stm.sciencemag.org/cgi/content/full/11/518/eaaw6635/DC1

Supplementary Materials for

Targeting redox heterogeneity to counteract drug tolerance in replicating

Mycobacterium tuberculosis

Richa Mishra, Sakshi Kohli, Nitish Malhotra, Parijat Bandyopadhyay, Mansi Mehta, MohamedHusen Munshi, Vasista Adiga, Vijay Kamal Ahuja, Radha K. Shandil, Raju S. Rajmani, Aswin Sai Narain Seshasayee, Amit Singh*

*Corresponding author. Email: asingh@iisc.ac.in

Published 13 November 2019, Sci. Transl. Med. 11, eaaw6635 (2019)

DOI: 10.1126/scitranslmed.aaw6635

The PDF file includes:

Materials and Methods Fig. S1. Phenotypic drug tolerance in Mtb during infection. Fig. S2. Flow cytometry–based quantification of redox heterogeneity in Mtb using Mrx1-roGFP2. Fig. S3. EMSH-reduced population is tolerant to Inh. Fig. S4. Transcriptome of Mtb from EMSH-reduced and EMSH-basal fractions. Fig. S5. Measuring phagosomal pH of THP-1 macrophages infected with Mtb/Mrx1-roGFP2. Fig. S6. The transcriptome of Mtb from the EMSH-reduced fraction overlaps with low pH–specific WhiB3 regulon. Fig. S7. WT Mtb generates H2S gas in a pH-dependent manner. Fig. S8. Generation and characterization of Mtb∆metB and Mtb∆sufR. Fig. S9. Deletion of metB and sufR does not impair growth and metabolism of Mtb. Fig. S10. Phagosomal acidification is required for the redox-dependent multidrug tolerance of Mtb. Fig. S11. Drug-tolerant EMSH-reduced population is replicative and has high efflux pump activity. Fig. S12. CQ counteracts drug tolerance in vivo to reduce lung tissue damage in chronic model of Mtb infection. Fig. S13. Long-term CQ treatment of chronically infected BALB/c mice deacidifies macrophage pH without affecting oxidative stress and necrosis. Fig. S14. Model depicting various mechanisms underlying redox-mediated drug tolerance in replicating Mtb. References (94–112)

Other Supplementary Material for this manuscript includes the following: (available at stm.sciencemag.org/cgi/content/full/11/518/eaaw6635/DC1)

Table S1 (Microsoft Excel format). List of differentially expressed genes from DESeq2 for EMSH-reduced, EMSH-basal, and in vitro control samples. Table S2 (Microsoft Excel format). List of differentially expressed genes from DESeq2 for WT Mtb, Mtb∆whiB3, and whiB3-Comp strains at pH 6.6 and pH 4.5 used to specify the low pH–inducible WhiB3 regulon. Table S3 (Microsoft Excel format). List of EMSH-reduced and EMSH-basal differentially expressed genes used to generate custom heat maps. Table S4 (Microsoft Excel format). List of strains and primers used in this study.

Materials and Methods

Bacterial and mammalian culture conditions.

All Mtb strains were cultured in Middlebrook 7H9 or 7H11 synthetic medium with 0.4% glycerol,

0.1% Tween-80 and 10% albumin-dextrose-sodium chloride (ADS), or 10% oleic acid-albumen-dextrose-

catalase (OADC) supplement, as required. For in vitro acid stress exposure, Mtb H37Rv grown to OD600nm

0.4 was exposed to pH 4.5, pH 6.2, and pH 6.6 in 7H9-ADS broth, adjusted to specified pH with 0.1 N

hydrochloric acid (HCl) and buffered with 100 mM MES buffer. For media adjusted to acidic pH, Tween

80 was replaced with 0.02% tyloxapol. Acid exposure for RNA isolation was done for 2 h at 37C at 180

rpm (82). For reductive stress exposure, Mtb H37Rv grown till OD600nm

0.4 was exposed to 2 mM

dithiothreitol (DTT) for 6 h in regular 7H9-ADS broth.

The human monocytic cell line THP-1 was cultured in RPMI-1640 synthetic medium with 10%

fetal bovine serum (FBS) supplement. Monocytes were differentiated by treatment with 20 ng/mL phorbol

12-myristate 13-acetate (PMA) for 18 h. Cells were rested for 2 days following chemical differentiation to

ensure that they reverted to a resting phenotype before infection (12). For isolation of primary peritoneal

macrophages, 4-6 weeks old BALB/c mice were injected intra-peritoneally (i.p.) with 1 mL of 8%

Brewer’s thioglycolate (HiMedia). After 4 days of thioglycolate injection, mice were sacrificed and

macrophages were isolated from the peritoneal sac by lavage with ice-cold phosphate buffered saline (1X

PBS). Cells were cultured in DMEM with 10% FBS supplement for 8-12 h, following which adherent cells

were used as peritoneal macrophages (91). The pro-monocytic cell lines U937 and U1 were grown

similarly, with the exception of 2 mM L-glutamine supplementation for U1 and U937 cells. For all HIV-

TB co-infection experiments in these cell lines, U1 cells were differentiated by treatment with 5 ng/mL

PMA for 18 h following which immediate infection was carried out, as mentioned earlier. U937 cells were

differentiated by PMA treatment at 2.5 ng/mL for 2 days followed by removal of PMA and rest for an

additional 24 h for cells to revert to a resting phenotype (54, 92).

Macrophage infections and survival assays.

PMA-differentiated THP-1 monocytes or primary peritoneal macrophages were infected at a

multiplicity of infection (moi) of 2 for drug tolerance experiments. After 4 h of incubation with bacteria,

infected cells were treated with 0.2 mg/mL amikacin for 2 h, following which infected cells were washed

three times with pre-warmed 1X PBS for complete killing and removal of extracellular bacteria. Washed

cells were re-incubated in complete RPMI-1640 or DMEM media at 37C with 5% CO2, for indicated

time-points of assays. For experiments involving the use of lysosomotropic agents (LAs) to perturb

phagosomal acidity, differentiated macrophages were pre-treated with non-toxic concentrations of LAs- 10

nM BafilomycinA1 (BafA1), 10 M chloroquine (CQ) or 10 mM ammonium chloride (NH4CL)- for 1 h

prior to infection. Cells were maintained in LAs through the course of the experiments (20).

For colony-forming unit (CFU)-based assays, infected cells were lysed in 0.06% sodium dodecyl

sulfate (SDS) in 7H9, diluted as required and plated on 7H11-OADC agar plates. Plates were incubated at

37C for 3 weeks before colonies were enumerated. For drug tolerance assays, percent survival at 48 h of

treatment with antibiotics was determined by quantifying change in CFUs from 0 h of antibiotic treatment

(6).

In vitro and intra-macrophage EMSH determination.

For EMSH determination of intra-macrophage WT Mtb and other strains, macrophages were infected

with Mtb strains expressing Mrx1-roGFP2 at moi 10, as described previously. Infected cells were treated

with 10 mM N-ethylmaleimide (NEM) for 5 minutes at room temperature (RT) followed by 4% para-

formaldehyde PFA for 15 minutes at RT. Fixed cells were then washed, re-suspended in 1X PBS and

gently scraped off for flow-cytometric analysis on BD FACS Verse or BD FACS Aria cytometer (BD

Biosciences). For flow cytometry, 10,000 GFP positive events were analyzed by excitation at 405 and 488

nm with a constant emission (510 nm) to determine EMSH

under different treatment conditions (12). The

BD FACS suite or BD FACSDiva software (BD Biosciences) was used to analyze the population

distribution of Mtb based on mycothiol (MSH) mid-point potential (EMSH): EMSH

-reduced, EMSH

-basal and

EMSH

-oxidized. Number of events per population was counted and representative percentage of each

population was estimated. Gating strategies were devised on the basis of complete oxidation (by 10

mM CHP) and complete reduction (by 50 mM DTT) of the biosensor, as developed previously (12).

For EMSH determination in vitro, Mtb strains expressing Mrx1-roGFP2 were grown to OD600nm 0.4

in 7H9-ADS medium or released from infected macrophages and cultured in RPMI-1640 medium. For

measurement, bacteria were treated with 10 mM NEM followed by 4% PFA at RT, as described earlier.

Fixed bacteria were washed, re-suspended in 1X PBS, and analyzed at 405/488 nm excitations and 510 nm

emission using BD FACS Aria flow cytometer (BD Biosciences) to calculate EMSH (12).

Gating strategy for flow-sorting of redox-diverse fractions.

Using Mrx1-roGFP2, we have previously investigated the redox physiology of Mtb inside

macrophages (12). Confocal microscopy revealed the presence of Mtb cells with a gradient of intra-

mycobacterial EMSH inside a single macrophage (12). Also, the relative proportion of redox-diverse Mtb

shows variations at an individual macrophage level such that the infected macrophage population can be

classified into fractions that predominantly harbor Mtb displaying EMSH-basal (-275 ± 5 mV), EMSH-reduced

(-300 ± 6) mV, or EMSH-oxidized (> -240 mV) (12). These findings are in agreement with other studies

showing that drug-tolerant bacteria originate within a subset of macrophages (9).

In addition to confocal microscopy, which provides EMSH readout at a level of individual bacterium

inside macrophages, we also applied flow cytometry to analyze Mtb/Mrx1-roGFP2 inside macrophages. In

flow cytometry, the readout is derived from averaging median fluorescence intensity from relatively high

number of Mtb inside macrophages. Despite minor variations in measurements, both confocal and flow

cytometry consistently demonstrated the presence of two major fractions of macrophages enriched with

either EMSH-basal or EMSH-reduced bacteria and a minor fraction having EMSH-oxidized bacteria (12). Flow

cytometry data confirm that the relative proportion of macrophages dominated by bacteria showing EMSH-

reduced, EMSH-basal, and EMSH-oxidized was 55-60%, 25-35%, and 5%, respectively, at 24 h p.i. (12). In

sum, Mtb displays redox heterogeneity such that a subset of macrophages is enriched with either EMSH-

reduced, EMSH-basal, or EMSH-oxidized bacteria.

Based on these observations, we followed a flow cytometry based gating strategy to sort

macrophages enriched with EMSH-basal or EMSH-reduced bacteria as described previously (12). THP-1

macrophages infected with Mtb/Mrx1-roGFP2 for 24 h were subjected to ratiometric fluorescence analysis

by flow cytometry. The Mrx1-roGFP2 biosensor was excited by subjecting infected macrophages to 405

and 488 nm laser wavelength at a fixed emission wavelength of 510 nm. We first confirmed that

Mtb/Mrx1-roGFP2 inside macrophages responds ratiometrically to oxidant cumene hydroperoxide (CHP)

and reductant dithiothreitol (DTT) (fig. S2). Gates for EMSH-oxidized and EMSH-reduced bacteria were set

based on nearly complete oxidation or reduction of the biosensor upon treatment of Mtb/Mrx1-roGFP2

infected macrophages with 10 mM CHP or 50 mM DTT, respectively (fig. S2). The gate for the EMSH-basal

population with an intermediate EMSH lies between the oxidized and reduced gates (fig. S2). EMSH for all

oxidation/reduction controls and test samples was additionally quantified using a modified Nernst equation

to ensure stringent analysis of redox potential of individual populations. At 24 h p.i., 405/488 ratio of 60%

of Mtb/Mrx1-roGFP2 infected THP-1 overlapped with the DTT gate (i.e. EMSH-reduced; below -290 mV),

whereas 35% remained in the intermediate gate (i.e. EMSH-basal; -269 mV to -279 mV). The 405/488 ratio

of only 5% bacteria coincided with the CHP gate (fig. S2). The Mtb/Mrx1-roGFP2 infected THP-1

macrophages were sorted into EMSH-basal or EMSH-reduced bins based on the gates set by CHP and DTT

treatment for further analysis. Only samples that maintained reduced (below -290 mV) and basal (-269 mV

to -279 mV) EMSH in post-sort analysis as well, were considered for further downstream processing to

characterize the two populations.

Flow sorting of macrophages harboring redox-altered Mtb populations.

PMA-differentiated THP-1 monocytes were infected with Mtb/Mrx1-roGFP2 at moi 10, as

described previously. At 24 h p.i., infected macrophages were washed once with pre-warmed RPMI-1640,

centrifuged at 800 rpm for 5 minutes and the pellet re-suspended in 3 mL phenol-free RPMI-1640 with

10% FBS supplementation. This cell suspension was passed through a 40 m nylon cell strainer (BD

Falcon) to remove cell clumps. The de-clumped suspension was sorted using a BD FACS Aria flow

cytometer equipped with 405 nm and 488 nm lasers, under the four-way purity sort setting. Voltage

settings for FSC, SSC, 405 nm and GFP channels were kept constant for all experiments. Ratio-metric

changes of excitation at 405 nm and 488 nm upon 10 mM CHP (complete bio-sensor oxidation) and 50

mM DTT (complete bio-sensor reduction) treatment of infected macrophages were utilized to gate and sort

macrophages enriched in EMSH-reduced (below -290 mV) and EMSH-basal Mtb (-269 mV to -279 mV),

along with manual gating strategies to allow for optimal separation between redox populations (12).

Infected macrophages sorted into both bins were analysed individually through post-sort runs to determine

overall EMSH of sorted populations. Only samples that maintained reduced (below -290 mV) and basal (-

269 mV to -279 mV) EMSH in post-sort analysis were considered for further downstream processing to

characterize the two populations. Each analysed population was scored for 10,000 Mrx1-roGFP2 positive

events.

RNA isolation, amplification and library preparation for RNA-seq.

Flow-sorted macrophages harboring EMSH-reduced or EMSH-basal Mtb were collected directly in 4M

GTC buffer containing 1% 2-mercaptoethanol for differential lysis of macrophages and fixation of

bacterial RNA. Fixed bacteria were then centrifuged at 5000 rpm for 5 minutes. Total RNA was isolated as

previously described (93). Each replicate of RNA samples for EMSH-reduced and EMSH-basal populations

was prepared from pooled total RNA, extracted from bacteria collected in 5 different experiments.

Exponentially-grown cultures of Mtb, Mtb∆whiB3 and whiB3-complemented strains were exposed

to 7H9-tyloxapol broth acidified to pH 4.5 or at neutral pH (6.6) for 2 h. Total RNA was isolated as

mentioned earlier.

The concentration and quality of total extracted RNA 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 (15 ng mRNA per sample), first and second cDNA synthesis and library

preparation was 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 (2nM) 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.

Differential gene expression and statistical analysis for RNA-Seq.

A custom (. gff) annotation file was made for the reference genome sequence (.fna) for the wild

type strain Mtb H37Rv (accession number: NC_000962.3) downloaded from the NCBI website

("https://www.ncbi.nlm.nih.gov/nuccore/448814763"). Raw reads from sequencing were obtained as .fastq

files. Raw read quality was checked using the FastQC software (version v0.11.5.) (102). BWA (version

0.7.12-r1039) was used to index the reference genome. Reads with raw read quality ≥ 20 were aligned

using BWA aln -q option (94). SAMTOOLS (version 0.1.19-96b5f2294a) was used to filter out multiply

mapped reads (95). BEDTOOLS (version 2.25.0) was used to calculate the reads count per gene using the

annotation file (.bed) (96).The format of the annotation file (.gff) was changed to .bed using an in-house

python script. The normalization and differential gene expression analysis for the conditions were carried

out using DESeq2 (97). Genes with at least 5 reads were selected for each comparative analysis.

Normalization for heat map generation was done using DESeq2. Differentially expressed genes

(DEGs) were determined based on the cut-off fold-change ≥ 1.5 and FDR ≤ 0.05. The top 100 genes from

the 'reduced vs in vitro control' comparison (log2 fold change between 5.2 and 2.2; FDR ≤ 0.05) and their

related values from the ‘basal vs in vitro control’ comparison were plotted to generate the heat map of top

100 DEGs. A dendrogram for hierarchical clustering in the heatmap was computed by using the heatmap.2

function with default parameters for Euclidean Distance and complete linkage method. Principal

Component Analysis (PCA) was performed using the plotPCA function of DESeq2 in R on the variance

stabilizing transformation (vst) of normalized data obtained. All Volcano and Scatter plots were generated

in RStudio (1.1.447) with R version 3.4.4 using ggplot2 of DESeq2 package in R (103). The p-value of

differential expression analysis was obtained through DeSeq2 after correction for multiple hypothesis

testing using false discovery rate (padj or FDR at a cut-off ≤ 0.05). Transcriptomic data are uploaded to the

NCBI GEO database, accession number GSE123267.

For overlap analysis, the list of genes for the reference study was matched with the gene annotation

file used in this study and only common genes were considered for analysis.

For identification of the acidic pH-inducible WhiB3 regulon, up-regulated DE genes in the WT Mtb

pH 4.5 versus pH 6.6 comparison which were simultaneously repressed in the Mtb∆whiB3 pH 4.5 versus

WT Mtb pH 4.5 comparison were extracted. This list of genes was cross-checked against genes in the

whiB3-Comp pH 4.5 versus pH 6.6 for partial or complete complementation (including non-significant

genes [FDR > 0.05], in certain cases). All genes short-listed from the above comparisons were selected

based on a cut-off of FDR ≤ 0.05 (tables S2A-D).

Inh efflux assay.

A logarithmically growing culture of Mtb/Mrx1-roGFP2 was incubated with [14

C]-Inh (ViTrax Inc.)

at a concentration of with 0.5 Ci/mL, as reported previously (51, 99). After 2 h of treatment, bacteria

were pelleted, extensively washed to remove residual [14

C]-Inh and used for infection of PMA-

differentiated THP-1 monocytes at moi 10, as described earlier. At 24 h p.i., equal number of infected cells

were flow-sorted with respect to the EMSH of resident bacteria and lysed in 0.06% SDS-7H9. The

supernatant from macrophage lysis was collected and the bacterial pellet was further subjected to lysis by

bead-beating. Lysates from the macrophage and bacterial disruption steps were concentrated by speed-vac,

loaded on Whatman No. 3 filter paper strips and suspended in 1,4-Bis(5-phenyl-2-oxazolyl)benzene

solution in toluene (or POPOP luminophore, Sigma). Radioactivity was measured by a liquid scintillation

counter (WALLAC 1410, Perkin Elmer) and counts for each fraction were calculated as a percentage of

total counts from the EMSH-reduced and EMSH-basal samples individually.

Measuring phagosomal pH of macrophages enriched with EMSH-reduced or EMSH-basal bacteria.

The Mtb/Mrx1-roGFP2 strain was labeled with 20 M pHrodo Red succinimidyl ester (Molecular

Probes), an amine-reactive pH-sensitive dye, which emits at 585 nm in an acidic environment (21). The

pHrodo labeled Mtb/Mrx1-roGFP2 was used to infect THP-1 macrophages at moi 10 and at 6 h post-

internalization mutli-parametric flow analysis was used to measure pHrodo fluorescence in infected

macrophages. The EMSH-reduced and EMSH-basal fractions were determined from the infected macrophage

population. Infected macrophages pre-treated with 10 µM CQ or 10 nM BafA1 were used as controls for

phagosomal alkalinization. For calculating phagosomal pH, pHrodo SE median fluorescence intensities

(MFI) were acquired for infected macrophages incubated in buffers of pH 7.0, 6.2, 5.0 and 4.5 in presence

of 10 µM nigericin and 10 µM valinomycin (to equilibrate phagosomal pH with external pH) in CO2-

independent medium for 1h before flow acquisition. A calibration curve was generated by finding the

linear relation between pHrodo SE fluorescence intensities in the range of pH 4.5-7.0 and this was used to

calculate the phagosomal pH of untreated, CQ- and BafA1- treated infected macrophages, as well as for the

EMSH-reduced and EMSH-basal fractions at 6 h p.i.

Identification of acidic pH inducible WhiB3 regulon through RNA-seq data.

For identification of the acidic pH-inducible WhiB3 regulon, up-regulated DE genes in the WT Mtb

pH 4.5 versus pH 6.6 comparison which were simultaneously repressed in the Mtb∆whiB3 pH 4.5 versus

WT Mtb pH 4.5 comparison were extracted. This list of genes was cross-referenced against genes in the

whiB3-Comp pH 4.5 versus pH 6.6 comparison for partial or complete complementation. All genes short-

listed from the above comparisons were selected based on a cut-off of FDR ≤ 0.05 (tables S2A-D).

Cloning and knock-out validation.

1kb left and right flanking regions of the genomic locus of sufR (Rv1460) and metB (Rv1079) were

amplified and cloned upstream and downstream, respectively, of the loxP-hyg-gfp-loxP cassette in the

mycobacterial sacB-based suicide vector pML523 (a kind gift from Michael Niederweis at the University

of Alabama at Birmingham). The complete construct of the flanking sequences and the hyg-gfp construct

was then amplified and cloned into the pRSF-duet vector (Clontech). The pRSF-sufR and pRSF-metB

clones were pre-treated with ultraviolet light, as described previously (20), and electroporated into WT Mtb

H37Rv for allelic exchange. Colonies were screened for positive clones (Hygr-Kan

s-GFP+) for either

knock-out strain by antibiotic selection and genomic DNA PCR. Disruption of sufR and metB genes were

also confirmed by quantitative RT-PCR (qRT-PCR). To unmark the knock-out strains, the pCRE-ZEO-

SacB plasmid (a kind gift from Amit K. Pandey at the Translational Health Science and Technology

Institute, Haryana, India) was electroporated into both MtbsufR and MtbmetB knock-out strains to allow

for loss of the loxP-hyg-gfp-loxP cassette from the genome. The resulting unmarked strains were confirmed

by the loss of GFP fluorescence and antibiotic selection.

qRT-PCR analysis.

Total RNA from EMSH-reduced or EMSH-basal Mtb populations growing inside macrophages was

isolated, as described earlier. Purified total RNA obtained was treated with DNase and 100 ng of purified

RNA was then subjected to amplification using MessageAmp II RNA amplification kit (Ambion) as

previously described (17). 200 ng of amplified RNA (aRNA) was used for cDNA synthesis for subsequent

qRT-PCR analysis. For in vitro cultures, logarithmically grown Mtb H37Rv and other strains at OD600nm

0.4 was harvested untreated or upon exposure to 250 M cumene hydroperoxide (CHP) for 2h, 2 mM

dithiothreitol (DTT) for 6 h or acidic pH exposure in 7H9-tyloxapol broth for 2 h, and total RNA was

isolated as described earlier (20) Total RNA obtained was purified, DNase treated and quantified. 200 ng of

purified RNA was used for cDNA synthesis. Random oligonucleotide primers were used from the iScript

Select cDNA Synthesis Kit (BioRad) for this purpose. Gene-specific primers were selected for qRT-PCR

(CFX96 RT-PCR System, BioRad) and iQ SYBR Green Supermix (BioRad) was used for gene expression

analysis (table S4). Ct value for 16s rRNA was used as an internal normalization control in all cases (20,

93).

Bismuth Chloride assay for H2S determination.

To quantify the H2S-generation capacity of Mtb strains, bismuth chloride (BiCl3) test was used

(104). Briefly, bacteria were grown to OD600nm 0.6 and pre-treated for 1 h in 7H9-tyloxapol broth acidified

to pH 4.5 and 6.2, as described earlier. 108 cells were taken from each pre-treatment condition and

incubated with 100 μL BiCl3 buffer for an additional 3 h. BiCl3 buffer was composed of 0.4 M

triethanolamine-HCl, pH 8.0; 10 mM bismuth(III)chloride; 20 μM pyridoxal 5-phosphate monohydrate, 20

mM EDTA and 40 mM l-cysteine. Absorbance was measured at OD405nm at the indicated time points. The

absorbance of cells suspended in BiCl3 buffer without cysteine was taken as blank and subtracted from all

the other readings.

Growth curves for Mtb strains.

Freezer stocks of WT Mtb, mutant and complemented strains were diluted 1:10 in 7H9-ADS broth

and grown for 1 week. The one week old cultures were inoculated in 7H9-ADS broth at OD600nm 0.05 and

incubated at 37ºC at 180 rpm for 7 days. OD600nm was recorded every 24 h till cultures reached stationary

phase.

OCR and ECAR measurements of Mtb strains by Seahorse XF Flux Analyser.

Exponentially grown WT Mtb and other strains were starved for 20 h in 7H9 supplemented with

0.01% tyloxapol. Single cell suspension of the strains was individually prepared and adhered to the bottom

of cell-tak (1 μg per well; Corning) coated wells of XF cell culture microplate (Seahorse Biosciences) at a

density of 2 × 106 bacilli per well. Assays were conducted in unbuffered 7H9 without carbon sources. Basal

oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured for ∼21 min (3

readings) before and after the addition of 2 mg/mL glucose automatically through the drug ports of the

sensor cartridge (Wave Software, Seahorse Biosciences) (105). Percentage changes in OCR and ECAR

were calculated as a percentage of the third baseline values for all strains. Figures indicate the point of

addition of glucose in the assay.

Isolation and culture of peritoneal macrophages and Mtb infection.

For isolation of primary peritoneal macrophages, 4-6 weeks old BALB/c mice were injected intra-

peritoneally (i.p.) with 1 mL of 8% Brewer’s thioglycolate (HiMedia). After 4 days of thioglycolate

injection, mice were sacrificed and macrophages were isolated from the peritoneal sac by lavage with ice-

cold phosphate buffered saline (1X PBS). Cells were cultured in DMEM with 10% FBS supplement for 8-

12 h, following which adherent cells were used as peritoneal macrophages (91). Infection of peritoneal

macrophages, untreated or pre-treated with LAs, with Mtb strains for EMSH-determination and antibiotic

tolerance were conducted, as described earlier.

Alamar Blue Assay.

To determine percentage inhibition, microplate Alamar blue assay (MABA) was performed in a 96

well flat bottom plate (106). Mtb strains were cultured in 7H9-ADS and grown till exponential phase

(OD600nm 0.4). Approximately 1*105 bacteria were taken per well in a total of 200 μL of media. After 5

days of incubation at 37C, 20 μL of Alamar blue was added and the plates were re-incubated for 24 h. The

fluorescence readings were recorded in a Spectramax M3 microplate reader with excitation at 530 nm and

emission at 590 nm. Percentage inhibition was calculated based on the relative fluorescence units and the

minimum concentration that resulted in at least 90% inhibition (Minimum Inhibitory Concentration or

MIC).

CV staining.

NEM-fixed, flow-sorted EMSH-reduced and EMSH-basal Mtb containing macrophages were lysed in

0.06% SDS and re-suspended in 1X PBS. The Calcein Violet Acetoxy Methyl ester-based dye (CV-AM)

(Invitrogen, Life Technologies) was freshly dissolved in 25 L of DMSO for each experiment. 1 L of this

stock solution was added to 100 L of sample (containing 5 X106 bacteria obtained either from lysis of

infected macrophages or in vitro control cultures) and the sample was incubated at 37C while shaking at

220 rpm (107). As a control for metabolic inactivity, an in vitro cultured sample at similar bacterial density

was stained at 4C for the same duration. Post-incubation, cells were washed with 1X PBS and analyzed

for CV positive events on the BD FACS Aria flow cytometer, through excitation at 405 nm and emission at

450 nm. Each analyzed population was scored for 10,000 CV positive events.

Replication clock plasmid.

The Mtb/Mrx1-roGFP2 strain was electroporated with the kanamycin-resistant (Kanr) unstable

clock plasmid pBP10 (generous gift from Dr. David R. Sherman, Seattle Children’s Research Institute,

Seattle, USA) and the resulting strain was used to calculate replication dynamics of EMSH-reduced and

EMSH-basal intra-macrophage Mtb populations. Briefly, PMA-differentiated THP1 monocytes were infected

at moi 10 with the Mtb/Mrx1-roGFP2 strain harboring pBP10, as described earlier. At 0, 24 and 72 h p.i.,

equal number of infected macrophages enriched with EMSH-reduced and EMSH-basal bacteria were flow-

sorted. The sorted samples were lysed in 0.06% SDS to release intra-macrophage Mtb, followed by dilution

and plating on 7H11 agar plates with and without 25 g/mL kanamycin. Based on loss of kanamycin

resistance over time, replication and death rates as well as cumulative bacterial burden (CBB) were

determined using the mathematical model, as previously described (58).

Modified histopathology-scoring system.

The table illustrates the basis of a modified system for scoring lung lesions in mice and guinea pigs

infected with Mtb, based on number of lesions and area involved in granuloma. The modified system

ranked granuloma score on a scale of 0-4.

Characteristics Granuloma score Modified score

Heavy involvement >50 4

Moderate involvement 11-50 3

Scanty involvement 4-10 2

Minimal involvement 1-3 1

No involvement 0 0

Mice lung tissue preparation and macrophage staining.

BALB/c mice (4-6 weeks old) were chronically infected with WT Mtb for 4 weeks, beyond which

animals were segregated into 4 groups- untreated, only CQ treated, only Inh treated or combination

treatment with CQ and Inh- as described earlier. At 6 weeks of treatment, animals were sacrificed and

whole lungs were aseptically removed to prepare a single-cell suspension for flow cytometry. Lungs were

minced and digested in serum-free RPMI containing 0.2 mg/mL Liberase DL (Roche) and 0.1 mg/mL

DNase I (Roche) for 60 minutes at 37ºC at 180 rpm. This was followed by mechanical disruption of the

suspension using the GentleMACS dissociator (Militenyi Biotech) and de-clumping by passage through 70

M nylon cell strainers (BD Falcon). For removal of all red blood cells, the single cell suspension was

incubated in RBC lysis buffer for 15 mins with gentle vortexing (108).

For staining of surface markers, lung cell suspensions were blocked with anti-mouse CD16/CD32

(Fc Block, BD Pharmingen) followed by anti-mouse CD64 (FCRI, APC-conjugated; BioLegend) and

MerTK (DS5MMER, Alexa Fluor 700-conjugated; eBioscience) antibodies. The CD64 and MerTK double

positive cells were gated to analyse total lung macrophage population in mice lungs (109).

pH, ROS and necrosis measurements in lung macrophages.

It is known that CQ functions as a weak base, which diffuses across a pH gradient into acidic sub-

cellular compartments such as endosomes/phagosomes and becomes di-protonated at lower pH (50). This

results in alkalization of endosomal/phagosomal pH. However, it is not clear if the observed decrease in

drug tolerance is associated with de-acidification of phagosomal pH in vivo. To clarify this, lung resident

macrophages of BALB/c mice, chronically infected with Mtb and treated with CQ for 6 weeks, were

isolated and stained with MerTK and CD64 (pan-macrophage surface markers to identify macrophages in

mice lungs).

To assess change in pH of lung macrophages, we utilized MR Cathepsin B (Immunochemistry

Technologies) which is a fluorogenic protease substrate that fluoresces upon hydrolysis only under acidic

pH (73). The fluorogenic substrate was re-suspended in DMSO, as recommended in the manufacturer’s

protocol. Total lung cell suspension was seeded in a 96-well plate in RPMI-1640 supplemented with 10%

FBS at a density of 50,000 cells per well and incubated for 1 h at 37ºC in 5% CO2, for adherence of cells,

after which complete media was removed and replaced with RPMI-1640 with 1X dilution of MR Cathepsin

B substrate for additional 1 h under the same conditions. After 1 h of incubation, media was removed, cells

were washed twice with PBS and stained for cell surface macrophage markers. Flow cytometry analysis of

MR Cathepsin B labeled lung macrophages was done at 560 nm excitation and 585 nm emission.)

To examine if the long treatment duration of CQ plus Inh used in our animal experiments triggered

other influences of CQ such as ROS generation and necrosis, we treated mice chronically infected with

Mtb with CQ, CQ plus Inh, and Inh alone for additional 6 weeks. Lung macrophages were isolated stained

with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA; excitation: 495 nm and emission: 529 nm) to

measure ROS and propidium iodide (PI; excitation: 535 nm and emission: 617 nm) to examine macrophage

death by flow cytometry. For H2DCFDA (Molecular Probes) staining, total macrophages were incubated

with 10 M dye at 37ºC for 30 minutes. Extracellular stain was washed off post-incubation and suspension

was stained for cell surface markers (72). For propidium iodide (PI) staining, total macrophages were

incubated with 10 M PI at RT for 15 minutes in dark. Extracellular dye was washed off post-incubation

and suspension was stained for cell surface markers (72). All samples were analyzed by multi-parametric

flow cytometry analysis using BD FACS Aria (BD Biosciences).

PK-DDI studies.

4-6 weeks old BALB/c mice (n = 6 per group) were dosed with CQ, first-line anti-TB therapy

regimen HREZ or a combination of both, as follows: CQ, 10 mg/kg body weight i.p. injection; Inh (H) 25

mg/kg body weight by mouth or per os (p.o.); Rif (R) 10 mg/kg body weight p.o.; Emb (E) 200 mg/kg

body weight p.o. and Pza (Z) 150 mg/kg body weight p.o. All drug formulations were made in 0.5%

hydroxypropyl methyl cellulose and 0.1% Tween 80. Care was taken to pre-dose Rif 2 h prior to

administration of CQ and other antibiotics to minimize drug-drug interactions during adsorption

stage(110). Blood samples were collected from animals in the three treatment groups at regular intervals

over a period of 24 h from dosing and plasma was isolated. All plasma samples along with drug controls

were analysed by LC-MS (Waters Acquity UPLC system, flow rate 0.300 l/mL with gradient elution;

Waters Acquity-TQD Triple Quadrupole Mass Spectrometer). Calibration curves were generated for each

analyte and accepted if 67% of calibration points were within ± 20% nominal concentration.

Pharmacokinetics profile was generated for each drug in terms of mean plasma concentration over 24 h,

maximum concentration achieved (Cmax) and net exposure (AUClast) (111).

Fig. S1. Phenotypic drug tolerance in Mtb during infection. Diagram illustrating various mechanisms

involved in mediating phenotypic drug tolerance in Mtb. Hostile stresses faced by Mtb upon immune-

activation (Interferon-) induce a non-growing metabolically active (NGMA; growth-restrictive) state

wherein bacteria are tolerant to multiple anti-TB drugs. Various host and bacterial factors involved in

mobilizing drug tolerance and replication/metabolic slow down are indicated. Mtb also tolerates antibiotics

during active multiplication in a naïve macrophage (growth-permissive stage) by up-regulating drug efflux

pumps. However, the identity of host factors, bacterial determinants, and the physiology of drug-tolerant,

actively-replicating bacteria remain uncharacterized. In the present study, we performed a mechanistic

dissection of the link between limited phagosomal acidification and redox physiology of Mtb in tolerating

antibiotics inside non-activated macrophages.

Fig. S2. Flow cytometry–based quantification of redox heterogeneity in Mtb using Mrx1-roGFP2.

THP-1 macrophages infected with Mtb/Mrx1-roGFP2 were treated with an oxidant cumene hydroperoxide

(CHP) and a reductant dithiothreitol (DTT), independently. Biosensor median fluorescence ratio (405/488

nm excitation with fixed emission at 510 nm) of 10,000 GFP-positive macrophages was analysed by flow

cytometry for each control. Dot plots show a complete shift in population towards oxidized (high 405/488;

EMSH-oxidized) or reduced (low 405/488; EMSH-reduced) after treatment with 10 mM CHP (yellow dots)

(A) and 50 mM DTT (blue dots) (B), respectively. (C) Dot plot shows an overlay of EMSH-oxidised and

EMSH-reduced fractions of infected THP-1 cells. Infected macrophages that fall neither in the EMSH-oxidised

nor in the EMSH-reduced gates are classified as EMSH-basal (red dots). (D) Bar plot shows the ratiometric

biosensor response. (E) Dot plot shows THP-1 macrophages infected with Mtb/Mrx1-roGFP2 at 24 h p.i.

(F) Bar plot shows percentage of infected macrophages enriched with EMSH-reduced (Red), EMSH-oxidized

(Ox), EMSH-basal (Bas) bacteria at 24 h p.i. based on the overlap of these gated cells with the CHP

(oxidized) or DTT (reduced) treated cells (supplementary methods).

Fig. S3. EMSH-reduced population is tolerant to Inh.

(A) THP-1 macrophages enriched in EMSH-reduced and EMSH-basal fractions were collected by flow sorting

at 24 h p.i. (B) The individually isolated redox-diverse macrophage fractions were exposed to Inh (2.18

µM, 3X of in vitro MIC) or left unexposed, for an additional 48 h. Bacillary load was determined by CFU

enumeration and percent survival was quantified by normalizing the CFU in drug-treated samples at 48 h

against untreated samples at 0 h for each fraction. *p < 0.05, **p < 0.01 by Mann-Whitney Test. Data

shown are the result of three independent experiments performed in duplicate (mean ± S.D.).

Fig. S4. Transcriptome of Mtb from EMSH-reduced and EMSH-basal fractions.

(A) Principal component analysis (PCA) plot shows all RNA-seq samples- EMSH-reduced, EMSH-basal, and

in vitro control- clustering with their biological replicates. (B) Heat map for log values of normalized

counts, re-scaled between +2 and -2, along with hierarchical clustering dendrogram indicates gene

expression profile for top 100 differentially expressed genes (DEGs) in the EMSH-reduced sample compared

to EMSH-basal and in vitro control. All DEGs are based on false detection rate (FDR) ≤ 0.05 and fold-

change 1.5 (tables S1A-C).

Fig. S5. Measuring phagosomal pH of THP-1 macrophages infected with Mtb/Mrx1-roGFP2. (A)

Exponentially grown Mtb/Mrx1-roGFP2, stained with 20 µM pHrodo SE, was used for infecting THP-1

macrophages. At 6 h post-internalization, infected macrophages were analyzed by multi-parameter flow

cytometric analysis for quantifying redox-altered fractions as well as for pHrodo fluorescence.

Lysosomotropic agents like BafilomycinA1 (BafA1, 10 nM) and Chloroquine (CQ, 10 µM) alkalinize

intra-cellular compartments by blocking vacuolar ATPases (19) and by undergoing protonation themselves

(66) respectively and were, therefore, used as controls. (B) Calibration curve for calculating phagosomal

pH was generated by analyzing pHrodo median fluorescence intensities of infected macrophages incubated

in buffers of increasing acidity in CO2-independent medium for 1 h before flow analysis. (C) Flow spectra

showing pHrodo fluorescence emitted by EMSH-reduced and EMSH-basal fractions. (D) Estimated value of

phagosomal pH of indicated samples based on the calibration curve. A difference in phagosomal pH was

observed for the EMSH-reduced and EMSH-basal fraction. This is consistent with an earlier report of intra-

phagosomal Mtb displaying variations in induction of the low pH-sensitive aprA locus, indicative of

heterogeneity in acidification of unstimulated macrophages (112). *p < 0.05 by Mann-Whitney test. Data

shown are a representative of two independent biological experiments (mean ± S.D. of three technical

replicates).

Fig. S6. The transcriptome of Mtb from the EMSH-reduced fraction overlaps with low pH–specific

WhiB3 regulon. In vitro grown WT Mtb, Mtb∆whiB3 and whiB3-Comp were exposed to 7H9-tyloxapol

broth acidified to pH 4.5 for 2 h. RNA was isolated and RNA-seq analysis conducted using DeSeq2. Genes

were considered under WhiB3 regulation during low pH stress if found induced in WT Mtb pH 4.5 versus

pH 6.6 comparison and simultaneously reduced in the Mtb∆whiB3 pH 4.5 versus WT Mtb pH 4.5

comparison. Complementation of the mutant with WhiB3 partially or fully restored this effect. Heat map

indicates log2 fold changes of in vitro acidic pH-inducible genes in the WhiB3 regulon as well as in the

transcriptome of EMSH-reduced (A) and EMSH-basal fractions (B). All genes short-listed from the above

comparisons were selected based on a cut-off of FDR ≤ 0.05 (tables S2A-D).

Fig. S7. WT Mtb generates H2S gas in a pH-dependent manner. Exponentially grown WT Mtb was pre-

treated for 1 h in 7H9-tyloxapol broth acidified to pH 4.5 and 6.2 and then assayed for H2S generation by

colorimetric bismuth chloride assay. Changes in OD405nm absorbance indicative of pH-dependent H2S

generation by Mtb are shown in the bar plot. *p < 0.05 by Mann-Whitney test. Data shown are the results

of three experiments performed in triplicate (mean ± S.D.).

Fig. S8. Generation and characterization of Mtb∆metB and Mtb∆sufR. (A) Schematic depicts genomic

locus of metB (Rv1079) and double crossover recombination event for replacing the gene with a loxP-hyg-

gfp-loxP cassette. Colour-coded arrows depict primer pairs used for knock-out confirmation by PCR from

genomic DNA isolated from potential clones. (B) Genomic DNA was isolated from WT Mtb and putative

Mtb∆metB colony (KO1) and replacement of metB allele with Hyg-GFP cassette was confirmed using

PCR. An increase in amplicon size from 1.16 to 4.48 kb due to insertion of the loxP-hyg-gfp-loxP cassette

was observed in case of mutant clones, confirming the double crossover event. (C) RNA was isolated from

exponentially grown WT Mtb and the putative Mtb∆metB clone. qRT-PCR for metB was done using metB-

specific oligonucleotides and Ct values were plotted to assess the expression. (D) Schematic depicts

genomic locus of sufR (Rv1460) and double cross-over recombination event for replacing the gene with a

loxP-hyg-gfp-loxP cassette. Colour coded arrows depict primer pairs used for clone confirmation by PCR

from genomic DNA of clones. (E) Genomic DNA was isolated from WT Mtb (WT) and putative Mtb∆sufR

colonies (KO1, KO2, KO3, KO4, and KO5) and replacement of sufR allele with Hyg-GFP cassette was

confirmed using PCR. An increase in amplicon size from 2.6 to 4.8 kb due to insertion of the loxP-hyg-gfp-

loxP cassette was observed in case of mutant clones, confirming the double crossover event. (F)

Exponentially grown WT Mtb, Mtb∆metB and metB-complement (metB-Comp) were pre-treated for 1 h in

7H9-tyloxapol broth acidified to pH 4.5 and 6.2 and then assayed for H2S generation by colorimetric

bismuth chloride assay. Changes in OD405nm absorbance, indicative of pH-induced H2S generation in a

metB-dependent manner, are quantified in the bar plot. *p<0.05, **p < 0.01 by Mann-Whitney test. (G)

Exponentially grown WT Mtb and Mtb∆sufR were exposed to 250 μM of cumene hydroperoxide (CHP) for

2 h or left untreated and the expression of genes belonging to suf operon (Rv1460-Rv1466) was examined

by qRT-PCR. Fold change is plotted upon normalization with 16S rRNA transcript. Consistent with the

role of suf operon in repair of Fe-S clusters upon oxidative stress, the operon was induced in Mtb under

oxidative stress. Mtb∆sufR did not show induction of suf operon in response to CHP exposure, indicating

reduced ability to generate Fe-S clusters. */#p < 0.05, **/##p < 0.01 by Mann-Whitney test. ‘*’ and ‘#’

compare relative expression in CHP treated and untreated samples for WT Mtb and Mtb∆sufR, respectively.

Data shown are the result of three independent experiments performed in triplicate (mean ± S.D.).

Fig. S9. Deletion of metB and sufR does not impair growth and metabolism of Mtb. (A, B) Growth

curves (OD600nm) for WT Mtb, Mtb∆metB and Mtb∆sufR and their complemented strains over 7 days of

culturing in 7H9-ADS broth are depicted in the line plots. P values were determined using Kruskal-Wallis

test with Dunn’s correction across strains at each time-point. Data shown are the result of three independent

experiments performed in triplicate (mean ± S.D.). No significant difference was observed between WT

Mtb and the mutant/complement strains (p > 0.05). (C, D) Changes in Oxygen Consumption Rate (or

OCR) and Extracellular Acidification Rate (or ECAR), for indicated strains, were quantified upon addition

of 2 mg/mL glucose following overnight glucose starvation by Seahorse XF flux analyser. P values were

determined using Kruskal-Wallis test with Dunn’s correction. Data shown are the result of two independent

experiments performed in triplicate (mean ± S.D.). ‘ns’, no significant difference among indicated strains

in each plot (p > 0.05) post glucose addition.

Fig. S10. Phagosomal acidification is required for the redox-dependent multidrug tolerance of Mtb.

(A) Peritoneal macrophages isolated from BALB/c mice, untreated or pre-treated with 10 nM BafA1 or 10

µM CQ, were infected Mtb/Mrx1-roGFP2 and percent distribution of redox-diverse fractions was

measured at 24 h p.i. **p < 0.01 compares EMSH-reduced fractions between untreated and BafA1/CQ

treated samples by Mann-Whitney test. (B) Peritoneal macrophages isolated from BALB/c mice, untreated

or pre-treated with 10 nM BafA1 or 10 µM CQ, were infected with WT Mtb for 24 h and exposed to Inh

(2.18 µM, 3X of in vitro MIC) or left unexposed for an additional 48 h. Bacillary load was determined by

CFU enumeration and the percent survival was quantified by normalizing the CFU in drug-treated samples

at 48 h against untreated samples at 0 h. *p < 0.05, **p < 0.01 by Mann-Whitney test. (C) EMSH-reduced

bacteria released from THP-1 macrophages at 24 h p.i., were used to determine changes in MIC of Inh

using the microplate Alamar blue assay (MABA). Mtb H37Rv grown in 7H9 broth was used as a control.

Both samples showed comparable MIC. Data shown in each panel are the result of three independent

experiments performed in triplicate (mean ± S.D.).

Fig. S11. Drug-tolerant EMSH-reduced population is replicative and has high efflux pump activity. (A)

THP-1 macrophages were infected with Mtb/Mrx1-roGFP2 and Mtb/Mrx1-roGFP2 containing pBP10. At

indicated time-points, redox-diverse fractions were calculated for both strains. P values were calculated

using Mann-Whitney test. ‘ns’, no significant difference (p > 0.05) from the EMSH-reduced population in

the pBP10 negative strain. Exponentially grown WT Mtb was exposed to 7H9-tyloxapol broth acidified to

pH 6.2 and 4.5 for 2 h (B) or to 2 mM DTT in 7H9 broth for 6 h (C). Bacterial RNA was isolated and

expression of efflux pumps was measured by qRT-PCR for WT Mtb. Expression was compared with 7H9

broth-grown, untreated WT Mtb at pH 6.6 and fold change was quantified after normalizing with 16S

rRNA transcript. (D) Exponentially grown WT Mtb and MtbΔmshA was exposed to 7H9-tyloxapol broth

acidified to pH 6.2 and 4.5 for 2 h. Bacterial RNA was isolated and expression of efflux pumps was

measured by qRT-PCR. Expression was compared with untreated WT Mtb at pH 6.6 and fold change was

quantified after normalizing with 16S rRNA transcript. *p < 0.05, **p < 0.01 by Mann-Whitney test. ‘*’

compares gene expression of all treated strains with untreated WT Mtb. Data shown in each panel are the

results of at least two independent experiments performed in triplicate (mean ± S.D.).

Fig. S12. CQ counteracts drug tolerance in vivo to reduce lung tissue damage in chronic model of

Mtb infection. Scatter plots indicate modified granuloma score (supplementary methods) for lung section

histopathology of chronically-infected mice (A) and guinea pigs (B) across experimental groups at 8 weeks

of treatment. *p < 0.05 by Mann-Whitney test. Data shown are representative of two experiments with n =

4 animals per group (median ± inter-quartile range).

Fig. S13. Long-term CQ treatment of chronically infected BALB/c mice deacidifies macrophage pH

without affecting oxidative stress and necrosis. (A) Dot plots indicate the sequential gating strategy for

identifying macrophages from lung homogenates of infected and uninfected BALB/c mice at 6 weeks of

antibiotic and/or CQ treatment. Pan-macrophage surface markers (double positive for CD64+MerTK

+)

were stained for identifying total lung macrophage population. (B) Representative histogram for MR

Cathepsin B fluorescence and quantification of lung macrophages positive for intra-cellular cathepsin B

protease activity in chronically infected mice from the untreated and CQ-treated groups. *p < 0.05 by

Mann-Whitney test. (C) Representative histogram and quantification for DCFDA fluorescence in lung

macrophages of chronically infected mice across experimental groups. (D) Representative histogram for

propidium iodide (PI) fluorescence and quantification of PI-positive lung macrophages in chronically

infected mice across experimental groups. P values were calculated using Mann-Whitney test. Data shown

are representative of two independent experiments with n = 4-6 animals per group (median ± inter-quartile

range). ‘ns’, no significant difference (p > 0.05). Abbreviations: ‘UI’, uninfected; ‘UT’, untreated; ‘CQ’,

CQ treatment only; ‘Inh’ Isoniazid treatment only; ‘CQ+Inh’, CQ and isoniazid combination treatment.

Fig. S14. Model depicting various mechanisms underlying redox-mediated drug tolerance in

replicating Mtb. Phagosomal acidification inside resting macrophages serves as a cue to induce redox

heterogeneity and drug tolerance in Mtb. Gene expression data indicate that low pH induces accumulation

of cysteine (CySH) causing reductive stress. Elevation of CySH is known to generate reactive oxygen

species (ROS) through metal (Fe/Cu) catalysed oxidation to cystine (CyS2). Acidic pH also increases

solubility of metals such as Fe and Cu, which drives generation of ROS via Fenton chemistry. Also, various

anti-TB drugs induce oxidative stress inside Mtb during infection. To manage these stresses, Mtb efficiently

channelizes the flux of CySH in reverse transsulfuration pathway (H2S generation), Fe-S cluster biogenesis

(SufR/WhiBs), and MSH production, resulting a reductive shift in the EMSH of Mtb. All of these

mechanisms are known to protect bacteria from drugs and oxidative stress by metal sequestration and

activation of antioxidants. Increased expression of metal and drug efflux pumps further remediate

metal/antibiotic triggered redox stress in Mtb. Induction of SAM-dependent methyl-transferase in EMSH-

reduced bacteria can directly inactivate drugs by N-methylation. Impaired ability of metB, sufR, and whiB3

mutants in tolerating antibiotics suggests management of CySH flux as an important bacterial strategy to

protect from drugs. Pharmacological inhibition of phagosomal acidity by CQ restores redox heterogeneity

to subvert drug tolerance and post-therapeutic relapse.