Research
Profiling the Proteome of Mycobacterium tuberculosis during Dormancy and Reactivation*

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Tuberculosis, caused by Mycobacterium tuberculosis, still remains a major global health problem. The main obstacle in eradicating this disease is the ability of this pathogen to remain dormant in macrophages, and then reactivate later under immuno-compromised conditions. The physiology of hypoxic nonreplicating M. tuberculosis is well-studied using many in vitro dormancy models. However, the physiological changes that take place during the shift from dormancy to aerobic growth (reactivation) have rarely been subjected to a detailed investigation. In this study, we developed an in vitro reactivation system by re-aerating the virulent laboratory strain of M. tuberculosis that was made dormant employing Wayne's dormancy model, and compared the proteome profiles of dormant and reactivated bacteria using label-free one-dimensional LC/MS/MS analysis. The proteome of dormant bacteria was analyzed at nonreplicating persistent stage 1 (NRP1) and stage 2 (NRP2), whereas that of reactivated bacteria was analyzed at 6 and 24 h post re-aeration. Proteome of normoxially grown bacteria served as the reference. In total, 1871 proteins comprising 47% of the M. tuberculosis proteome were identified, and many of them were observed to be expressed differentially or uniquely during dormancy and reactivation. The number of proteins detected at different stages of dormancy (764 at NRP1, 691 at NRP2) and reactivation (768 at R6 and 983 at R24) was very low compared with that of the control (1663). The number of unique proteins identified during normoxia, NRP1, NRP2, R6, and R24 were 597, 66, 56, 73, and 94, respectively. We analyzed various biological functions during these conditions. Fluctuation in the relative quantities of proteins involved in energy metabolism during dormancy and reactivation was the most significant observation we made in this study. Proteins that are up-regulated or uniquely expressed during reactivation from dormancy offer to be attractive targets for therapeutic intervention to prevent reactivation of latent tuberculosis.

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Author contributions: V.G., S.R., R.L.G., L.J., and R.A.K. designed research; V.G., S.R., L.J., A.S., R.R., A.R.P., and R.L.G. performed research; V.G., S.R., R.L.G., L.J., A.S., and A.J. contributed new reagents or analytic tools; V.G., S.R., R.L.G., L.J., R.R., A.R.P., and R.A.K. analyzed data; V.G., S.R., L.J., R.R., A.R.P., S.M., A.J., and R.A.K. wrote the paper.

Additional information: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001158 and DOI 10.6019/PXD001158. Supplemental data containing details such as uniquely expressed, up-regulated and down-regulated proteins, and their functional classification are available online through the MCP website.

    1 The abbreviations used are:

    TB

    Tuberculosis

    MTB

    Mycobacterium tuberculosis

    BCG

    Bacillus Calmette–Guérin

    NRP1

    Nonreplicating persistent stage 1

    NRP2

    Nonreplicating persistent stage 2

    R6

    6 hours after Reactivation

    R24

    24 hours after Reactivation

    LC/MS/MS

    Liquid chromatography – tandem mass spectrometry

    GO

    Gene Ontology

    DAVID

    Database for Annotation, Visualization and Integrated Discovery

    PBS

    Phosphate buffered saline

    MPDS

    MassPREP™ Digestion Standard

    CFU

    Colony forming unit.

*

This study has been funded by the Department of Biotechnology, Government of India [BT/PR5361/MED/29/507/2012 (RAK)]. SR thanks Department of Science and Technology, Government of India, for INSPIRE fellowship. RLG and LJ acknowledge Council for Scientific and Industrial Research, Government of India, and RR acknowledges University Grants Commission, Government of India, for research fellowship. We are grateful to the PRIDE Team for assistance in MS/MS data distribution.

This article contains supplemental Lists S1 to S5.

Vipin Gopinath and Sajith Raghunandanan have contributed equally to this work.