Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis

  1. Somnath Shee
  2. Reshma T Veetil
  3. Karthikeyan Mohanraj
  4. Mayashree Das
  5. Nitish Malhotra
  6. Devleena Bandopadhyay
  7. Hussain Beig
  8. Shalini Birua
  9. Shreyas Niphadkar
  10. Sathya Narayanan Nagarajan
  11. Vikrant Kumar Sinha
  12. Chandrani Thakur
  13. Raju S Rajmani
  14. Nagasuma Chandra
  15. Sunil Laxman
  16. Mahavir Singh
  17. Areejit Samal
  18. Aswin N Seshasayee
  19. Amit Singh  Is a corresponding author
  1. Indian Institute of Science Bangalore, India
  2. National Centre for Biological Sciences, India
  3. Institute of Mathematical Sciences, India
  4. Institute for Stem Cell Science and Regenerative Medicine, India
  5. Institute for Stem Cell Biology and Regenerative Medicine, India

Abstract

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species but shows vulnerability to an increase in endogenous ROS (eROS). Since eROS is an unavoidable consequence of aerobic metabolism, understanding how Mtb manages eROS levels is essential yet needs to be characterized. By combining the Mrx1-roGFP2 redox biosensor with transposon mutagenesis, we identified 368 genes (redoxosome) responsible for maintaining homeostatic levels of eROS in Mtb. Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Mtb. Disruption of rv0158 (rv0158 KO) impaired growth, redox balance, respiration, and metabolism of Mtb on glucose but not on fatty acids. Importantly, rv0158 KO exhibited enhanced growth on propionate, and the Rv0158 protein directly binds to methylmalonyl-CoA, a key intermediate in propionate catabolism. Metabolite profiling, ChIP-Seq, and gene-expression analyses indicate that Rv0158 manages metabolic neutralization of propionate toxicity by regulating the methylcitrate cycle. Disruption of rv0158 enhanced the sensitivity of Mtb to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, rv0158 KO showed poor survival in macrophages and persistence defect in mice. Our results suggest that Rv0158 is a metabolic integrator for carbon metabolism and redox balance in Mtb.

Data availability

All data generated or analysed during this study are included in the manuscript (in the materials and methods section). The TraDIS data has been submitted to NCBI's Gene Expression Omnibus (Submission ID: SUB11081305; BioProject ID: PRJNA807454). RNA Sequencing data have been submitted to NCBI's Gene Expression Omnibus (GEO, accession number GSE196844). Source Data have been provided for all Figures. Source Data contain the numerical data used to generate the figures.

The following data sets were generated

Article and author information

Author details

  1. Somnath Shee

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  2. Reshma T Veetil

    National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  3. Karthikeyan Mohanraj

    Institute of Mathematical Sciences, Chennai, India
    Competing interests
    The authors declare that no competing interests exist.
  4. Mayashree Das

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  5. Nitish Malhotra

    National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8580-9623
  6. Devleena Bandopadhyay

    Molecular Biophysics Unit, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  7. Hussain Beig

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  8. Shalini Birua

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  9. Shreyas Niphadkar

    Institute for Stem Cell Science and Regenerative Medicine, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  10. Sathya Narayanan Nagarajan

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  11. Vikrant Kumar Sinha

    Molecular Biophysics Unit, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  12. Chandrani Thakur

    Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  13. Raju S Rajmani

    Centre for Infectious Disease Research, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  14. Nagasuma Chandra

    Department of Biochemistry, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9939-8439
  15. Sunil Laxman

    Regulation of Cell fate, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0861-5080
  16. Mahavir Singh

    Molecular Biophysics Unit, Indian Institute of Science Bangalore, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  17. Areejit Samal

    Institute of Mathematical Sciences, Chennai, India
    Competing interests
    The authors declare that no competing interests exist.
  18. Aswin N Seshasayee

    National Centre for Biological Sciences, Bangalore, India
    Competing interests
    The authors declare that no competing interests exist.
  19. Amit Singh

    Department of Microbiology and Cell Biology, Indian Institute of Science Bangalore, Bangalore, India
    For correspondence
    asingh@iisc.ac.in
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6761-1664

Funding

Wellcome Trust- DBT India Alliance (IA/S/16/2/502700)

  • Amit Singh

DBT (BT/PR13522/COE/34/27/2015)

  • Amit Singh

Indian Institute of Science (Graduate Student Fellowship)

  • Somnath Shee

DBT-IISc Partnership Program (grant 22-0905-0006-05-987 436)

  • Amit Singh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

Animal experimentation: All animal studies were executed as per guidelines prescribed by the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, with approval from the Institutional Animal Ethical Committee (CAF/Ethics/544/2017- Institute animal ethical clearance number) and Biosafety Level-3 Committee.

Copyright

© 2023, Shee et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Somnath Shee
  2. Reshma T Veetil
  3. Karthikeyan Mohanraj
  4. Mayashree Das
  5. Nitish Malhotra
  6. Devleena Bandopadhyay
  7. Hussain Beig
  8. Shalini Birua
  9. Shreyas Niphadkar
  10. Sathya Narayanan Nagarajan
  11. Vikrant Kumar Sinha
  12. Chandrani Thakur
  13. Raju S Rajmani
  14. Nagasuma Chandra
  15. Sunil Laxman
  16. Mahavir Singh
  17. Areejit Samal
  18. Aswin N Seshasayee
  19. Amit Singh
(2023)
Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis
eLife 12:e80218.
https://doi.org/10.7554/eLife.80218

Share this article

https://doi.org/10.7554/eLife.80218

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