
==== Front
Redox Biol
Redox Biol
Redox Biology
2213-2317
Elsevier

S2213-2317(24)00263-5
10.1016/j.redox.2024.103285
103285
Review Article
Intracellular peroxynitrite perturbs redox balance, bioenergetics, and Fe–S cluster homeostasis in Mycobacterium tuberculosis
Dewan Arshiya a
Jain Charu b
Das Mayashree a
Tripathi Ashutosh a
Sharma Ajay Kumar b
Singh Harshit b
Malhotra Nitish c
Seshasayee Aswin Sai Narain c
Chakrapani Harinath harinath@iiserpune.ac.in
b⁎⁎
Singh Amit asingh@iisc.ac.in
a⁎
a Department of Microbiology and Cell Biology, Centre for Infectious Disease Research, Indian Institute of Science, Bengaluru, 560012, India
b Department of Chemistry, Indian Institute of Science Education and Research, Pune, 411008, India
c National Center for Biological Sciences, Bengaluru, 560065, India
⁎ Corresponding author. asingh@iisc.ac.in
⁎⁎ Corresponding author. harinath@iiserpune.ac.in
31 7 2024
9 2024
31 7 2024
75 10328526 6 2024
21 7 2024
23 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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).

Graphical abstract

Image 1

Keywords

Peroxynitrite
Tuberculosis
Fe–S clusters
Redox
Bioenergetics
==== Body
pmc List of abbreviations

Abbreviation	Definition	
Mtb	Mycobacterium tuberculosis	
Msm	Mycobacterium smegmatis	
ONOO−	Peroxynitrite	
•NO	Nitric oxide	
O2•−	Superoxide	
iNOS	Inducible nitric oxide synthase	
AD	Authentic donor; sodium salt of peroxynitrite	
MDR	Multi-drug resistant	
XDR	Extensively-drug resistant	
MIC	Minimum inhibitory concentration	
MBC	Minimum bactericidal concentration	
OCR	Oxygen consumption rate	
ECAR	Extracellular acidification rate	
SRC	Spare respiratory capacity	
NaC	N-acetyl cysteine	
BMDMs	Bone-marrow derived macrophages	

1 Introduction

Most people infected with Mycobacterium tuberculosis (Mtb) remain asymptomatic, indicating that host immunity effectively suppresses the pathogen's ability to multiply without eradicating the bacterium. In this context, the ability to produce reactive nitrogen species (RNS), such as nitric oxide (•NO), nitrogen dioxide (•NO2), and peroxynitrite (ONOO−), via host-inducible nitric oxide synthase (iNOS) contributes to tuberculosis (TB) control in mice [[1], [2], [3], [4]]. Failure to express iNOS correlates with Mtb multiplication and early death of infected mice [2]. Moreover, host-derived RNS diminish Mtb metabolism, reducing the efficacy of anti-TB drugs in murine macrophages [5]. However, evidence for RNS controlling Mtb in humans remains inconclusive. For example, alveolar macrophages of Mtb-infected humans express functional iNOS and correlative evidence indicates that iNOS may contribute to the human control of TB [5]. However, human macrophages produce low levels of RNS that, rather than inhibiting growth, promote Mtb survival [6]. Moreover, a recent study proposed that Mtb-derived nitrite, rather than host-derived RNS, is able to suppress the growth and metabolism of Mtb during infection [7]. Thus, additional work is needed to understand how Mtb responds to RNS and their role in TB.

During infection, macrophages target intracellular pathogens by assembling NADPH-oxidase (NOX2) at the interior of the phagosome membrane to produce superoxide (O2•−) [8]. Concurrently, induction of iNOS generates •NO in the cytosol, which readily diffuses into phagosomes [8]. O2•− is a charged molecule that cannot cross the cytoplasmic membrane of the intracellular pathogen, unlike •NO. However, •NO mainly inhibits respiration by reversible targeting of the heme enzymes [9,10]. •NO arrests the aerobic respiration and slows the growth of Mtb without killing the pathogen, even at a concentration of 5 mM [10]. Moreover, Mtb can survive millimolar concentrations of hydrogen peroxide (H2O2) that, unlike O2•−, easily crosses biological membranes and is generated by the dismutation of O2•− by superoxide dismutases (SODs) [11]. Therefore, it is plausible that the toxic effects of O2•− and •NO are mediated by their reaction by-products. For example, •NO reacts with O2•− in close proximity within the phagosome at a diffusion-controlled reaction (equation (1)) in which k1 = 1.9 × 1010 M−1s−1 for generation of ONOO− [12].… (equation 1) •NO + O2•− → ONOO− … … … … … … … … … … … … … … … … … … … …

ONOO− readily crosses cytoplasmic membranes, and unlike •NO promptly kills bacterial cells [[13], [14], [15], [16], [17], [18], [19]]. Consistent with this assertion, mice deficient in producing ONOO− (gp91phox−/−/NOS2−/−) die of spontaneous infections with commensal organisms at elevated frequency [20]. Surprisingly, Mtb is not killed by exogeneous donors of ONOO− anion [21]. These findings suggest that the ability to cope with exogenous ONOO− could be why host-derived RNS do not eradicate Mtb during infection. In accordance with this, cell wall-associated lipids (PDIMs), antioxidant buffers (ergothioneine, mycothiol, and thioredoxins), enzymes (KatG), peroxiredoxins (AhpD), hemoglobin (GlbO and HbN), and the Fe–S cluster repair system protect mycobacteria from RNS [[22], [23], [24], [25], [26], [27], [28]]. While such findings indicate that Mtb is naturally equipped to mitigate toxicity associated with exogenous RNS, its exceptional sensitivity towards the anti-TB drug pretomanid (PA-824), which delivers des-nitroimidazole and •NO to the mycobacterial cytoplasm, exposes the pathogen's vulnerability to agents that deliver RNS intracellularly [29]. Until now, majority of studies on RNS remain focussed on identifying mechanisms exploited by Mtb to counteract host-generated exogeneous RNS. Therefore, we have very little information on how Mtb responds to elevation of intracellular RNS levels. Filling this knowledge gap is important to identify new pathways specifically targeted by ONOO− to induce lethality in Mtb. This will open up fresh avenues of redox research about the development of RNS-generating anti-TB drugs.

We have previously shown that Mtb is uniquely sensitive to agents that generate intracellular O2•− [30]. Using a non-invasive biosensor of mycobacterial redox potential (Mrx1-roGFP2), we reported that the cytoplasmic redox balance of Mtb strains, including drug-resistant patient isolates, is susceptible to perturbation by intracellular O2•− [24,25]. Here, we explored how Mtb responds to intracellular ONOO−. Studies in this area are difficult due to the short half-life, poor bioavailability, and uncontrolled spatio-temporal generation of ONOO− by commercially available ONOO− donors (e.g., SIN-1 (3-morpholinosydnonimine) and authentic donor (AD) i.e. the sodium salt of ONOO−) [21,[31], [32], [33], [34]]. Moreover, culture media components scavenge oxidants, thereby precluding precise assessment of cellular responses toward ONOO− donors [35]. In addition, the outer membrane permeability barrier limits the permeability of ONOO− donors into Mtb. To circumvent these issues, we previously reported that a novel small molecule, HyPR-1, generates ONOO− upon activation by a cellular enzyme, NADH:quinone reductase, in mammalian cells [36]. This compound has a quinone moiety that undergoes either one-electron (1e−) or two-electron (2e−) bio-reduction processes, forming intermediates I and II, respectively. Subsequent oxidation of these intermediates leads to the formation of O2•−. Additionally, intermediate II undergoes a rearrangement reaction, producing free diazeniumdiolate, which releases up to 2 moles of •NO. The rapid combination of •NO and O2•− within the cellular environment results in the formation of ONOO− (Scheme 1). However, HyPR-1 was relatively inefficient in delivering ONOO− into bacterial cells including mycobacteria. We have now modified HyPR-1 to generate a novel enzyme-coupled ONOO− donor CJ067 that shows enhance fluxes of ONOO− inside mycobacteria.Scheme 1 Design of ONOO−donors: Compounds in this series are expected to permeate cells to undergo either 1 or 2 e−reduction to produce superoxide radical anion(s) and intermediate II, which can undergo rearrangement to produce the free diazeniumdiolate that generate • NO. The combination of • NO and O2•− is rapid and should produce ONOO−. The rate and efficiency of ONOO− generation will depend on cell permeability, rate of bioreduction and rate of • NO generation. HyPR-1 is R = Me.

Scheme 1

The present work used chemical, enzymatic, genetic, and expression-based assays to investigate the ability of CJ067 to (i) enhance intracellular ONOO− levels, (ii) perturb redox homeostasis and bioenergetics, (iii) remodel the bacterial transcriptome, and (iv) identify mechanisms of ONOO− resistance in mycobacteria. These mechanistic findings open new avenues for the study for RNS that can be exploited to augment iNOS-dependent host immunity against Mtb.

2 Materials and methods

2.1 Bacterial strains and culture conditions

Mycobacterium tuberculosis (Mtb-H37Rv), drug-resistant patient isolates [30,37], Mycobacterium smegmatis (Msm mc2155) and other mutant strains were cultured in Middlebrook 7H9-media (Becton, Dickinson and Company, USA) supplemented with 10 % ADS (albumin, dextrose & sodium chloride), 0.2 % glycerol and 0.05 % tween-80 (with 50 μg/mL hygromycin (MP Biomedicals, Santa Ana, CA) and 25 μg/mL kanamycin (Amresco, USA) or as required) with shaking at 180 rpm in a shaker incubator (Lab Therm LT-X, Kuhner, Basel, Switzerland) at 37°C till exponential phase or till OD600nm specified for each experiment. The Mtb ΔiscS strain was a gift from S. T. Cole (Pasteur Institute, Paris, France). Mtb sufS-KD and Mtb ΔiscS-sufS-KD were generated in the lab in a previous publication [38]. Drug-resistant clinical isolates Jal 1934, Jal 2287, Jal 2261, Myc 431 and BND 320 were kind gifts from Dr. Kanury V.S. Rao (International Centre for Genetic Engineering and Biotechnology, Delhi, India).

2.2 Intracellular peroxynitrite detection using Dye-1

Time course of peroxynitrite generation was measured using a boronic ester probe (Dye-1) [39,40]. Mtb-H37Rv was grown till exponential phase (OD600nm ∼ 0.6). Mtb culture was diluted to an OD600nm of 0.4 and incubated with Dye-1 (25 μM). Bacteria alone and bacteria (treated with the dye) were incubated at 37°C for 15 min in the dark. 200 μL of culture was dispensed in a 96-well flat bottom plate and treated with 25 μM of CJ067, JCH, Menadione (Sigma-Aldrich, St. Louis, MO), SIN-1 (Thermo Fisher Scientific), sodium salt of peroxynitrite (authentic donor; AD) or other controls in triplicates. CJ067, JCH, Menadione, SIN-1 was prepared in DMSO. 10 mM stock solution of AD was prepared using the reported method [41] and stored in 1N NaOH. Wells containing bacteria with only dye were taken as a control. Fluorescence intensity was measured immediately at 1 min intervals for 30 min in a SpectraMax M3 plate reader in top-reading mode with excitation at 315 nm and emission at 460 nm.

2.3 Confocal microscopy

Mtb-H37Rv was grown till OD600nm of 0.4 and treated with 25 μM Ds-DAB for 30 min at 37°C. The culture was then treated for 1 h at 37°C with different concentrations of CJ067, taking SIN-1 as a positive control. After treatment, the cells were pelleted down at 5000 rpm for 5 min. The pellet was resuspended in 1X PBS. The mixture was then fixed with an equal volume of 4 % PFA. Cells were pelleted and finally resuspended in 1X PBS. After preparing single-cell suspension, 20 μL of the sample was applied to glass slides. The spot was air-dried before application of antifade (ProLong™ Diamond Antifade Mountant, Thermo Scientific). Cover slips were then applied and sealed. The samples were imaged at 63X magnification on Confocal Zeiss LSM880 (Airyscan) at an excitation of 350 nm and emission at 505 nm.

2.4 Resazurin microtiter assay (REMA)

The minimum inhibitory concentration (MIC) for all bacterial strains was determined by REMA performed in 96-well flat bottom plates as previously described [37,38]. Mtb and Msm strains were cultured in 7H9-ADS medium till the exponential phase (OD600 ∼0.6). 100 μL of 7H9-ADS media (without tween-80) was dispensed in all wells and serial dilutions (two-fold) for the compounds were made. 105 cells were dispensed in each well. The plate was incubated at 37°C for 5 days for Mtb or 1 day for Msm. Post-incubation, 20 μL of resazurin (0.002 %) (Sigma-Aldrich, catalog no. R7017) was added per well and incubated for an additional 24 h for Mtb or 12 h for Msm. Fluorescence intensity was measured at 530 nm (excitation) and 590 nm (emission) on SpectraMax M3 plate reader (Molecular Devices, San Jose, CA). Media and cell controls were appropriately taken to account for autofluorescence and as growth control respectively. From the fluorescence values obtained, MIC was considered the minimum drug concentration that yielded at least 90 % reduction in fluorescence compared to the untreated growth control.

2.5 Determination of minimum bactericidal concentration (MBC)

10 μL of the cells treated with various concentrations of compounds in the REMA MIC plates were spotted on solid 7H11 plates supplemented with 10 % OADC (oleic acid, bovine serum albumin, dextrose, catalase and sodium chloride) and incubated at 37°C for 2–3 weeks. The concentration at which there was no visible bacterial colonies was taken as the MBC.

2.6 Estimation of oxidative shift in EMSH in Mtb using redox biosensor Mrx1-roGFP2

The protocol was adapted from Refs. [30,38,42]. Mtb-H37Rv expressing the biosensor Mrx1-roGFP2 was grown till the early log-phase (OD600nm–0.4) in 7H9-ADS medium in presence of 50 μg/mL hygromycin. The cultures were individually treated with various compounds and the biosensor response was measured by ﬂowcytometry (on BD FACS Aria) by exciting at 405 and 488 nm lasers recording constant emission at 510 nm over time. The EMSH and 405/488 ratio was calculated as previously described [43].

2.7 Growth kinetics and survival assays

Mtb-H37Rv was grown till OD600nm of 0.6, diluted to 0.06 and treated with CJ067 at various concentrations, as indicated in the results. The OD600nm of the culture was determined over a week. The cultures were plated at various time points on 7H11-OADC to determine colony-forming units (CFUs). Colonies were counted at 2–3 weeks of incubation at 37°C and enumerated. The log-fold change was calculated.

2.8 RNA isolation

Mtb-H37Rv was grown till OD600nm of 0.4 and treated with 2X MIC of CJ067 for 2 h. To both untreated and CJ067-treated cultures, 3 times the volume of 4M guanidium thiocyanate (GTC) buffer containing 1% 2-mercaptoethanol was added for fixation of bacterial RNA. Fixed bacteria were then pelleted by centrifugation at 5000 rpm for 5 min. Total RNA was isolated as previously reported [44]. Purified total RNA obtained was treated with TURBO DNA-free™ Kit (Invitrogen) to get rid of any DNA contamination.

2.9 Ribosomal RNA-depletion, cDNA library preparation and RNA-sequencing

Isolation and purification of total RNA was carried out as described previously. Concentration and quality of total RNA extracted was checked spectrophotometrically using a NanoDrop ND-1000 (Thermo Scientific). Total RNA was then subjected to mRNA enrichment by depletion of 16s and 23s rRNA using the MICROBExpress Kit (Life Technologies) and concentration of ribo-depleted RNA was quantified by QuBit RNA HS Assay Kit (Life Technologies). After fragmentation and random priming of samples, first and second strand cDNA synthesis and library preparation were carried out using NEBNextUltra Directional RNA Library Prep Kit for Illumina (New England Biolabs), according to manufacturer's protocol. The library size distribution and quality were assessed using a high sensitivity DNA Chip (Agilent Technologies). Equimolar quantities (2 nM) of all libraries were pooled and sequenced in a high throughput run on the Illumina HiSeq 2500 sequencer using 1X50 bp single-end reads and a 1 % PhiX spike-in control.

2.10 RNA-sequencing analysis

Raw reads (single end; read length = 50 bp) obtained for samples as ‘.fastq’ files. The reference genome sequence (.fna) and annotation (.gff) files for Mtb-H37Rv (accession number: NC_000962.3) were downloaded from the ncbi ftp website (“ftp.ncbi.nlm.nih.gov”). The format of the annotation file (.gff) was changed to ‘.bed’ using an in-house python script. The raw read quality was checked using the FastQC software (version v0.11.5) (Andrews S. (2010), URL:http://www.bioinformatics.babraham.ac.uk/projects/fastqc). BWA (version 0.7.12-r1039) [45] was used to index the reference genome. Reads with raw read quality≥20 were aligned using BWA aln-q option. SAMTOOLS (version 0.1.19–96b5f2294a) [46] was used to filter out the multiply mapped reads. BEDTOOLS (version 2.25.0) [47] was used to calculate the reads count per gene using the annotation file (.bed). The differential gene expression (DGE) analysis and clustering analysis (MDS plot) for the conditions were carried out using edgeR [48] as explained in the paper [49]. Genes with 10 reads were selected for each comparative analysis. The whole analysis and heatmap generation were done using RStudio version 1.1.447 with R version 3.4.4. Gene list can be found in the “Excel 1_ CJ067 RNA sequencing genes and heat map data” document in the supplementary information.

The overlap analysis of the differentially expressed genes (DEGs) with data from this paper and previous studies [4,30], the significance testing and odds ratio calculation was determined by Fisher's exact test using a two-by-two contingency table. The universal genes set was calculated based on the intersection of the total genes analyzed in all three studies. For identifying overlap between all three conditions, only those genes that were analyzed by all three studies (“common gene set” spreadsheet in the “Excel 2_Transcriptome comparison” document in the supplementary information) were considered for overlap.

2.11 Protein lysate preparation

Mtb-H37Rv was grown till OD600nm of 0.4 and treated with 2X MIC of CJ067 for 1 h. The treated culture was pelleted down and washed with 1X PBS. Further, the pellet was resuspended in 1 mL of lysis buffer (1 tablet of cOmplete™, Mini, EDTA-free Protease Inhibitor Cocktail (Roche) in 50 mL 1X PBS). Resuspended cells were subjected to bead-beating at 6.5 m/s for 45 s for a total of 7 times. Between bead-beating cycles, the vials were cooled on ice for 5 min. The lysate was centrifuged at 14000 rpm for 10 min at 4°C and the supernatant was transferred to a cryovial and snap-frozen in liquid nitrogen. Protein concentration was estimated using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific) as per the manufacturer's instructions.

2.12 Aconitase assay

Aconitase activity was measured spectrophotometrically by monitoring the time-dependent conversion of cis-aconitate to isocitrate at 25°C in a UV/visible light spectrometer at 240 nm (Thermo Fisher Scientific, Biomat 3S, USA) [47,48]. 25 mM Tris-HCl (pH 8.0), 100 mM NaCl and 50 μg Mtb protein lysate were added to a reaction volume of 1 mL. Reactions were initiated by adding 0.15 mM cis-aconitate. The reaction monitored the disappearance of cis-aconitate at λ 240 nm after every 15 s for 30 min. Absorbance at λ 240 nm was plotted against time. An extinction coefficient of 3500 M−1s−1cm−1 was used to calculate the enzymatic activity [3,38].

2.13 Immuno-blotting

Mtb-H37Rv lysate (20 μg) was mixed with Laemmli buffer, heated at 95°C for 5 min, chilled on ice for 5 min and loaded onto 12 % acrylamide gel and transferred on a PVDF membrane. The samples were subjected to western blotting with antiserum (1:5000 dilution in skimmed milk) raised by injecting rabbits with purified Aconitase [3] or Cystathionine-beta synthase [50] as an internal control. Goat anti-Rabbit IgG HRP (CST-7074) (1:10000 dilution in skimmed milk) was used as the secondary antibody. Image acquisition and analysis was done using ImageLab Software (Version 5.2.1).

2.14 Seahorse extracellular flux analysis

Protocol adapted from Ref. [51]. Mtb-H37Rv was grown till OD600nm of 0.4 in 7H9 supplemented with 0.01 % tyloxapol. Single cell suspension of the culture was prepared and adhered to the bottom of cell-tak (1 μg per well; (Corning, Corning, NY)) coated wells of XF cell culture microplate (Agilent/Seahorse Biosciences, Santa Clara, CA) at a density of 2 × 106 bacilli per well. Assays were conducted using a Seahorse XFp analyzer (Agilent Technologies, CA) in unbuffered 7H9 with 2 mg/mL glucose. Basal oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured for ∼21 min (3 readings) before the addition of indicated concentrations of CJ067 automatically through the drug ports of the sensor cartridge (Wave Software, Seahorse Biosciences). Readings were taken for ∼42 min (6 readings) post which CCCP (Sigma-Aldrich, St. Louis, MO) was injected consecutively into the ports. OCR and ECAR were measured for ∼21 min (3 readings) on each CCCP addition. Percentage changes in OCR are calculated as a percentage of the third baseline values. Figures indicate the point of addition of CJ067/CCCP in the assay.

2.15 BMDM isolation and infection

8–12 week old WT and iNOS−/− C57BL/6 female mice were scarified via cervical dislocation. BMDMs were obtained by flushing cells from the femurs and tibias of mice using 10 mL 1X PBS and 25-gauge needle under aseptic conditions, adapted from a previously described method [52]. Extracted cells were washed at 250 g for 5 min and the supernatant was discarded. Cell pellets were resuspended in 2 mL of media (DMEM high glucose with 10 mM HEPES, 1 mM Sodium Pyruvate, 2 mM Glutamine, and 10% FBS with 25 μg/mL recombinant M-CSF and Pen-strep antibiotic) and mixed gently to disaggregate clumps into single cells. Finally total cells were made up to 20 mL media volume and seeded in tissue culture Petri dishes (100 mm) and cultured for 5 days. BMDMs were given additional media on day 3 of culturing. BMDMs were plated into 96-well plates with 4 × 104 macrophages/well and were allowed to adhere for 24 h. BMDMs were then infected with different strains of Mtb-H37Rv (WT, MtbΔiscS, Mtb sufS-KD (+ATc), and Mtb ΔiscS-sufS-KD (+ATc)) at a multiplicity of infection of 5. After a 4 h phagocytosis period, infected BMDMs were washed with 1X PBS before replacing it with fresh media. For CFU enumeration at different time points, infected BMDMs were washed with 1X PBS and lysed in water containing 0.06 % SDS for 10 min. Suitable serial dilutions of lysed cells were prepared in 7H9 media with 0.05 % Tween-80 and plated onto 7H11 plates with for 3 weeks.

3 Results

3.1 Synthesis of intracellular ONOO− donors and enzyme-catalyzed peroxynitrite generation

Compounds of the series 1 were prepared using a two-step protocol (Scheme 2). First, bromide 3 was reacted with diazeniumdiolate salt under neutral conditions to produce compound 2. Reaction of compound 2 with ceric ammonium nitrate gave the desired quinone-diazenoumdiolate adduct 1. The compounds synthesized were next tested for their ability to generate ONOO− in the presence of DT-Diaphorase, a bioreductive enzyme. Its activity was measured using a dye that is based on its boronate ester functional group being sensitive to cleavage by ONOO− (Fig. 1A) [36,53]. The initial screen revealed 1d (CJ067) as the most efficient ONOO− generator. The t-Boc group was deprotected under acidic conditions, and the resulting adduct 1e was next tested for ONOO− generation. It was found to be less efficient than CJ067. The time course of ONOO− generation revealed that CJ067 was able to generate ONOO− efficiently over 15 min (Fig. 1C). Our data suggests that the yield of OONO− is about 0.6 molecules per molecule of CJ067. Based on these results, CJ067 was identified as the lead compound.Scheme 2 Synthesis of potential donors of ONOO−: The compounds synthesized were next tested for their ability to generate ONOO− in the presence of DT-Diaphorase, a bioreductive enzyme. Its activity was measured using a dye that is based on its boronate ester functional group being sensitive to cleavage by ONOO− (Fig. 1A). The initial screen revealed 1d (CJ067) as the most efficient ONOO− generator. The t-Boc group was deprotected under acidic conditions, and the resulting adduct 1e was next tested for ONOO− generation. It was found to be less efficient than CJ067. The time course of ONOO− generation revealed that 1e was able to generate ONOO− efficiently over 15 min (Fig. 1C). Based on these results, CJ067 was identified as the lead compound.

Scheme 2

Fig. 1 Peroxynitrite generation by CJ067: (A) Boronate-ester dyes used for ONOO− detection (B and C) •NO generating diazeniumdiolate group is linked to a 1,4-Naphthoquinone which releases O2•− upon bio-reduction by DT-Diaphorase (DT-D)/NAPDH-Quinone reductase/NADH-dehydrogenase which gives ONOO− in buffer [53].

Fig. 1

3.2 Intracellular delivery of ONOO− by CJ067 to mycobacteria

CJ067 was synthesized to release O2•− and •NO upon bio-reductive activation by cellular enzymes such as NADH-dependent oxidoreductases. Once generated, O2•− and •NO react at near diffusion-limited rates to produce ONOO− [33]. To demonstrate this reaction, we examined ONOO− generation from CJ067 in aerobic buffer using an ONOO−-specific fluorescent dye, Dye-1 (Fig. 1A) [36]. This dye is not intrinsically fluorescent, but upon oxidation with ONOO− it generates a strongly fluorescent compound (Fig. 1A). When CJ067 was incubated in buffer at pH 7.4, fluorescence emission at 460 nm showed no increase; however, in the presence of DT-diaphorase (DT-D), a bio-reductive enzyme, a significant increase in fluorescence was observed (Fig. 2A). Since arylboronic acid-based dyes react with H2O2 with lower rates than ONOO− [39,40], we co-incubated CJ067 with DT-D in the presence of an H2O2 quencher (catalase) and found a minimal decrease in fluorescence emission (Fig. 2A). This result confirmed ONOO− as the major reactive species produced by CJ067.Fig. 2 CJ067 generates ONOO−in buffer and within mycobacteria: (A) Increase in fluorescence intensity of Dye-1 on incubation with CJ067 (100 μM), DT-Diaphorase (DT-D) enzyme & cofactor NADH in phosphate buffer of pH 7.4. Here, ns is non-significant, ***P < 0.001 and ****P < 0.0001 (One-way ANOVA test) as compared to CJ067 only treatment. (B) Time-course generation of ONOO− within Msm (pre-treated with 25 μM of Dye-1) upon incubation with various compounds at 10 μM each. *P < 0.05 (Unpaired t-test) at 20 min comparing CJ067 to other compounds. (C) Quenching of ONOO− released from CJ067 using Uric Acid in Msm. *P < 0.05 (One-way ANOVA test) (D) Time-course generation of ONOO− within Mtb-H37Rv (pre-treated with 25 μM of Dye-1) upon incubation with various compounds at 25 μM each. Compounds used in assay are CJ067 (intracellular ONOO− donor), SIN-1 (3-morpholinosydnonimine, exogenous ONOO− donor), Authentic donor (AD) (Sodium salt of ONOO−), JCH & Menadione (intracellular O2•− donors), AJ0279 (structural analogue of CJ067 that only releases O2•−), •NO donor (esterase-activated •NO donor) and DMSO (solvent control). Error bars represent standard deviation from the mean. Data shown are representative of three independent experiments done in triplicates. **P < 0.01 (One-way ANOVA test) as compared to no compound control at 30 min.

Fig. 2

Next, using the Dye-1 probe, we determined whether CJ067 penetrates mycobacterial cells to generate ONOO−. A non-pathogenic mycobacterial species, Mycobacterium smegmatis (Msm), when treated with 10 μM CJ067 and stained with Dye-1, revealed a time-dependent increase in the fluorescence signal (Fig. 2B). Importantly, treatment of Msm with 10 μM of ONOO− salt (AD) or O2•− donors (menadione and JCH) or esterase-activated •NO donor [54] failed to increase the fluorescent signal (Fig. 2B). Finally, co-treatment of Msm with uric acid (a ONOO− quencher) completely abolished the signal generated by CJ067-derived ONOO− (Fig. 2C) . As with Msm, treatment of pathogenic Mtb with 25 μM of CJ067 but not with SIN-1, AD, menadione, and JCH reliably increased the ONOO− fluorescent signal (Fig. 2D). Importantly, AJ0279, a structural analogue of CJ067 that does not generate •NO but is expected to enhance only O2•−, failed to increase fluorescence (Fig. 2D) [36], confirming that CJ067 raises ONOO− levels inside mycobacteria.

We confirmed the formation of intracellular ONOO− upon treatment of mycobacteria with CJ067 using another ONOO−-specific dye N (2-aminophenyl)-5-(dimethylamino)-1-naphthalene sulfonic amide (Ds-DAB) (Fig. 3A) [55]. Using Mtb preincubated with Ds-DAB, confocal microscopy revealed a significant enhancement in the fluorescent signal at 505 nm when incubated with 20 μM and 50 μM CJ067 (Fig. 3B). Interestingly, SIN-1 also produced a fluorescent signal, but only at a higher concentration (200 μM) (Fig. 3B). Although accurate estimation of intracellular yield of ONOO− is not possible, CJ067 robustly enhances ONOO− levels inside mycobacteria, and in head-to-head comparisons, performed better than commonly used ONOO donors.Fig. 3 Intracellular peroxynitrite detection in Mtb-H37Rv by Ds-DAB: (A) Reaction scheme for Ds-DAB; Ds-DAB reacts specifically with ONOO− to give dansylsulfonic acid (in green) which fluoresces at excitation/emission of 350/505 nm. (B) Fluorescence (top panel); bright field Differential Interference Contrast (DIC, lower panel); Panels indicate untreated cells, cells treated with CJ067 (50 μM) only, Ds-DAB (25 μM) only, Ds-DAB (25 μM) with CJ067 (20 μM) or CJ067 (50 μM) or with SIN-1 (200 μM) (from left to right). Confocal imaging was done at 63X magnification. Images were analyzed on ZEN2 Blue software.

Fig. 3

3.3 CJ067 perturbs mycothiol redox homeostasis in Mtb

Thus far, we have shown generation of ONOO− in buffer and within mycobacterial cells using redox active fluorogenic dyes. Intra-bacterial detection of ONOO− by fluorogenic dye-based techniques require manipulation, such as cell fixation by paraformaldehyde, that can induce oxidation artifacts and prohibit dynamic, real-time measurements. In Mtb, the mycothiol antioxidant system serves as a biologically relevant pathway to reduce proteins oxidized by ONOO− [56]. Based on this idea, ONOO− exposure is likely to induce dynamic changes in the redox potential of mycothiol (EMSH), which can be measured using a genetically encoded, non-invasive, highly sensitive and specific biosensor of mycobacterial EMSH (Mrx1-roGFP2) (Fig. 4A) [42]. Changes in EMSH due to depletion of the total mycothiol pool (MSH + MSSM) or oxidation of reduced mycothiol (MSH) increases the biosensor 405/488 nm ratio, whereas reductive stress decreases the 405/488 ratio (Fig. 4A) [42].Fig. 4 Detection of transient redox changes in mycobacteria by ONOO−donors using Mrx1-roGFP2: (A)Genetic coupling of Mrx1 with roGFP2 allows reversible transfer of electrons between the mycothiol redox couple and thiol groups on roGFP2, which leads to formation and release of disulphide bridges of the roGFP2. Oxidation of Mrx1-roGFP2 increases fluorescence intensity for excitation at 405 nm and decreases it for excitation at 488 nm (B) Comparison of oxidation in Msm expressing Mrx1-roGFP2 using CJ067 and Authentic donor (AD) at 10 μM. (C) Comparison of oxidation in Mtb-H37Rv expressing Mrx1-roGFP2 using CJ067 and AD at 50 μM. (D) Oxidation of Mtb BND320 expressing Mrx1-roGFP2 treated with 50 μM CJ067. Error bars represent standard deviation from the mean. Data shown are representative of three independent experiments [50].

Fig. 4

Msm expressing the Mrx1-roGFP2 biosensor was separately exposed to 10 μM of CJ067 and AD, and the fluorescence ratio was monitored over time using flow cytometry. Treatment with AD induces biosensor oxidation (Fig. 4B). However, the response was transient, as the biosensor ratio returns to baseline values by 18 min. Treatment with CJ067 induces greater biosensor oxidation that results in slowly recovery to baseline values as compared to AD (Fig. 4B). In the case of Mtb, we found that 50 μM of AD showed a dynamic biosensor response comparable to 10 μM in Msm. Therefore, we monitored biosensor response upon exposure of Mtb to 50 μM of CJ067 and AD. Treatment with 50 μM CJ067 treatment of Mtb expressing the Mrx1-roGFP2 biosensor induces a more enduring oxidation of the biosensor, as compared to 50 μM AD (Fig. 4C). We also expressed the biosensor in a drug-resistant patient isolate of Mtb (BND320) and confirmed that exposure to CJ067 induces enduring oxidative stress (Fig. 4D).

3.4 Mtb is sensitive to intracellular ONOO−

Having demonstrated that CJ067 efficiently releases ONOO− within Mtb and induces oxidative stress, we next determined the effect of CJ067 on mycobacterial growth. As a control, we used various reactive oxygen species (ROS) donors including AJ0279. We measured the minimum inhibitory concentration (MIC) of CJ067 against Msm, Mtb, and several drug-resistant patient isolates of Mtb using the resazurin microtiter assay (REMA). Resazurin is an oxidation-reduction indicator dye that has been routinely used to assess the inhibitory action of anti-TB drugs against Mtb [57,58]. Consistent with our earlier findings, Msm showed remarkable resistance to various ROS donors (Table 1). Surprisingly, despite oxidation of the biosensor by 10 μM of CJ067, Msm did not show growth inhibition towards CJ067, even at a concentration greater than 800 μM (Table 1). Similarly, ROS donors did not have adverse effects on the growth of Msm (Table 1). In contrast, Mtb strains, including the drug-resistant clinical isolates, exhibited growth inhibition by CJ067 at concentrations ranging from 12.5 to 50 μM (Table 1). Pre-treatment of Mtb with ONOO− scavengers, such as uric acid and N-acetyl cysteine (NaC), increased MIC of CJ067 by 2–4 fold (Table 2), indicating that the growth inhibition was caused by enhanced ONOO− levels. These findings were consistent with the Mrx1-roGFP2 data in which CJ067 induces long-lasting oxidative stress in Mtb. Similar to results with CJ067, Mtb strains were sensitive to intracellular donors of O2•− (JCH and menadione). However, Mtb strains remained relatively more resilient to exogeneous ONOO− donors (AD and SIN-1) (Table 1).Table 1 MIC of peroxynitrite and ROS donors with various mycobacterial strains: Minimum inhibitory concentration (MIC) for the compounds were measured using the Resazurin Microtiter Assay (REMA) at concentration that resulted in growth inhibition of Msm mc2155, Mtb-H37Rv, BND 320 (Isoniazid-resistant), Jal 2287/Jal2261/Jal1934 (MDR) and Myc 431 (XDR).

Table 1Strains	MIC (μM)	
CJ067	AD	SIN-1	Menadione	JCH	AJ0279	
Msm mc2155	>800	400	>800	>800	>800	>800	
Mtb-H37Rv	25–50	400	200–400	50	3.125	100–200	
Jal 2287	50	400	200	50	3.125	200	
Jal 2261	50	400	400	50	3.125	200	
Jal 1934	12.5	400	200	25	3.125	200	
BND 320	25	400	200	50	3.125	100	
Myc 431	12.5	400	200	25	3.125	100	

Table 2 MIC for CJ067 after treatment with peroxynitrite scavengers: Minimum inhibitory concentrations (MICs) for CJ067 treatment were determined using the Resazurin Microtiter Assay (REMA) at concentration that resulted in growth inhibition after pre-treatment with ONOO− scavengers (N-acetyl cysteine (NaC) and Uric Acid) for 1 h.

Table 2Strain	Peroxynitrite scavenger	Scavenger concentration	MIC for CJ067 (μM)	
Mtb-H37Rv	N-acetyl cysteine (NaC)	0 μM	50	
80 μM	100	
1 mM	200	
Uric Acid	0 μM	50	
100 μM	100	

It can be argued that the use of redox-cycling compounds, such as CJ067, can influence oxidation-reduction of the resazurin dye to compromise the MIC estimation. To rule out this possibility, we determined the killing efficiency of CJ067 by estimating minimum bactericidal concentration (MBC). As shown in Fig. 5A, 50 μM of CJ067 resulted in absence of growth on the agar plate. In contrast, similar extent of killing required 400 μM of AD (Fig. 5B). We further measured killing kinetics by measuring OD600 and CFU at various doses of CJ067 over time (Fig. 5C and D). We observed that 0.5X MIC and 1X MIC of CJ067 did not decrease viability of Mtb (Fig. 5C and D). However, 2X MIC, 5X MIC and 10X MIC of CJ067 killed Mtb in a time-dependent manner, with 2X MIC resulting in the loss of viability as early as 6 h post-treatment (Fig. 5D). Overall, this revealed that Msm and Mtb strains differ in their abilities to respond to intracellular increases in ONOO−. More importantly, pathogenic drug-resistant strains of Mtb showed similar or higher sensitivity to CJ067 as compared to drug-sensitive Mtb (Table 1).Fig. 5 Mtb is sensitive to intracellular ONOO−: Minimal bactericidal concentration (MBC) was assessed by spotting 10 μl (∼105 cells) volume from a range of concentrations of the MIC 96-well plates and observing growth on 7H11-OADC solid-media for (A) CJ067 and (B) AD. Mid-log phase grown Mtb-H37Rv adjusted to an OD600nm of 0.06 was treated with indicated MICs of CJ067. At the indicated time points (C) OD600nm was measured. Data shown are the average of two independent experiments. (D) CFUs were enumerated by plating cultures on 7H11-OADC solid media. Data shown are indicative of three biological experiments done in triplicates. Error bars represent standard deviation from mean. **P < 0.01, ***P < 0.001 & ****P < 0.0001 (Unpaired t-test) as compared to untreated control at each time point.

Fig. 5

CJ067 modulates Mtb expression of pathways involved in antioxidant defence, lipid biosynthesis, Fe-starvation, and metal homeostasis.

To further define Mtb's response to intracellular ONOO−, we performed RNA-sequencing (RNA-seq) of Mtb exposed to a sublethal 2X MIC of CJ067 for 2 h as determined by MBC (Fig. S2A). Multidimensional scaling (MDS) and hierarchical clustering confirmed that the samples from the two conditions clustered with their biological replicates (Fig. S2B and Fig. S2C). RNA-seq analysis confirmed that 637 genes were differentially regulated in response to CJ067 (FDR ≤0.05 and 2-fold cut-off) (Fig. 6A). Since ONOO− generally targets metal-containing proteins, Fe–S clusters, cysteine thiols, and lipids, the transcriptome on CJ067 treatment is largely composed of genes involved in metal homeostasis, Fe–S cluster biogenesis, sulphur metabolism, and redox-regulators (Fig. 6). Given that CJ067 induces substantial oxidative stress, expression of katG was induced (∼3 log fold-change) by treatment (Fig. 6B). Interestingly, in addition to exhibiting canonical catalase/peroxidase activity, KatG also functions as a peroxynitritase by catalysing the decomposition of ONOO− with a second-order rate constant of 1.4 × 105 M−1 s−1 [59]. A thioredoxin-like protein has been shown to protect Mtb from ONOO− stress [60]. Furthermore, ONOO− is known to oxidize thiols, which Mtb mitigates via the thioredoxin system [26]. Consistent with these observations, CJ067 induces the expression of the thioredoxin system (trxB2, trxC and trxB1) in Mtb (Fig. 6B). We also observed that Mtb responds to CJ067 by inducing redox-sensitive transcription factors/sigma factors (e.g., whiB3, sufR, sigE, sigB and sigH) involved in providing resistance to oxidative, nitrosative, and thiol-disulfide stress (Fig. 6C) [[61], [62], [63], [64]].Fig. 6 RNA-sequencing of Mtb-H37Rv in response to CJ067:Mtb-H37Rv was grown till an OD600nm of 0.4 and treated with 2X MIC of CJ067 for 2 h. Total RNA was isolated from three biological replicates and subjected to RNA-sequencing analysis. Genes were considered differentially expressed based on FDR≤0.05 and log2fold change ≥2. (A) Volcano plot denoting differentially expressed genes (DEGs); upregulated (blue), downregulated (orange) and unchanged (grey). Heat maps denote log2fold changes of DEGs in CJ067 treated Mtb-H37Rv as compared to untreated control in functional categories such as (A) Antioxidant machinery (B) Transcriptional regulators (C) Iron homeostasis (D) Sulphur metabolism, SUF-operon & Copper homeostasis (E) Lipid biosynthesis. GEO Submission (GSE246347).

Fig. 6

ONOO− is known to primarily damage Fe–S clusters of dehydratases inside bacterial and mammalian cells with high second order rate constants [65]. The Fe–S cluster damage caused by ONOO− could be repaired by Fe–S cluster repair systems such as SUF and ISC [66,67]. As expected, CJ067 induced expression of the Mtb Suf operon, which is involved in Fe–S cluster repair (Fig. 6E) [68,69]. Suf-mediated Fe–S cluster biogenesis is dependent on a continuous supply of iron and cysteine [45,47]. An increased demand of cysteine could also be due to a known role of ONOO− in depleting the cytoplasmic pool of cysteine due to oxidation of cellular thiols [[70], [71], [72], [73]]. Consistent with this, CJ067 treatment resulted in upregulation of iron-siderophore biosynthesis (mbt operon), iron-siderophore secretion (mmpL4/5 and mmpS4/5), and iron uptake machinery (irtA/irtB) (Fig. 6D) [74,75]. Down-regulation of the iron storage gene bacterioferritin (bfrB) further indicated release of stored iron for the repair of Fe–S clusters. Induction of genes encoding de novo cysteine biosynthesis pathway is consistent with increased requirement of cysteine for repairing Fe–S clusters and recovering reduced form of oxidized thiols in response to ONOO− in Mtb (Fig. 6E). ONOO− is known to release copper from metal-containing proteins [70,76,77], increasing the free copper pool which can damage DNA [78,79]. Mtb treated with CJ067 exhibits a signature of copper toxicity as evident by the induction of transcriptional regulators involved in copper homeostasis (csoR and ricR) along with genes encoding a copper efflux pump (ctpV) and copper binding metallothionein (mymT) (Fig. 6E) [80,81].

Moreover, ONOO− induces membrane lipid peroxidation [73,82]. As expected, CJ067 induces the expression of genes associated with lipid biosynthesis pathways in Mtb (e.g., mycolic acids) [83] (Fig. 6F). Given that ONOO− is formed from the reaction involving O2•− and •NO, we cannot completely rule out the contributions of O2•− and •NO in affecting transcriptomic signature. To begin understanding this possibility, we compared ONOO− transcriptome with the expression changes induced by •NO and O2•−. While our findings reveal a significant overlap between the datasets, a substantial fraction of genes exhibits dysregulation specifically in CJ067-treated Mtb (Fig. S3). In sum, our results indicate that Mtb responds to the elevation of intracellular ONOO− levels by mobilizing antioxidant machinery, Fe–S cluster repair pathway, cysteine biogenesis, and copper detoxification systems.

3.5 CJ067 damages Fe–S clusters

In vitro and in vivo studies confirm that Fe–S clusters of the dehydratase enzyme class are the primary targets of ONOO− [84]. The TCA cycle enzyme aconitase (Acn) belongs to the family of dehydratases that co-ordinate 4Fe–4S cluster for isomerization of citrate to iso-citrate [85,86]. To determine whether an intracellular increase in ONOO− damages Fe–S clusters of Mtb Acn, we evaluated Acn activity in Mtb treated with CJ067. We consistently observed a ∼30–40 % decrease in the Acn activity, presumably due to the disruption of its Fe–S cluster (Fig. 7A) without any change in Acn expression levels as compared to treatment and loading control (Fig. 7B and C and Fig. S4).Fig. 7 Inactivation of aconitase activity by CJ067 treatment:Mtb-H37Rv (OD600nm–0.4) was subjected to 2XMIC of CJ067 for 1 h. (A) The aconitase activity was measured from the crude lysate by measuring the disappearance of 0.15 mM cis-aconitate at 240 nm over time. Error bars represent standard deviation from mean. Results are mean of three independent experiments done in duplicates. ****P < 0.0001 (Unpaired t-test) as compared to untreated control. Aconitase protein levels were estimated by Western blot. Equal amount (20 μg) of whole-cell lysate from untreated and CJ067-treated Mtb was loaded in respective lanes. Western blotting was performed via primary antibodies against aconitase (102.45 kDa) and the loading control Cbs (cystathione-β-synthase; 59.64 kDa). Blots are representative of three independent experiments. Comparative densitometric quantification of Western blots for (B) Aconitase and (C) Cbs between untreated and 2XMIC CJ067 treated groups. Error bars represent standard deviation from mean. Results are mean of three independent experiments. ns = not significant (Unpaired t-test) as compared to untreated control.

Fig. 7

In Mtb, repair of Fe–S clusters depends on two Fe–S cluster assembly systems, Suf and IscS. The Suf system is essential for survival of Mtb, whereas the loss of IscS causes a slow-growth phenotype [3,4,38,87]. An Mtb strain lacking IscS (Mtb ΔiscS) exhibits diminished activity of Acn [38]. We also showed that Mtb strains with reduced expression of suf operon (Mtb ΔsufR and [sufT-KD]), failed to repair oxidatively damaged Fe–S clusters of Acn [3,4]. On this basis, we hypothesize that Suf and/or IscS pathways protect Mtb from the inhibitory effects of CJ067 by repairing Fe–S clusters of dehydratases such as Acn. We tested this idea by estimating MIC of CJ067 for Mtb ΔsufR, Mtb sufS-KD, Mtb sufT-KD, Mtb ΔiscS, and Mtb ΔiscS-sufS-KD (Table 3). Surprisingly, MIC of CJ067 for the Fe–S cluster assembly/repair mutants were similar to those of wild-type (WT) Mtb. We have recently shown that the Suf and IscS systems can compensate for the loss of each other in Mtb [38,67]. Therefore, we reduced the expression of sufS in Mtb ΔiscS (Mtb ΔiscS-sufS-KD); the double mutant was 4-fold more sensitive to CJ067 relative to WT Mtb or the single-mutant counterparts (Table 3).Table 3 MIC for CJ067 for Fe–S cluster mutants of Mtb: Minimum inhibitory concentrations (MICs) for CJ067 treatment were determined using the Resazurin Microtiter Assay (REMA). MIC values depicted are from at least two independent experiments.

Table 3Strain	MIC for CJ067(μM)	
Mtb-H37Rv	50	
Mtb ΔsufR	50	
Mtb sufS-KD	50	
Mtb sufT-KD	50	
Mtb ΔiscS	50	
Mtb ΔiscS sufS-KD	12.5	

3.6 CJ067 inhibits respiration and metabolism in Mtb

Our data indicate that Fe–S cluster-containing metabolic enzymes and Fe–S cluster assembly pathways (Suf and IscS) are likely to be important for responding to intracellular ONOO−. Since Fe–S cluster enzymes are crucial for central carbon metabolism (e.g., TCA cycle) and oxidative phosphorylation (OXPHOS) in Mtb [3,4,88], we reasoned that ONOO− exposure could affect bioenergetics of Mtb. Therefore, we exploited extracellular flux (XF) technology to examine the influence of CJ067 on oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), quantifiable readouts of OXPHOS and carbon metabolism, respectively. The basal and maximal rates of OCR and ECAR were measured by culturing Mtb in 7H9-glucose in an XF microchamber, followed by exposure to various concentrations of CJ067, and finally to the uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Addition of CCCP stimulates respiration to the maximal capacity manageable by Mtb. The difference between basal and CCCP-induced OCR provides an estimate of the respiratory reserves available for tolerating unfavourable respiratory conditions i.e. spare respiratory capacity (SRC) [4]. For measurements, Mtb cultures were diluted, and an equal number of Mtb cells (2 × 106 cells/well) adhered to XF cell culture plates. Under standard growth conditions, Mtb displayed a basal OCR of ∼25 pmol/min, which increased to ∼70 pmol/min upon uncoupling by the first injection of CCCP (Fig. S5A). We found that Mtb displayed a biphasic response to CJ067. Low concentrations of CJ067 (0.125X and 0.25X of MIC) stimulated OCR and ECAR (Fig. 8A and B signifying normalized data, Fig. S5A and Fig. S5C; empirical values), whereas further increase (0.5X MIC) inhibited Mtb OCR and ECAR (Fig. 8A and B). These observations suggest that Mtb mobilizes respiratory and metabolic reserves to induce respiration and carbon catabolism at lower concentrations of CJ067. However, higher concentrations lead to overwhelming damage to respiratory and metabolic enzymes, resulting in the inhibition of OCR and ECAR. Consistent with this, we found that pre-treatment with CJ067 progressively reduced the ability of Mtb to further increase OCR upon uncoupling by CCCP (Fig. 8A). Consequently, CJ067 treatment exhausts the SRC in a dose-dependent manner (Fig. 8C). Importantly, when CJ067 was co-incubated with N-acetyl cysteine (NaC), a quencher of ONOO−, we found a nearly complete restoration of OCR (Fig. 8D). Since Suf and IscS systems protect from CJ067-derived intracellular ONOO−, we examined the changes in the OCR and ECAR of Mtb ΔiscS-sufS-KD in response to CJ067. The OCR of Mtb ΔiscS-sufS-KD in response to 0.125X MIC of CJ067 remain suppressed and did not increase upon treatment with CCCP (Fig. 8E). Interestingly, ECAR of Mtb ΔiscS-sufS-KD was not significantly affected by 0.125X MIC of CJ067 (Fig. 8F). These data suggest that defects in Fe–S cluster biogenesis cause Mtb to switch from OXPHOS to glycolysis in response to intracellular OONO−, perhaps in an effort to sustain metabolism and energy homeostasis. Furthermore, diminishing Fe–S cluster biogenesis/repair pathways increases the vulnerability of Mtb to bioenergetic exhaustion by intracellular ONOO−.Fig. 8 CJ067 blocks oxygen consumption in Mtb: (A) Oxygen consumption rate (OCR) and (B) Extracellular Acidification Rate (ECAR) of 2 × 106 wildtype Mtb cells were determined using XF technology. Three basal OCR measurements were obtained before treatment with varying concentrations of CJ067 MIC (as indicated). (C) Plot depicting mycobacterial respiratory reserve capacity, which is the difference between maximal and basal respiration. Seven measurements were then taken before two consecutive treatments with mitochondrial uncoupler CCCP (2 μM). (D) OCR was studied using 1 mM NaC treated or untreated 2 × 106Mtb cells. (E) OCR and (F) ECAR for wildtype Mtb and MtbΔiscS-sufS-KD on treatment with 0.125X MIC CJ067 with untreated (UT) control. Three basal OCR measurements were obtained before treatment with CJ067. Seven measurements were then taken before two consecutive treatments with mitochondrial uncoupler CCCP (2 μM). Data shown are representative of three biological experiments done in duplicates.

Fig. 8

3.7 Fe–S cluster homeostasis coordinates Mtb survival in response to RNS in macrophages

Our data indicate that ONOO− damages Fe–S clusters and the Fe–S cluster biogenesis/repair by Suf and IscS systems protect Mtb from adverse consequences of intracellular increases in ONOO− level. To test his hypothesis, we measured survival of WT Mtb and Mtb ΔiscS-sufS-KD in bone marrow-derived macrophages (BMDMs) from WT C57BL/6 mice, which were expected to expose the bacteria to RNS, and also in BMDMs derived from iNOS−/− C57BL/6 mice that lacked RNS. As expected, WT Mtb showed unrestrictive growth in WT BMDMs and iNOS−/− BMDMs (Fig. 9A and B). In contrast, Mtb ΔiscS-sufS-KD showed a survival defect inside WT BMDMs, and growth inhibition was significantly restored in iNOS−/− BMDMs lacking the ability to generate RNS (Fig. 9A and B). Taken together, these data suggest that Suf and IscS systems provide resistance to RNS stress in vitro and inside macrophages.Fig. 9 Fe–S cluster deficient Mtb showed defective survival in macrophages in an iNOS-dependent manner: BMDMs were isolated from WT and iNOS−/− C57BL/6 mice and were infected at 1:5 MOI with MtbΔiscS-sufS KD (+Atc) and Mtb-H37Rv strains and survival was monitored at the indicated days by enumerating (A) CFU/well (wherein ● indicates H37Rv infecting WT BMDMs, Image 1 indicates H37Rv infecting iNOS−/− BMDMs, Image 2 indicates ΔiscS- sufS-KD infecting WT BMDMs and Image 3 indicates ΔiscS- sufS-KD infecting iNOS−/− BMDM. *P < 0.05, **P < 0.01 and ***P < 0.001 (One-way ANOVA test) as compared to H37Rv control in WT or iNOS−/− BMDMs (B) Percentage survival (at Day 4) of bacteria infecting BMDMs. ****P < 0.0001 (Two-way ANOVA test) as compared to H37Rv control in WT (black bars) and iNOS−/− BMDMs (red bars). Data shown are representative of three biological experiments. Error bars represent standard deviation from mean.

Fig. 9

4 Discussion

The phagosomes of phagocytic cells, such as macrophages, generate both O2•− and •NO, but it is unlikely that these species are directly lethal to Mtb. However, the simultaneous production of these radical species in close proximity drives them to react together, forming ONOO−, whereas some fraction of O2•− dismutates to generate H2O2 [70,73]. Both ONOO− and H2O2 damage intracellular targets such as proteins, lipids, and DNA. ONOO− is exceptionally potent in sterilizing bacteria owing to its extremely high oxidation potential (+1.2–1.4 mV) and the “non-specificity” with which it attacks all macromolecules [70]. Such wide-ranging effects prevent resistance that would otherwise come up, as the pathogen cannot possibly dispense with such essential targets [89]. Surprisingly, Mtb resisted commercially available ONOO−, presumably due to the anatomical barrier of a complex array of cell wall-associated lipids. Therefore, how Mtb responds to ONOO− remained poorly studied. We circumvent this issue by synthesizing CJ067 that penetrates Mtb and reliably generates ONOO− upon bio-reductive activation by mycobacterial enzymes. The specific detection of ONOO− inside mycobacteria confirms our chemical synthesis approach.

In contrast to previous studies showing exceptional resistance of Mtb towards •NO and ONOO− administered exogenously [21], our study revealed that Mtb is vulnerable to donors that permeate inside Mtb to elevate intracellular ONOO− levels and perturb redox homeostasis. These findings are consistent with intracellular production of RNS by an anti-TB drug pretomanid contributing to the killing of Mtb under aerobic and anaerobic conditions [29,90,91]. Furthermore, drug-sensitive Mtb (H37Rv) and drug-resistant strains have shown exceptional sensitivity to intracellular ROS produced by redox cycling drugs (ATD-3169 and clofazimine), vitamin C, and small molecule thiols [30,37,[92], [93], [94], [95]]. In contrast to Mtb, a non-pathogenic mycobacterium Msm was comparatively less affected by intracellular ROS and RNS donors (e.g., ATD-3169, CJ067, and PA-824) [30,96]. While the reasons for these differences are unclear, a more reductive cytoplasm of Msm (EMSH = −300 mV) relative to Mtb (EMSH = −275 mV), greater retention of antioxidant enzymes in the cytoplasm, expression of truncated hemoglobins, differences in permeability, and induction of a protective redox stress response in Msm could contribute to better resistance towards intracellular ROS and RNS [23,30,[97], [98], [99]].

Our RNA sequencing data provided insights into how Mtb responds to intracellular ONOO−. CJ067 appears to function by disrupting redox balance and damaging the Fe–S cluster of metabolic enzymes such as dehydratases. The induction of the Fe–S cluster biogenesis/repair system (suf operon) and the genes associated with cysteine biosynthesis and iron transport indicate heightened demand for Fe–S clusters in response to CJ067. Since CJ067 generates •NO and O2•− during the formation of ONOO−, we cannot completely rule out the effect of these radicals on the suf operon and mycobacterial bioenergetics. O2•− or ONOO−-mediated destabilization of Fe–S clusters of metabolic enzymes is known to perturb central carbon metabolism and respiration in other organisms, such as E. coli [100]. Our XF-flux analysis showed diminished ECAR, OCR, and reserve respiration capacity in response to CJ067, recapitulating these bioenergetic defects in Mtb exhibiting elevated intracellular ONOO−. In addition to Fe–S clusters, heme-containing cytochromes and other metal centers in respiratory complexes I,II,III, and IV are sensitive to attack by ONOO− [[101], [102], [103]]. The oxidative phosphorylation pathway's (OXPHOS) substrates, such as NADH and cytochrome c, are also depleted by ONOO− [104,105]. Further, lipid peroxidation mediated by ONOO− leads to disruption of the integrity of the lipid bilayer [106]. Additionally, ONOO− is also known to introduce 3-nitrotyrosine modifications within the mitochondria, resulting in altered mitochondrial functions [107,108]. Similar mechanisms could deplete the bioenergetic efficiency of Mtb in response to CJ067.

Iron availability influences the rate and the extent to which the Fe–S cluster can be repaired [65]. In this context, it appears that in E. coli, iron for repairing Fe–S clusters is mainly provided by the iron storage proteins [65]. Our RNA-seq data showing down-regulation of iron storage proteins and significant induction of iron-transporters and siderophores suggest that Mtb will likely rely on importing exogenous iron for repairing Fe–S clusters. Uniform expression of iron-transporters in response to CJ067 mimics Mtb's transcriptional response to iron starvation, suggesting that intracellular ONOO− induces a state of iron deficiency in Mtb. This is surprising given that ONOO− leaches iron from Fe–S clusters and increases the free iron pool in the cytoplasm [65]. However, in E. coli, the labile iron released upon ONOO− exposure is transient and rapidly sequestered (within 30 s), resulting in iron deprivation [65]. Sequestering of labile iron could be a mechanism to prevent DNA damage by the iron-catalyzed generation of harmful hydroxyl radicals by the Fenton reaction.

Furthermore, sequestering/clearance of iron was faster than the rate of Fe–S cluster repair, ruling out the possibility that the leached iron was re-utilized for repairing Fe–S clusters damaged by ONOO− [65]. Our RNA-seq data support this hypothesis; we propose that exogenous iron, rather than intracellular labile iron is a likely source of iron for repairing ONOO−-damaged Fe–S clusters. Despite the ideas, the reason for the iron starvation response upon ONOO− by Mtb and E. coli is unclear. In this regard, a recent finding in mammalian cells shows that ONOO− removes the pool of labile iron by oxidizing the labile ferrous iron pool to generate a less toxic hypothetical by-product oxyferryl (FeIV=O) [109]. In doing so, the labile iron pool mitigates the nitrosative and oxidative potential of ONOO− [109]. Therefore, bacteria such as E. coli and Mtb may prioritize the utilization of labile iron to reduce intracellular levels of ONOO−, carrying out this task alongside repairing the Fe–S clusters. We tested these speculations by examining the phenotype of Mtb mutants lacking Fe–S cluster biogenesis/repair pathways SUF and IscS. We confirmed that both systems jointly contribute to Mtb's ability to tolerate intracellular ONOO−, a finding consistent with our recent finding of compensatory roles for SUF and IscS in ensuring Mtb persistence in mice [3,4,38,67]. Based on the present results, we propose that inhibitors of Fe–S cluster biogenesis systems are likely to potentiate the iNOS-dependent host immunity against Mtb and the efficacy of anti-TB drug pretomanid (PA-824). These hypotheses will become testable when these inhibitors are identified.

Fundings

This work was supported by 10.13039/501100009053 India Alliance grant IA/S/16/2/502700 (AS) and in part by 10.13039/501100001407 DBT grants BT/PR29098/Med/29/1324/2018 (AS), DST-10.13039/501100001843 SERB grant SPR/2021/000175 , 10.13039/501100001843 SERB /10.13039/100017169 CRG /2022/002009 (AS), UGC-10.13039/501100001502 DAE Consortium for Scientific Research, University Grants Commission 10.13039/501100001501 UGC special Assistance (AS), Crypto Relief grant ODAA/INT/20-21 (AS), Department of atomic energy (10.13039/501100001502 DAE ) 12-R&D-TFR-5.04-0800 (ASNS) and the Revati and Satya Nadham Atturi Chair Professorship (AS). MD was supported by GATE Ph. D fellowship from 10.13039/100007780 Indian Institute of Science (10.13039/100007780 IISc ). The funders had no role in study design, data collection and analysis, or preparation of the manuscript.

Data and materials availability

All data associated with this study are present in the paper or the Supplementary Materials. RNA-seq data generated and analysed in this study have been uploaded to the NCBI Gene Expression Omnibus under accession number GSE246347.

CRediT authorship contribution statement

Arshiya Dewan: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Charu Jain: Methodology, Investigation, Formal analysis. Mayashree Das: Methodology, Investigation. Ashutosh Tripathi: Methodology, Investigation. Ajay Kumar Sharma: Methodology, Investigation. Harshit Singh: Methodology. Nitish Malhotra: Methodology, Investigation, Formal analysis, Data curation. Aswin Sai Narain Seshasayee: Supervision, Software, Resources, Funding acquisition, Formal analysis, Data curation. Harinath Chakrapani: Writing – review & editing, Writing – original draft, Supervision, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualization. Amit Singh: Writing – review & editing, Writing – original draft, Supervision, Project administration, Investigation, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following are the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Multimedia component 2

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Multimedia component 3

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Data availability

Data will be made available on request.

Acknowledgments

We thank Karl Drlica for providing critical comments on the manuscript. We are thankful to Awadhesh Pandit and Next Generation Genomics Facility (NGGF) at National Centre for Biological Sciences (NCBS), Bangalore for conducting the RNA-sequencing experiment. We acknowledge the BSL-3 facilities at CIDR, IISc, Bangalore.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.redox.2024.103285.
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