生理耐药性直接相关，这使根除潜在形式的结核病的努力变得复杂。为了找到新的抗潜伏性结核病化合物，应表征在休眠状态和转变为活性状态（再活化）过程中可能发生的代谢过程。目标在当前的研究中，我们在全球范围内分析了休眠和活化的耻垢分枝杆菌细胞（一种具有MTB的许多常见生理特征的模型微生物）的非靶向代谢组学概况，因为对代谢物动力学的表征和分析将提供细菌对应激条件的全球生化反应的全面概述。方法采用流式细胞术，通过比率测量法测量膜电位值，追踪再活化过程。选择关键的时间点，并将细菌取样进行LC-MS代谢分析。结果储存60天后，这些细胞的重新激活显示该过程分两个阶段进行：（I）持续10小时，其特征在于CFU数恒定，细胞大小不变，呼吸活动小幅增加和膜电位值明显增加，表明在该时间间隔内开始了新陈代谢过程；第二阶段（10-26小时）的特征是内源性呼吸加快，细胞大小变化，并随着细胞分裂的开始而结束。对带有KEGG注释的代谢物的相对丰度变化的分析表明，大量的代谢物（例如硬脂酸，甘油，D-葡萄糖，海藻糖6-磷酸酯）在重新活化时间内会降低其浓度，而与此相反，在重新活化过程中会合成十二烷基酸，霉菌素S和PG / AG生物合成的其他化合物等代谢产物。代谢谱的差异分析揭示了在重新活化的早期阶段许多代谢途径的激活：次级代谢产物的生物合成，嘌呤和嘧啶代谢，甘油磷脂和脂肪酸代谢等。结论：获得的数据表明，尽管长期处于休眠状态的细胞处于最小的代谢活性状态，但根据代谢谱，它们仍保留了大量的代谢产物。在重新激活的过程中，会发生完整代谢途径的增量随机组装。