Phenotypically drug-tolerant Mycobacterium tuberculosis ( Mtb ) subpopulations within macrophages delay bacterial clearance, contributing to prolonged therapy and treatment failure. Here, we identify phagosomal acidification as a metabolic control point linking host lipid metabolism, bacterial redox homeostasis, and antibiotic tolerance. Acidic phagosomes promote lipid droplet (LD) biogenesis in macrophages, increasing lipid availability to intracellular Mtb . Access to host lipids enables Mtb to maintain a reductive cytoplasmic redox state that supports drug tolerance. Chloroquine (CQ)-mediated phagosomal alkalinization disrupted this pH–LD axis, reducing LD accumulation, bacterial lipid access, and redox-associated drug tolerance in both H37Rv and the multidrug-resistant clinical isolate NHN1664. Transcriptomic profiling of intraphagosomal Mtb identified the Fe–S cluster transcription factor WhiB6 as a candidate regulator linking phagosomal pH, host lipid availability, and reductive stress. In C3HeB/FeJ mice infected with NHN1664, CQ monotherapy attenuated lung fibrosis and improved pulmonary function without affecting bacterial burden. In this model, where necrotic and fibrotic lesions limit anti-tuberculosis drug efficacy, moxifloxacin (MXF) alone had little effect on bacterial burden, whereas CQ–MXF combination therapy significantly improved bacterial clearance. Collectively, these findings identify phagosomal acidification as a central regulator of lipid-driven redox adaptation and establish CQ as a promising host-directed adjunct to improve tuberculosis chemotherapy.

Treatment of tuberculosis (TB) is faced with several challenges including the long treatment duration, drug toxicity and tissue pathology. Host-directed therapy provides promising avenues to find compounds for adjunctively assisting antimycobacterials in the TB treatment regimen, by promoting pathogen eradication or limiting tissue destruction. Eicosanoids are a class of lipid molecules that are potent mediators of inflammation and have been implicated in aspects of the host response against TB. Here, we have explored the blood transcriptome of pulmonary TB patients to understand the activity of leukotriene B4, a pro-inflammatory eicosanoid. Our study shows a significant upregulation in the leukotriene B4 signalling pathway in active TB patients, which is reversed with TB treatment. We have further utilized our in-house network analysis algorithm, ResponseNet, to identify potential downstream signal effectors of leukotriene B4 in TB patients including STAT1/2 and NADPH oxidase at a systemic as well as local level, followed by experimental validation of the same. Finally, we show the potential of inhibiting leukotriene B4 signalling as a mode of adjunctive host-directed therapy against TB. This study provides a new mode of TB treatment along with mechanistic insights which can be further explored in pre-clinical trials.

Viruses exploit host metabolism for replication and immune-regulation. 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), resulting in diminished endogenous H2S levels. Inhibition of CTH resulted in SARS-CoV-2 proliferation. A slow-releasing H2S donor, GYY4137, diminished virus replication by inducing Nrf2/Keap1 pathway, restoring redox balance and mitochondrial bioenergetics. Treatment of SARS-CoV-2-infected animals with GYY4137 suppressed viral replication, ameliorated respiratory pathology, and restored antioxidant gene expression. Notably, whole-body plethysmography showed improved pulmonary function variables, including pulmonary obstruction and end-expiratory pause upon GYY4137 treatment in vivo. Data 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. ### Competing Interest Statement The authors have declared no competing interest.

The ability of Mycobacterium tuberculosis (Mtb) to tolerate nitric oxide (•NO) and superoxide (O2•−) produced by phagocytes contributes to its success as a human pathogen. Recombination of •NO and O2•− generates peroxynitrite (ONOO−), a potent oxidant produced inside activated macrophages causing lethality in diverse organisms. While the response of Mtb toward •NO and O2•− is well established, how Mtb responds to ONOO− remains unclear. Filling this knowledge gap is important to understand the persistence mechanisms of Mtb during infection. We synthesized a series of compounds that generate both •NO and O2•−, which should combine to produce ONOO−. From this library, we identified CJ067 that permeates Mtb to reliably enhance intracellular ONOO− levels. CJ067-exposed Mtb strains, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates, exhibited dose-dependent, long-lasting oxidative stress and growth inhibition. In contrast, Mycobacterium smegmatis (Msm), a fast-growing, non-pathogenic mycobacterial species, maintained redox balance and growth in response to intracellular ONOO−. RNA-sequencing with Mtb revealed that CJ067 induces antioxidant machinery, sulphur metabolism, metal homeostasis, and a 4Fe–4S cluster repair pathway (suf operon). CJ067 impaired the activity of the 4Fe–4S cluster-containing TCA cycle enzyme, aconitase, and diminished bioenergetics of Mtb. Work with Mtb strains defective in SUF and IscS involved in Fe–S cluster biogenesis pathways showed that both systems cooperatively protect Mtb from intracellular ONOO− in vitro and inducible nitric oxide synthase (iNOS)-dependent growth inhibition during macrophage infection. Thus, Mtb is uniquely sensitive to intracellular ONOO− and targeting Fe–S cluster homeostasis is expected to promote iNOS-dependent host immunity against tuberculosis (TB).

ABSTRACT Mycobacterium tuberculosis employs several signaling pathways to regulate its cellular physiology and survival within the host. Mycobacterial genomes encode multiple adenylyl cyclases and cAMP effector proteins, underscoring the diverse ways in which these bacteria utilize cAMP. We have earlier identified universal stress proteins (USP), Rv1636 and MSMEG_3811 in M. tuberculosis and M. smegmatis respectively, as abundantly expressed, novel cAMP-binding proteins. In this study, we show that these USPs may function to regulate cAMP signaling by direct sequestration of the second messenger. In slow-growing mycobacteria, concentrations of Rv1636 were equivalent to the amounts of cAMP present in the cell, and overexpression of Rv1636 in M. smegmatis increased levels of ‘bound’ cAMP. Rv1636 is secreted via the SecA2 secretion system in M. tuberculosis but is not directly responsible for the efflux of cAMP from the cell. While msmeg_3811 could be readily deleted from the genome of M. smegmatis , we find that the rv1636 gene is essential for growth of M. tuberculosis , and this functionality depends on the cAMP-binding ability of Rv1636. This is the first evidence of a ‘sink’ for any second messenger in bacterial signaling that would allow mycobacterial cells to regulate the available intracellular ‘free’ pool of cAMP.

Mining large-scale data to discover biologically relevant information remains a challenge despite the rapid development of bioinformatics tools. Here, we have developed a new tool, PathTracer, to identify biologically relevant information flows by mining genome-wide protein-protein interaction networks following integration of gene expression data. PathTracer successfully mines interactions between genes and traces the most perturbed paths of perceived activities under the conditions of the study. We further demonstrated the utility of this tool by identifying adaptation mechanisms of hypoxia-induced dormancy in Mycobacterium tuberculosis (Mtb).

Iron-sulfur (Fe-S) biogenesis requires multiprotein assembly systems, SUF and ISC, in most prokaryotes. M. tuberculosis ( Mtb ) encodes a complete SUF system, the depletion of which was bactericidal. The ISC operon is truncated to a single gene iscS (cysteine desulfurase), whose function remains uncertain. Here, we show that Mtb Δ iscS is bioenergetically deficient and hypersensitive to oxidative stress, antibiotics, and hypoxia. Mtb Δ iscS 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 Mtb , Mtb Δ iscS could not enter a stable persistent state, continued replicating in mice, and showed hypervirulence. The suf operon was overexpressed in Mtb Δ iscS during infection in a NO-dependent manner. Suppressing suf expression in Mtb Δ iscS either by CRISPR interference or upon infection in inducible NO-deficient mice arrests hypervirulence. Together, Mtb redesigned the ISC system to “fine-tune” the expression of SUF machinery for establishing persistence without causing detrimental disease in the host.

Mycobacterium tuberculosis (Mtb) is evolutionarily equipped to resist exogenous reactive oxygen species (ROS) 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.