Biosensor-integrated transposon mutagenesis reveals rv0158 as a coordinator of redox homeostasis in Mycobacterium tuberculosis
- Indian Institute of Science Bangalore, India
- National Centre for Biological Sciences, India
- Institute of Mathematical Sciences, India
- Institute for Stem Cell Science and Regenerative Medicine, India
- 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.
Article and author information
Author details
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.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
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
Further reading
-
- Microbiology and Infectious Disease
Non-inheritable antibiotic or phenotypic resistance ensures bacterial survival during antibiotic treatment. However, exogenous factors promoting phenotypic resistance are poorly defined. Here, we demonstrate that Vibrio alginolyticus are recalcitrant to killing by a broad spectrum of antibiotics under high magnesium. Functional metabolomics demonstrated that magnesium modulates fatty acid biosynthesis by increasing saturated fatty acid biosynthesis while decreasing unsaturated fatty acid production. Exogenous supplementation of unsaturated and saturated fatty acids increased and decreased bacterial susceptibility to antibiotics, respectively, confirming the role of fatty acids in antibiotic resistance. Functional lipidomics revealed that glycerophospholipid metabolism is the major metabolic pathway remodeled by magnesium, where phosphatidylethanolamine biosynthesis is reduced and phosphatidylglycerol production is increased. This process alters membrane composition, increasing membrane polarization, and decreasing permeability and fluidity, thereby reducing antibiotic uptake by V. alginolyticus. These findings suggest the presence of a previously unrecognized metabolic mechanism by which bacteria escape antibiotic killing through the use of an environmental factor.
-
- Microbiology and Infectious Disease
Because of high mutation rates, viruses constantly adapt to new environments. When propagated in cell lines, certain viruses acquire positively charged amino acids on their surface proteins, enabling them to utilize negatively charged heparan sulfate (HS) as an attachment receptor. In this study, we used enterovirus A71 (EV-A71) as model and demonstrated that unlike the parental MP4 variant, the cell-adapted strong HS-binder MP4-97R/167G does not require acidification for uncoating and releases its genome in the neutral or weakly acidic environment of early endosomes. We experimentally confirmed that this pH-independent entry is not associated with the use of HS as an attachment receptor but rather with compromised capsid stability. We then extended these findings to another HS-dependent strain. In summary, our data indicate that acquisition of capsid mutations conferring affinity for HS come together with decreased capsid stability and allow EV-A71 to enter the cell via a pH-independent pathway. This pH-independent entry mechanism boosts viral replication in cell lines but may prove deleterious in vivo, especially for enteric viruses crossing the acidic gastric environment before reaching their primary replication site, the intestine. Our study thus provides new insight into the mechanisms underlying the in vivo attenuation of HS-binding EV-A71 strains. Not only are these viruses hindered in tissues rich in HS due to viral trapping, as generally accepted, but our research reveals that their diminished capsid stability further contributes to attenuation in vivo. This underscores the complex relationship between HS-binding, capsid stability, and viral fitness, where increased replication in cell lines coincides with attenuation in harsh in vivo environments like the gastrointestinal tract.
