Abstract
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.
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Acknowledgements
The work was supported by the Russian Foundation for Basic Research (VDN Grant No. 17-04-00564). Preparation of dormant bacteria and their microbiological characterization was financially supported by the Russian Science Foundation (ASK Grant 16-15-00245-P).
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VDN and ASK designed and VDN supervised the project. MOS carried out cultivation of mycobacteria and supported the study with the dormant bacteria. VDN carried out the experiments on mycobacteria reactivation, membrane potential assessment, microscopy, measurements of respiratory activity, prepared the samples for further metabolic profiling. GRD carried out the CFU, MPN, radiolabel incorporation assays. ST carried out the metabolic profiling, metabolites identification. VDN, ST carried out statistical analysis. VDN visualized, analyzed data and prepared figures. VDN, ASK—wrote the manuscript. All authors read, commented and approved the final version of the manuscript.
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Nikitushkin, V.D., Trenkamp, S., Demina, G.R. et al. Metabolic profiling of dormant Mycolicibacterium smegmatis cells’ reactivation reveals a gradual assembly of metabolic processes. Metabolomics 16, 24 (2020). https://doi.org/10.1007/s11306-020-1645-8
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DOI: https://doi.org/10.1007/s11306-020-1645-8