@article {161133, title = {Bioenergetic reprogramming of macrophages reduces drug tolerance in Mycobacterium tuberculosis.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Oct 23}, pages = {9370}, abstract = {

Effective clearance of Mycobacterium tuberculosis (Mtb) requires targeting drug-tolerant populations within host macrophages. Here, we show that macrophage metabolic states govern redox heterogeneity and drug response in intracellular Mtb. Using a redox-sensitive fluorescent reporter (Mrx1-roGFP2), flow cytometry, and transcriptomics, we found that macrophages with high oxidative phosphorylation (OXPHOS) and low glycolysis harbor reductive, drug-tolerant Mtb, whereas glycolytically active macrophages generate mitochondrial ROS via reverse electron transport, imposing oxidative stress on Mtb and enhancing drug efficacy. Computational and genetic analyses identified NRF2 as a key regulator linking host metabolism to bacterial redox state and drug tolerance. Pharmacological reprogramming of macrophages with the FDA-approved drug meclizine (MEC) shifted metabolism towards glycolysis, suppressed redox heterogeneity, and reduced Mtb drug tolerance in macrophages and mice. MEC exhibited no adverse interactions with frontline anti-TB drugs. These findings demonstrate the therapeutic potential of host metabolic reprogramming to overcome Mtb drug tolerance.

}, keywords = {Animals, Antitubercular Agents, Drug Tolerance, Energy Metabolism, Female, Glycolysis, Humans, Macrophages, Mice, Mice, Inbred C57BL, Mitochondria, Mycobacterium tuberculosis, NF-E2-Related Factor 2, Oxidation-Reduction, Oxidative Phosphorylation, Oxidative Stress, Reactive Oxygen Species, Tuberculosis}, issn = {2041-1723}, doi = {10.1038/s41467-025-64407-w}, author = {Yadav, Vikas and Sahoo, Sarthak and Malhotra, Nitish and Mishra, Richa and Sreedharan, Sreesa and Rajmani, Raju S and Shanmugam, Siva and Shandil, Radha K and Narayanan, Shridhar and Thacker, Vivek V and Laxman, Sunil and Jolly, Mohit Kumar and Seshasayee, Aswin Sai Narain and Singh, Amit} } @article {157491, title = {Hydrogen sulfide (H2S) coordinates redox balance, carbon metabolism, and mitochondrial bioenergetics to suppress SARS-CoV-2 infection.}, journal = {PLoS Pathog}, volume = {21}, year = {2025}, month = {2025 May 19}, pages = {e1013164}, abstract = {

Viruses modulate various aspects of host physiology, including carbon metabolism, redox balance, and mitochondrial bioenergetics to acquire the building blocks for replication and regulation of the immune response. Understanding how SARS-CoV-2 alters the host metabolism may lead to treatments for COVID-19. We report that a ubiquitous gaseous molecule, hydrogen sulfide (H2S), regulates redox, metabolism, and mitochondrial bioenergetics to control SARS-CoV-2. Virus replication is associated with down-regulation of the H2S-producing enzymes cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CTH), and 3-mercaptopyruvate sulfurtransferase (3-MST) in multiple cell lines and nasopharyngeal swabs of symptomatic COVID-19 patients. Consequently, SARS-CoV-2-infected cells showed diminished endogenous H2S levels and a protein modification (S-sulfhydration) caused by H2S. Genetic silencing or chemical inhibition of CTH resulted in SARS-CoV-2 proliferation. Chemical supplementation of H2S using a slow-releasing H2S donor, GYY4137, diminished virus replication. Using a redox biosensor, metabolomics, transcriptomics, and XF-flux analyzer, we showed that GYY4137 blocked SARS-CoV-2 replication by inducing the Nrf2/Keap1 pathway, restoring redox balance and carbon metabolites and potentiating mitochondrial oxidative phosphorylation. Treatment of SARS-CoV-2-infected mice or hamsters with GYY4137 suppressed viral replication and ameliorated lung pathology. GYY4137 treatment reduced the expression of inflammatory cytokines and re-established the expression of Nrf2-dependent antioxidant genes in the lungs of SARS-CoV-2-infected mice. Notably, non-invasive measurement of respiratory functions using unrestrained whole-body plethysmography (uWBP) of SARS-CoV-2-infected mice showed improved pulmonary function variables, including pulmonary obstruction (Penh), end-expiratory pause (EEP), and relaxation time (RT) upon GYY4137 treatment. Together, our findings significantly extend our understanding of H2S-mediated regulation of viral infections and open new avenues for investigating the pathogenic mechanisms and therapeutic opportunities for coronavirus-associated disorders.

}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1013164}, url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1013164}, author = {Agrawal, Ragini and Pal, Virender Kumar and K S, Suhas and Menon, Gopika Jayan and Singh, Inder Raj and Malhotra, Nitish and C S, Naren and Ganesh, Kailash and Rajmani, Raju S and Narain Seshasayee, Aswin Sai and Chandra, Nagasuma and Joshi, Manjunath B and Singh, Amit} } @article {133186, title = {Biosensor-integrated transposon mutagenesis reveals as a coordinator of redox homeostasis in .}, journal = {Elife}, volume = {12}, year = {2023}, month = {2023 Aug 29}, abstract = {

() 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 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 . Integrating redoxosome with a global network of transcriptional regulators revealed a hypothetical protein (Rv0158) as a critical node managing eROS in Disruption of ( KO) impaired growth, redox balance, respiration, and metabolism of on glucose but not on fatty acids. Importantly, 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 enhanced the sensitivity of to oxidative stress, nitric oxide, and anti-TB drugs. Lastly, 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 .

}, issn = {2050-084X}, doi = {10.7554/eLife.80218}, author = {Shee, Somnath and Veetil, Reshma T and Mohanraj, Karthikeyan and Das, Mayashree and Malhotra, Nitish and Bandopadhyay, Devleena and Beig, Hussain and Birua, Shalini and Niphadkar, Shreyas and Nagarajan, Sathya Narayanan and Sinha, Vikrant Kumar and Thakur, Chandrani and Rajmani, Raju S and Chandra, Nagasuma and Laxman, Sunil and Singh, Mahavir and Samal, Areejit and Seshasayee, Aswin N and Singh, Amit} } @article {137347, title = {Cysteine desulfurase (IscS)-mediated fine-tuning of bioenergetics and SUF expression prevents hypervirulence.}, journal = {Sci Adv}, volume = {9}, year = {2023}, month = {2023 Dec 15}, pages = {eadh2858}, abstract = {

Iron-sulfur (Fe-S) biogenesis requires multiprotein assembly systems, SUF and ISC, in most prokaryotes. () encodes a complete SUF system, the depletion of which was bactericidal. The ISC operon is truncated to a single gene (cysteine desulfurase), whose function remains uncertain. Here, we show that Δ is bioenergetically deficient and hypersensitive to oxidative stress, antibiotics, and hypoxia. Δ resisted killing by nitric oxide (NO). RNA sequencing indicates that IscS is important for expressing regulons of DosR and Fe-S-containing transcription factors, WhiB3 and SufR. Unlike wild-type , Δ could not enter a stable persistent state, continued replicating in mice, and showed hypervirulence. The operon was overexpressed in Δ during infection in a NO-dependent manner. Suppressing expression in Δ either by CRISPR interference or upon infection in inducible NO-deficient mice arrests hypervirulence. Together, redesigned the ISC system to "fine-tune" the expression of SUF machinery for establishing persistence without causing detrimental disease in the host.

}, keywords = {Animals, Energy Metabolism, Escherichia coli, Iron-Sulfur Proteins, Mice, Mycobacterium tuberculosis}, issn = {2375-2548}, doi = {10.1126/sciadv.adh2858}, author = {Das, Mayashree and Sreedharan, Sreesa and Shee, Somnath and Malhotra, Nitish and Nandy, Meghna and Banerjee, Ushashi and Kohli, Sakshi and Rajmani, Raju S and Chandra, Nagasuma and Seshasayee, Aswin Sai Narain and Laxman, Sunil and Singh, Amit} } @article {98081, title = {Mycobacterium tuberculosis SufR responds to nitric oxide via its 4Fe-4S cluster and regulates Fe-S cluster biogenesis for persistence in mice.}, journal = {Redox Biol}, volume = {46}, year = {2021}, month = {2021 Jul 02}, pages = {102062}, abstract = {

The persistence of Mycobacterium tuberculosis (Mtb) is a major problem in managing tuberculosis (TB). Host-generated nitric oxide (NO) is perceived as one of the signals by Mtb to reprogram metabolism and respiration for persistence. However, the mechanisms involved in NO sensing and reorganizing Mtb{\textquoteright}s physiology are not fully understood. Since NO damages iron-sulfur (Fe-S) clusters of essential enzymes, the mechanism(s) involved in regulating Fe-S cluster biogenesis could help Mtb persist in host tissues. Here, we show that a transcription factor SufR (Rv1460) senses NO via its 4Fe-4S cluster and promotes persistence of Mtb by mobilizing the Fe-S cluster biogenesis system; suf operon (Rv1460-Rv1466). Analysis of anaerobically purified SufR by UV-visible spectroscopy, circular dichroism, and iron-sulfide estimation confirms the presence of a 4Fe-4S cluster. Atmospheric O and HO gradually degrade the 4Fe-4S cluster of SufR. Furthermore, electron paramagnetic resonance (EPR) analysis demonstrates that NO directly targets SufR 4Fe-4S cluster by forming a protein-bound dinitrosyl-iron-dithiol complex. DNase I footprinting, gel-shift, and in vitro transcription assays confirm that SufR directly regulates the expression of the suf operon in response to NO. Consistent with this, RNA-sequencing of MtbΔsufR demonstrates deregulation of the suf operon under NO stress. Strikingly, NO inflicted irreversible damage upon Fe-S clusters to exhaust respiratory and redox buffering capacity of MtbΔsufR. Lastly, MtbΔsufR failed to recover from a NO-induced non-growing state and displayed persistence defect inside immune-activated macrophages and murine lungs in a NO-dependent manner. Data suggest that SufR is a sensor of NO that supports persistence by reprogramming Fe-S cluster metabolism and bioenergetics.

}, issn = {2213-2317}, doi = {10.1016/j.redox.2021.102062}, author = {Anand, Kushi and Tripathi, Ashutosh and Shukla, Kaustubh and Malhotra, Nitish and Jamithireddy, Anil Kumar and Jha, Rajiv Kumar and Chaudhury, Susmit Narayan and Rajmani, Raju S and Ramesh, Arati and Nagaraja, Valakunja and Gopal, Balasubramanian and Nagaraju, Ganesh and Narain Seshayee, Aswin Sai and Singh, Amit} } @article {67836, title = {Targeting redox heterogeneity to counteract drug tolerance in replicating .}, journal = {Sci Transl Med}, volume = {11}, year = {2019}, month = {2019 Nov 13}, abstract = {

The capacity of () to tolerate multiple antibiotics represents a major problem in tuberculosis (TB) management. Heterogeneity in populations is one of the factors that drives antibiotic tolerance during infection. However, the mechanisms underpinning this variation in bacterial population remain poorly understood. Here, we show that phagosomal acidification alters the redox physiology of to generate a population of replicating bacteria that display drug tolerance during infection. RNA sequencing of this redox-altered population revealed the involvement of iron-sulfur (Fe-S) cluster biogenesis, hydrogen sulfide (HS) gas, and drug efflux pumps in antibiotic tolerance. The fraction of the pH- and redox-dependent tolerant population increased when infected macrophages with actively replicating HIV-1, suggesting that redox heterogeneity could contribute to high rates of TB therapy failure during HIV-TB coinfection. Pharmacological inhibition of phagosomal acidification by the antimalarial drug chloroquine (CQ) eradicated drug-tolerant , ameliorated lung pathology, and reduced postchemotherapeutic relapse in in vivo models. The pharmacological profile of CQ ( and AUC) exhibited no major drug-drug interaction when coadministered with first line anti-TB drugs in mice. Our data establish a link between phagosomal pH, redox metabolism, and drug tolerance in replicating and suggest repositioning of CQ to shorten TB therapy and achieve a relapse-free cure.

}, issn = {1946-6242}, doi = {10.1126/scitranslmed.aaw6635}, author = {Mishra, Richa and Kohli, Sakshi and Malhotra, Nitish and Bandyopadhyay, Parijat and Mehta, Mansi and Munshi, MohamedHusen and Adiga, Vasista and Ahuja, Vijay Kamal and Shandil, Radha K and Rajmani, Raju S and Seshasayee, Aswin Sai Narain and Singh, Amit} }