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Mycobacterium tuberculosis transcriptome on various phases of dormancy in potassium-deficient model

ABSTRACT: Dormant Mycobacterium tuberculosis bacilli play important role in latent tuberculosis infection. Previously we have demonstrated that cultivation of M. tuberculosis in potassium-deficient media resulted in the generation of dormant ‘non-culturable’ cells. Addition of a moderate concentration of rifampicin enabled to kill a minor subpopulation of actively replicating bacilli and obtain a homogeneous ‘zero-CFU’ population of dormant cells characterized by total inability to produce colonies on solid media and by high potential to reactivate after reintroducing of potassium. An RNA-seq based transcriptome analysis of this dormant ‘zero-CFU’ population revealed a 30-50 fold decrease of the total level of mRNA in the cells, indicating global shift-down of gene expression level. The residual scarce protein-coding transcriptome of the dormant cells showed decreased abundance of mRNAs encoding ribosomal proteins and enzymes of TCA cycle and respiratory chain, and increased abundance of mRNAs encoding PE-PGRS proteins. Interestingly, the transcriptome of dormant cells showed little changes during several days of persistence. This stability of ‘dormant’ transcripts may reflect their readiness for translation upon resuscitation process. Transition of M. tuberculosis cells to dormancy was accompanied by the cleavage of 23S ribosomal RNA at a specific point, located outside the ribosome catalytic center. Another feature of the ‘dormant’ transcriptome was an increased abundance of non-coding transcripts. Enrichment of "dormant transcriptome" by small non-coding RNAs playing regulatory function in the cell probably indicates their role in transition to and maintenance of dormant ‘non-culturable’ state.

ORGANISM(S): Mycobacterium tuberculosis

PROVIDER: GSE66408 | GEO | 2015/06/01

SECONDARY ACCESSION(S): PRJNA276810

REPOSITORIES: GEO

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Project description:Dormant cells of Mycobacterium tuberculosis, in addition to low metabolic activity and a high level of drug resistance, are characterized by ‘non-culturability’ – a specific reversible state of the inability of the cells to grow on solid media. The biochemical characterization of this physiological state of the pathogen is only superficial, pending clarification of the metabolic processes that may exist in such cells. In this study, applying LC-MS proteomic profiling, we report the analysis of proteins accumulated in dormant, ‘non-culturable’ M. tuberculosis cells in an in vitro model of self-acidification of mycobacteria in the post-stationary phase, simulating the in vivo persistence conditions. This approach revealed the preservation of 1379 proteins in cells after 5 months of storage in dormancy; among them, 468 proteins were statistically different from those in the actively growing cells and bore a positive fold change (FC). Differential analysis revealed the proteins of the pH-dependent regulatory system PhoP and allowed the reconstruction of the reactions of central carbon/glycerol metabolism, as well as revealing the salvaged pathways of mycothiol and UMP biosynthesis, establishing the cohort of survival enzymes of dormancy. The annotated pathways mirror the adaptation of the mycobacterial metabolic machinery to life within lipid-rich macrophages: especially the involvement of the methyl citrate and glyoxylate pathways. Thus, the current in vitro model of M. tuberculosis self-acidification reflects the biochemical adaptation of these bacteria to persistence in vivo. Comparative analysis with published proteins displaying antigenic properties makes it possible to distinguish immunoreactive proteins among the proteins bearing a positive FC in dormancy, which may include specific antigens of latent tuberculosis. Additionally, the biotransformatory enzymes (oxidoreductases and hydrolases) capable of prodrug activation and stored up in the dormant state were annotated. These findings may potentially lead to the discovery of immunodiagnostic tests for early latent tuberculosis and trigger the discovery of efficient drugs/prodrugs with potency against non-replicating, dormant populations of mycobacteria.

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Mycobacterium tuberculosis transcriptome on various phases of dormancy in potassium-deficient model

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