Enhanced respiration prevents drug tolerance and drug resistance in Mycobacterium tuberculosis

Catherine Vilchèze; Travis Hartman; Brian Weinrick; Paras Jain; Torin R. Weisbrod; Lawrence W. Leung; Joel S. Freundlich; William R. Jacobs

doi:10.1073/pnas.1704376114

Contributed by William R. Jacobs Jr., March 17, 2017 (sent for review October 7, 2016; reviewed by Courtney C. Aldrich, Meindert H. Lamers, and Alan Sher)

Significance

Tuberculosis (TB) patients would greatly benefit from shorter treatment options. The treatment of drug-susceptible TB, a disease caused by the bacillus Mycobacterium tuberculosis, is a lengthy and strenuous process. This long therapy is because of the ability of a small population of cells to become drug-tolerant. Here, we demonstrate that the addition of small thiols to drug-treated M. tuberculosis prevents the emergence of drug-tolerant but also drug-resistant cells leading to sterilization of the cultures in vitro. The thiols potentiate drug activity by preventing the cells from entering a persister state and shutting down their metabolism while generating an oxidative burst. This dual mechanism of killing could lead to novel approaches to shorten TB chemotherapy.

Abstract

Persistence, manifested as drug tolerance, represents a significant obstacle to global tuberculosis control. The bactericidal drugs isoniazid and rifampicin kill greater than 99% of exponentially growing Mycobacterium tuberculosis (Mtb) cells, but the remaining cells are persisters, cells with decreased metabolic rate, refractory to killing by these drugs, and able to generate drug-resistant mutants. We discovered that the combination of cysteine or other small thiols with either isoniazid or rifampicin prevents the formation of drug-tolerant and drug-resistant cells in Mtb cultures. This effect was concentration- and time-dependent, relying on increased oxygen consumption that triggered enhanced production of reactive oxygen species. In infected murine macrophages, the addition of N-acetylcysteine to isoniazid treatment potentiated the killing of Mtb. Furthermore, we demonstrate that the addition of small thiols to Mtb drug treatment shifted the menaquinol/menaquinone balance toward a reduced state that stimulates Mtb respiration and converts persister cells to metabolically active cells. This prevention of both persister cell formation and drug resistance leads ultimately to mycobacterial cell death. Strategies to enhance respiration and initiate oxidative damage should improve tuberculosis chemotherapies.

Multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) have become major threats to global public health (1). The emergence of MDR- and XDR-TB has been attributed primarily to the lengthy, 6-mo TB treatment. Failure to complete a full course of treatment can lead to the development of drug resistance and relapse, which requires up to 2 y to treat.

Resistance to the first-line TB drug isoniazid (INH) is most often a result of mutations in katG, which encodes a catalase peroxidase, the activator of INH (2), but other genes have also been associated with INH resistance in TB (3). In particular, genes involved in the biosynthesis of mycothiol, the main reducing thiol in mycobacteria and a storage molecule for cysteine (4), have been implicated in resistance to INH and the second-line TB drug ethionamide (ETH) in mycobacteria (5 ⇓⇓–8). We sought to explain the mechanisms by which mutations in the mshA-encoded glycosyltransferase, the first step in mycothiol biosynthesis, leads to a defect in mycothiol production and INH or ETH resistance in Mycobacterium tuberculosis (Mtb) (9). We hypothesized that mycothiol-deficient mshA mutants accumulated cysteine, leading to drug resistance, but surprisingly, we found that the addition of cysteine to INH-treated Mtb sterilized the cultures. In this report, we present the effects of adding cysteine to drug-susceptible and drug-resistant Mtb strains treated with INH. We performed a comprehensive analysis of the transcriptome, metabolites, production of reactive oxygen species (ROS), and cellular respiration to propose that sterilization of Mtb cultures can be obtained by coercing Mtb into a constant active metabolic state that renders the bacteria continuously susceptible to the cidal effects of the TB drugs.

Results

Addition of Cysteine to INH-Treated, Exponentially Growing Cultures of Mtb Results in Sterilization of Drug-Susceptible and Drug-Resistant Mtb.

Treatment of Mtb H37Rv cultures with INH in vitro is a triphasic event starting with a rapid initial killing of Mtb cells, followed by a stagnation step where the persister population is revealed, and finally the emergence of INH-resistant bacteria (10). Low-level INH resistance was previously observed in Mtb mutants that failed to produce mycothiol (9), a cysteine reservoir, leading us to hypothesize that these mutants might accumulate cysteine and that this accumulation mediated INH resistance. To test this hypothesis, increasing concentrations of cysteine (from 8 μM to 4 mM) were added to INH-treated Mtb H37Rv cultures. Rather than conferring INH resistance, the increasing concentrations of cysteine caused increasing delays in the growth of INH-resistant mutants (Fig. S1A), with their appearance completely inhibited at the highest concentration of cysteine tested (4 mM). Surprisingly, the combination of 4 mM of cysteine and INH had sterilized the culture (Fig. 1A). Both INH and the combination of INH and cysteine (INH/Cys) generated the same kinetics of killing for the first 7 d, but then the survival patterns with the two treatments diverged (Fig. 1A). Bacterial counts after 3 wk of INH/Cys treatment ranged between 0 and 10 CFU/mL, representing a 6-log killing of the initial culture (Fig. 1A), and none of these residual colonies were INH-resistant. This increased killing was not a result of a bactericidal effect of cysteine itself, because growth and viability of Mtb were not affected by the addition of cysteine alone, nor was the outcome specific to Mtb H37Rv; the same sterilizing activity was observed when either Mtb Beijing or Mtb CDC1551 was treated with INH/Cys (Fig. S1 B and C). Interestingly, the growth inhibition of Mtb by INH/Cys was observed only when cysteine was added at the same time or no more than 1 d following the addition of INH (Fig. S1D).

Fig. 1.

The combination INH/Cys sterilizes drug-susceptible and drug-resistant Mtb cultures. (A) Viability of Mtb treated with INH (7.3 μM, 20 times the MIC), cysteine (4.0 mM), or INH/Cys. The INH-treated culture was plated on 7H10 plates and 7H10-INH plates containing 3.7 μM of INH. (B) The RIF-resistant strain mc²4986 (Mtb H37Rv rpoB H445R) was treated with RIF (1.2 μM), INH (7.3 μM), cysteine (4.0 mM), or a combination. The combinations used the same concentrations as the individual treatments. Aliquots were taken at indicated times and plated to determine CFUs. Average with SD is plotted (n = 2–4).

Fig. S1.

The combination of cysteine with TB drugs sterilizes Mtb cultures in a strain-independent but TB drug-, concentration-, and time-dependent manner. (A) Growth kinetics of Mtb H37Rv treated with INH (7.3 μM) and different concentrations of cysteine (in millimolars) were followed by measuring optical density at 600 nm. (B) Viability of Mtb Beijing treated with INH (7.3 μM), cysteine (4.0 mM), or the combination INH/Cys was obtained by plating serially diluted aliquots to determine CFUs. (C) Viability of Mtb CDC1551 treated with INH (7.3 μM), cysteine (4.0 mM), or the combination INH/Cys was obtained by plating serially diluted aliquots to determine CFUs. (D) Effect of adding cysteine (4.0 mM) at different times on the growth kinetics of Mtb H37Rv cultures treated with INH (7.3 μM) and followed by measuring optical density at 600 nm. (E) Viability of Mtb H37Rv treated with rifampicin (RIF, 1.2 μM, 20 times the MIC), cysteine (4.0 mM), or the combination RIF/Cys was obtained by plating serially diluted aliquots to determine CFUs. (F) Viability of Mtb H37Rv treated with EMB (0.5 mM, 20 times the MIC), cysteine (4.0 mM), or the combination EMB/Cys was obtained by plating serially diluted aliquots to determine CFUs. The compounds combinations used the same concentrations of compounds as the individual treatments. In experiments in E and F, the colonies obtained on plates for CFUs determination at day 21 were picked and patched onto 7H10 plates containing or not RIF (1.2 μM) or EMB (0.5 mM), respectively, to check for drug resistance. Single replicates are shown in A and D, representative of two independent experiments. The graphs in B, C, E, and F represent the average of two independent experiments with error bars indicating SD.

Next, we investigated the possibility that INH/Cys might be beneficial for drug-resistant TB therapy by treating a rifampicin (RIF)-resistant Mtb strain (mc²4986, H37Rv rpoB H445R) with INH, cysteine, and INH/Cys (Fig. 1B). INH/Cys with or without the addition of RIF prevented the growth of drug-resistant populations and reduced the bacterial population by 6-log after 3 wk, similar to our observations with drug-susceptible Mtb strains.

A Free Thiol Group Is Required in Combination with INH for Sterilizing Activity.

To determine whether the observed sterilization activity of the INH/Cys combination was cysteine-specific, other thio-compounds or structurally similar amino acids were added to INH-treated Mtb cultures and the growth of the cultures was followed visually. The emergence of a growing population after 14 d was prevented only when Mtb was treated with a combination of INH and a compound having a free thiol group (Table S1). Interestingly, in combination with INH, DTT was effective at sterilizing cultures at just half the concentration used for cysteine (2 mM) (Fig. S2A), which substantiates the crucial role of the free-thiol group as DTT possesses two free-thiol groups, whereas cysteine has only one.

View this table:

Table S1.

Compounds (4.0 mM) tested for the ability to inhibit M. tuberculosis growth in combination with INH (7.3 μM)

Fig. S2.

The combinations INH/DTT and INH/NAC are bactericidal. Viability of Mtb H37Rv cultures treated with INH (7.3 μM) and either DTT (2.0 mM, A) or NAC (4.0 mM, B) was followed by taking aliquots of the cultures at the indicated time and plating for CFUs. The graphs represent the average of two independent experiments with error bars indicating SD.

The Combination of Cysteine and RIF Prevents the Emergence of RIF-Resistant Mutants.

To test whether the enhancing effect of cysteine was specific to INH, Mtb cultures were cotreated with cysteine and two other first-line TB drugs: RIF and ethambutol (EMB). Treatment with RIF resulted in a 3- to 4-log decrease in CFU after 2 wk followed by an increase in CFU because of the emergence of RIF-resistant mutants (Fig. S1E). When cysteine was added in combination with RIF, a 6-log decrease in CFUs was observed. However, a different pattern was observed for EMB (Fig. S1F), in that EMB treatment with or without cysteine resulted in a very slow decrease in CFU and no emergence of EMB-resistant mutants was observed. The colonies obtained after 3 wk of EMB treatment were all EMB-susceptible, whereas the majority of colonies isolated after 3 wk of INH or RIF treatment were INH-resistant or RIF-resistant, respectively. These results suggested a correlation between the emergence of a drug-resistant population and the beneficial effect of adding cysteine to a drug treatment. The remainder of this report will focus mostly on cysteine as a thiol delivery agent and its effects when combined with INH.

Transcriptional Profiling Reveals That INH/Cys Triggers an Oxidative Process.

To investigate the mechanisms involved in the INH/Cys effect on Mtb, we performed transcriptional profiling of Mtb H37Rv cultures treated for 4 h with INH or with INH/Cys. The comparison of treatment with INH/Cys to INH alone revealed an up-regulation (>twofold) of 143 genes, including many involved in the conservation of cation homeostasis (genes involved in the IdeR and Zur regulon, the regulatory genes furA and csoR) (Fig. 2 and Table S2). The DosR regulon, which is induced in a csoR knockout strain (11), was broadly induced, as was the DNA damage response stimulon (12). INH/Cys treatment led to the up-regulation of many genes observed to be induced in Mtb exposed to oxidative and nitrosative stresses (13, 14). INH/Cys treatment also repressed the expression of 79 genes (>twofold), several of which are involved in intermediary metabolism and respiration as well as cation transport (Table S3). Interestingly, although the zinc uptake regulator zur was down-regulated, its regulon was induced (11). These data suggested that the INH/Cys combination initiated a cation-dependent oxidative stress resulting in DNA damage.

Fig. 2.

Addition of cysteine to INH induces transcriptional responses to maintain cation homeostasis, counter oxidative, and genotoxic stresses. The transcriptional profile of Mtb H37Rv exposed for 4 h to the combination of INH (7.3 μM) and cysteine (4.0 mM) was compared with that of cells exposed to INH alone. Genes comprising the DosR regulon (35) (Left) were largely up-regulated in the cysteine-treated cells. The DNA damage response (12) was up-regulated (Center), except for the pyrR repressor. Likewise, the Zur regulon (11) was induced (Right), except for the putative repressor Rv0232, highlighting the effect of the cysteine treatment on cation homeostasis. Scale represents log₂ fold-change vs. INH alone; cells represent biological triplicate comparisons.

View this table:

Table S2.

Up-regulation of Mtb genes upon addition of cysteine to INH-treated Mtb cultures

View this table:

Table S3.

Down-regulation of Mtb genes upon addition of cysteine to INH-treated Mtb cultures

Cysteine Is Rapidly Oxidized into Cystine Leading to ROS Production.

High levels of intracellular cysteine have been shown to induce ROS production leading to DNA damage and oxidation of cysteine to cystine in Escherichia coli (15). To test whether oxidation of cysteine occurs in the cell cytoplasm or in the supernatant of Mtb cultures following addition of 4 mM cysteine, the free-thiol levels were measured in cultures treated with cysteine, INH, and INH/Cys. No increase in free thiols in the cell pellets was observed up to 24 h after the addition of cysteine (Fig. 3A). The thiol levels were further measured over the course of 4 d of treatment, and no significant difference in cell pellet thiol content was observed. In the supernatant of these cultures, the increase in free-thiol content was transient, lasting no more than 1 h after cysteine addition (Fig. 3B). We then tested whether cysteine was oxidized to cystine by analyzing the lysates of cell pellets by liquid chromatography coupled to a mass spectrometer (UPLC-MS). Cultures treated with cysteine, with or without INH, contained at least 100-fold more cystine in their cell pellets than did the cultures that did not receive cysteine (Fig. 3C). Because the oxidation of cysteine to cystine occurred rapidly in the cell pellet, the sterilization effect of the INH/Cys combination might result from cystine generation in situ. Viability of Mtb cultures treated with INH or cystine was therefore determined to assess whether cystine was the molecule responsible for sterilization by the INH/Cys combination (Fig. 3D). Although a growing bacterial population emerged more slowly in Mtb cultures cotreated with INH and cystine, we did not observe culture sterilization, and therefore cystine is not responsible for the bactericidal activity of the INH/Cys combination.

Fig. 3.

High levels of cystine but not cysteine are found in Mtb treated with cysteine or INH/Cys. Total thiol concentrations from cell pellets (A) or supernatants (B) of Mtb mc²6230 cultures treated with cysteine (4.0 mM), INH (7.3 μM), or INH/Cys were measured. (C) UPLC-MS determination of the relative amount of cystine in the cell pellets of Mtb mc²6230 treated with cysteine (4.0 mM), INH (7.3 μM), or INH/Cys compared with pellets from untreated cells. (D) Mtb was treated with cystine (2.0 mM), INH (7.3 μM), or the combination. Aliquots were taken at indicated times and plated to determine CFUs. The combination used the same concentrations as the individual treatments. Average with SD is plotted (n = 3).

Transition metals, such as copper or iron, are known to catalyze the oxidation of cysteine into cystine, leading to the production of hydrogen peroxide (H₂O₂), superoxide, and hydroxyl radicals (16). We hypothesized that high concentrations of cysteine could trigger a cationic stress, which could result in the formation of ROS and DNA damage (17, 18). Based on our transcriptional data indicating the possible involvement of cations in the INH/Cys combination activity, we first tested whether a cation scavenger, such as the iron chelator deferoxamine (DFO), could impair culture sterilization by INH/Cys. The addition of DFO to INH/Cys-treated Mtb cultures resulted in viability curves similar to those of INH-treated cultures, with the emergence of a growing population within 14 d of treatment (Fig. S3A). Iron is also implicated in the production of ROS by the Harber–Weiss and Fenton reactions, which rely on the reduction of ferric ions to ferrous ions in the presence of oxygen and a reductant such as cysteine. Thus, we compared the levels of free ferrous and ferric ions in Mtb cultures treated with cysteine, INH, and INH/Cys during a period of 2 d (Fig. S3 B and C). An increase in the ferrous ion levels was observed immediately in the cell pellet and supernatant of the INH/Cys-treated Mtb cultures, but the levels quickly returned to untreated levels. Interestingly, an increase in ferrous levels was subsequently observed after 24 h of treatment in both the INH/Cys and INH cell pellets. Next, superoxide levels and DNA breaks were measured in Mtb cultures treated with cysteine, INH, and INH/Cys during a period of 7 d by quantification using flow cytometry. Although INH/Cys treatment resulted in a clear increase in ROS formation in the first 24 h compared with treatment with cysteine or INH alone, the differences were less pronounced at later time points (Fig. 4A). However, this increase in ROS production correlated with a striking increase (up to 60%) in the level of double-stranded DNA breaks in the INH/Cys-treated samples after 7 d (Fig. 4B).

Fig. 4.

The combination INH/Cys increases intracellular ROS production and DNA damage. (A) Cell pellets of Mtb mc²6230 treated with INH (7.3 μM), cysteine (4.0 mM), or a combination of these using the same concentrations as the individual treatments, were stained with dihydroethidium. ROS levels were measured by flow cytometry for up to 7 d and compared with the untreated samples. (B) The samples in A were also used to determine the percentage of double-stranded DNA breakage. Average with SD is plotted (n = 3).

Fig. S3.

The sterilizing activity of INH/Cys is iron- and oxygen-dependent. (A) Mtb H37Rv was treated with INH (7.3 μM), cysteine (4.0 mM), and DFO (152 μM). Combinations used the same concentrations as the individual treatments. Aliquots were taken at the indicated times and plated for CFUs. Average with SD is shown (n = 2). (B and C) Ferrous and ferric ion concentrations were measured in the cell pellets (B) and supernatants (C) of Mtb mc²6230 cultures treated with INH (7.3 μM), cysteine (4.0 mM), or INH/Cys at the same concentration as the individual treatments for up to 3 d. At specific time, samples were taken and analyzed as described in SI Materials and Methods. Average with SD is shown (n = 2). (D) Mtb mc²6230 was treated with RIF (1.2 μM), cysteine (4.0 mM), and a RIF/Cys combination at the same concentration as the individual treatments, under anaerobic conditions (<0.0001% O₂, 5% CO₂, 10% H₂, 85% N₂). Aliquots were taken at indicated times and plated to determine CFUs. Average with SD is shown (n = 2).

To evaluate the association between enhanced ROS production with the INH/Cys and bactericidal activity, Mtb was treated with cysteine, RIF, and RIF/Cys in an anaerobic chamber. For this experiment, RIF was chosen because it retains bactericidal activity under anaerobic conditions, whereas INH does not (19). Under anaerobic conditions, the addition of cysteine to RIF-treated Mtb did not further increase killing of the bacterial population (Fig. S3D), indicating an oxygen-dependent etiology. These data suggests that the sterilization phenomenon observed in INH/Cys-treated Mtb cultures relies on the presence of cations, such as ferric ions, and oxygen, and leads to an oxidative process and DNA damage.

The INH/Cys Combination Generates a Transient Increase in H₂O₂ Concentration.

Autoxidation of 4 mM cysteine into cystine significantly increased H₂O₂ production in pig kidney cells (20). To assess whether H₂O₂ was generated concomitant with the oxidation of cysteine to cystine in Mtb, the concentration of H₂O₂ was measured in cultures treated with INH and/or cysteine. An increase in H₂O₂ was observed in the cultures receiving cysteine during the first 24 h of treatment: intracellular H₂O₂ levels were 1.5 and 6 times higher (Fig. 5A) and extracellular H₂O₂ levels were 12 and 28 times higher (Fig. 5B) in Mtb cultures treated with cysteine and INH/Cys, respectively, compared with untreated. The levels of H₂O₂ then decreased drastically after 24 h. To test whether the H₂O₂ burst was responsible for the INH/Cys sterilization effect, cultures were treated with a noninhibitory concentration of H₂O₂ (1 mM) with and without INH. No change in the growth kinetics of Mtb was observed in the presence of INH and H₂O₂ (Fig. S4 A and B), and a growing bacterial population emerged within 14 d of INH treatment, whether or not H₂O₂ was added to the cultures. To test whether the production of H₂O₂ and oxidation of cysteine could cause a redox unbalance, we measured the levels of NADH and NAD⁺ cofactors to assess whether the cell redox potential was changed upon addition of INH/Cys and found no significant changes over time (Fig. S4C). These results indicate that an oxidation process leading to H₂O₂ production does occur but that it may not be the primary factor in cell death.

Fig. 5.

The combination of cysteine and INH transiently increases H₂O₂ levels. Mtb mc²6230 was treated with cysteine (4.0 mM), INH (7.3 μM), alone or in combination, for 3 d at 37 °C. The combination used the same concentrations as the individual treatments. H₂O₂ concentration was determined in the cell pellet (A) and the supernatant (B) of the treated cells as described in the experimental section. Average with SD is shown (n = 2).

Fig. S4.

Hydrogen peroxide in combination with INH does not sterilize Mtb H37Rv sterilization and NADH/NAD⁺ ratio is not altered by INH/Cys-generated oxidative burst. (A) The growth of Mtb H37Rv cultures treated with INH (7.3 μM), hydrogen peroxide (H₂O₂, 1 mM), or their combination was followed by measuring optical density at 600 nm over time. A set of experiments was done where hydrogen peroxide (1 mM) was added every day for the first 5 d of the experiment and is annotated H₂O₂x. Single replicates are shown representative of two independent experiments. (B) H₂O₂ (1 mM) does not alter the killing of Mtb H37Rv by INH (7.3 μM). The individual treatments used the same concentration of compounds as the compound combination. Aliquots were taken at the indicated time points and plated to determine CFUs. Average with SD is shown (n = 2). (C) NADH and NAD⁺ concentrations were measured in Mtb treated with INH (7.3 μM), cysteine (4.0 mM), or INH/Cys at the same concentration as the individual treatments for up to 2 d, as described in SI Materials and Methods. Average with SD is shown (n = 3).

The INH/Cys Combination Increases Mtb Cellular Respiration and Prevents the Emergence of INH Persisters.

Recently, a correlation between antibiotic bactericidal activity and bacterial cellular respiration was established (21) through the observation that bacterial respiration increased in E. coli and Staphylococcus aureus following treatment with bactericidal drugs, whereas bacteriostatic drugs resulted in respiration arrest. Previously, we had shown that the addition of high concentrations (1–40 mM) of the thiol DTT induced oxygen consumption in nonrespiring Mtb cells (22). Because DTT in combination with INH sterilizes Mtb cultures similarly to the INH/Cys combination (Fig. S2A), we examined whether cysteine could also increase oxygen consumption of nonrespiring Mtb cells. Mtb cultures were grown to late stationary phase where minimal oxygen consumption was observed (Fig. 6A). The addition of cysteine to these nonrespiring cells had a modest effect on respiration, but the addition of INH/Cys provoked a rapid resumption of oxygen consumption (Fig. 6A).

Fig. 6.

INH/Cys induces Mtb respiration and prevents persister formation. (A) The levels of dissolved oxygen (DO₂) in Mtb were followed upon treatment with cysteine (4.0 mM), INH (7.3 μM), and INH/Cys. The first 100 s show the oxygen consumption of Mtb mc²6230 in stationary phase. Compounds were added at 100 s (left arrow), followed at 400 seconds (right arrow) by the addition of the respiration uncoupler CCCP. The curves represent a typical experiment from biological triplicates. (B) Four-day-old Mtb cultures treated with cysteine (4.0 mM), INH (7.3 μM), and INH/Cys (7.3 μM/4.0 mM) were infected with Φ²DRM9 and analyzed by flow cytometry. GFP expression after phage infection represents viable Mtb cells with higher GFP⁺ cells being more metabolically active (Left). The RFP population was back-gated to show GFP expression distribution in these cells (Right). The low-GFP/high-RFP (persister) population indicated by an arrow was absent in the INH/Cys-treated Mtb cultures (Right). (C) Representative microscopy images showing the presence and absence of persisters (low GFP/high RFP) in INH- (Upper) and INH/Cys-treated (Lower) Mtb cultures, respectively. (Scale bar, 10 μm.)

The stimulation of respiration by DTT in cells with previously reduced oxygen consumption was shown to be because of an increase in the menaquinol-9 (MKH₂) pool, which controls Mtb respiratory rate (22). We observed that the combination of INH/Cys further increases Mtb respiration above the increase generated by cysteine alone, suggesting a similar mechanism whereby the addition of high concentrations of cysteine shifts the ratio of MKH₂ to menaquinone-9 (MK) toward higher MKH₂ levels, resulting in enhanced bacterial respiration. To test this hypothesis, MK and MKH₂ were extracted from Mtb cultures treated with INH, cysteine, and INH/Cys, and analyzed by UPLC-MS (Fig. S5A). The addition of INH/Cys had the greatest impact on the MKH₂/MK ratio, with an increase up to 13 times that of untreated cells. This increase in the MKH₂/MK ratio occurred within the first 3 h of treatment, followed by a steady decrease over time.

Fig. S5.

Increased respiration is linked to alteration of menaquinol level and is also observed in Mtb sterilization phenotype with cidal compound not associated to a thiol. (A) The MKH₂/MK ratio in Mtb mc²6230 cells treated with INH (7.3 μM), cysteine (4.0 mM), or the combination at the same concentration, is plotted relative to the ratio in untreated cells. Average with SD is shown (n = 2). (B) Mtb mc²6230 grown to stationary phase was treated with inhibitory concentrations of vitamin C (4 and 20 mM) and Mtb resumption of oxygen consumption was followed over 500 s. The first 100 s show the oxygen consumption of Mtb mc²6230 in stationary phase. Vitamin C was added at 100 s followed at 400 s by the addition of the respiration uncoupler CCCP. The curves represent a typical experiment from biological duplicates.

Finally, we use the dual GFP/RFP-reporter mycobacteriophage Φ²DRM9 (10) to assess the effects of INH/Cys on metabolic rate and persister formation in Mtb. Expression level of GFP and RFP in Mtb infected with Φ²DRM9 depends on the metabolic status of the infecting cell. Actively growing cells express a higher level of GFP and a lower level of RFP in >90% of Mtb (Fig. S6). In contrast, INH persisters are characterized by low GFP/high RFP fluorescence after Φ²DRM9 infection (10). Mtb cultures treated with cysteine, INH, or INH/Cys for up to 4 d were infected with Φ²DRM9 (Fig. S6). Both microscopy and flow cytometry analysis showed the presence of the persister population (low GFP/high RFP) in the INH-treated samples at the end of day 4 (Fig. 6 B and C and Fig. S6). In contrast, this population was missing in the INH/Cys-treated samples (Fig. 6 B and C and Fig. S6). Furthermore, the INH/Cys-treated sample had a relatively greater population of cells expressing high GFP signal than the INH-treated samples, suggesting a higher metabolic rate of Mtb in this sample. These findings are consistent with a model in which a shift toward higher MKH₂ levels increases the Mtb respiratory rate and thus prevents cells from entering a persister or drug-tolerant state.

Fig. S6.

Determination of the persister population in Φ²DRM9-infected Mtb cells by flow cytometry. Mtb cultures were infected with Φ²DRM9 after 2, 3, and 4 d of treatment with cysteine (4.0 mM), INH (7.3 μM), or INH/Cys at the same concentration as the individual treatments, and analyzed by flow cytometry. GFP expression after phage infection represents viable Mtb cells (Upper). Higher GFP⁺ cells which represent metabolically active cells were expanding in untreated and cysteine-treated cells overtime. In the INH/Cys-treated cells, this population remained low while declining in the INH-treated cells. The RFP population was back-gated to show GFP expression distribution in these cells (Lower). The low GFP/high RFP population, marked with an arrow, was present at day two in all of the samples but was selectively enriched in the INH alone treated samples at days 3 and 4 while being undetectable in all of the other conditions.

The INH/Cys Combination Potentiates INH Activity in Macrophages.

Mtb is an intracellular pathogen that infects host macrophages, and so to be effective, a drug or drug combination needs to be transported through both the macrophage membrane and the mycobacterial cell wall. To assess whether the addition of a thiol to INH treatment would improve TB chemotherapy, we tested the combination of INH/thiol in Mtb-infected J774 murine macrophages. First, the cytotoxicity of cysteine for the host cells was determined, and the highest concentration of cysteine that allowed for at least 90% survival (LC₁₀) of macrophages was 0.0156 mM. In contrast, the thiol N-acetylcysteine (NAC) had an LC₁₀ of 5–10 mM in J774 macrophages and, in Mtb in vitro cultures, led to a 4- to 5-log decrease in CFUs within 3 wk when combined with INH (Fig. S2B). Mtb-infected macrophages were treated with NAC in combination with INH for 7 d (Fig. 7A). Although NAC did not impair the growth of Mtb in J774 macrophages, INH/NAC was significantly more effective at killing intracellular Mtb than INH alone (Fig. 7B). These observations demonstrate that the addition of NAC can enhance chemotherapy of Mtb in mammalian cells.

Fig. 7.

The combination of INH and NAC increases the intracellular killing of Mtb in murine macrophages. (A) J774 macrophages were infected with Mtb H37Rv at an MOI of 1 for 4 h before treatment with NAC (2.0 mM), INH (7.3 μM), or their combination at the same concentrations. (B) Intracellular Mtb killing upon treatment is represented by plotting bacterial load against titer at beginning of treatment. P was determined by two-tailed student’s test. Average with SD is shown (n = 4).

Discussion

Several mechanisms potentially underlie the sterilizing activity of the INH/Cys combination. The transcriptional data suggested mechanisms based on oxidative stress leading to DNA damage. Indeed, we showed that ROS concentrations were higher in INH/Cys-treated Mtb leading to DNA damage, which correlates with previously published work showing that high concentrations of intracellular cysteine increased DNA damage in E. coli (15). The lack of RIF/Cys activity under anaerobic conditions further suggests that oxygen plays an important role in our observed phenomenon. Furthermore, the increase in intracellular cystine concentration with no commensurate increase in cysteine concentration in the cysteine-treated cultures also points to an oxidative process, although cystine was not a key player in the sterilization phenotype. Although several lines of inquiries point to an oxidative process, the addition of a specific oxidant, H₂O₂, to INH-treated Mtb cultures did not mimic the sterilizing effect of INH/Cys in vitro.

Combining cysteine with INH treatment of exponentially growing Mtb cells prevented the emergence of drug-resistant mutants and ultimately led to sterilization of the cultures. Interestingly, this combination did not work when cysteine was added 2 d or more after the addition of INH to the Mtb cultures, implying that INH/Cys might prevent the formation of the persister population, which is often associated with treatment failure and the emergence of a drug-resistant population in TB-infected patients. Previously, the generation of INH persisters in Mtb cultures was associated with random on/off expression of the catalase/peroxidase KatG, the activator of INH (23). Those authors hypothesized that Mtb cells might alter KatG expression in response to ROS fluctuation during active respiratory metabolism. In our study, we observed a consistently higher ROS production in the INH/Cys-treated samples than in INH-treated samples, as well as up-regulation of katG (> fourfold). This finding suggests that higher respiratory metabolism leads to increased ROS production and KatG expression as a compensatory mechanism, which might result in enhanced INH activity and ultimately Mtb cell death. The mycobacterial persister population was also shown to be sensitive to oxygen concentration and could be killed at higher concentrations of dissolved oxygen that led to ROS production and sterilization of the culture (24). In our study, the persister population was absent in the INH/Cys-treated Mtb culture, suggesting that conditions or compounds that increase oxygen consumption would lead to the depletion of persister cells or the prevention of their appearance, which ultimately would result in sterilization of drug-treated Mtb cultures. Notably, vitamin C, which we had showed sterilized Mtb cultures (25), was also found to induce Mtb respiration (Fig. S5B), suggesting that this mechanism is not specific to the combination of thiols and INH.

NAC showed a similar sterilizing effect to cysteine when combined with INH in vitro (Fig. S2B). Recently, it was shown that when NAC was added to Mtb treated with the combination of electron transport chain inhibitors bedaquiline, Q203, and clofazimine, no colony could be detected after 5 d of treatment (26). In murine macrophages, INH/NAC reduced intracellular Mtb growth significantly better than INH alone (Fig. 7). NAC (10 mM) was also found to have moderate antimycobacterial activity in human THP-1 macrophages, resulting in a threefold decrease in bacterial load after 5 d (27). In the same study, the authors also showed that NAC was safe in mice and a dose of 400 mg/kg given by gavage daily to Mtb-infected mice could by itself reduce the mycobacterial lung burden by half a log (27). This result suggests that combining NAC to first-line TB drugs could further decrease organ bacterial loads in Mtb-infected mice and should be investigated. Furthermore, NAC is used for the treatment of drug-induced toxicity in TB patients (28, 29) and, when tested as an adjuvant to directly observed treatment short course, NAC significantly caused early sputum negativity and radiological improvement in pulmonary TB patients (30). Along with the increased bactericidal activity of the INH/NAC combination in our murine macrophage model, these data suggest that NAC could potentiate the efficacy of TB chemotherapy.

We show that INH/Cys enhances Mtb oxygen consumption. We propose that INH/Cys works by preventing Mtb from entering a low respiratory and low metabolic state, which might be found in persisters, and allows bactericidal drugs such as INH to continue to exert their activity. Furthermore, without persisters there is no opportunity for mutagenesis leading to the emergence of drug resistance. A consequence of increased oxygen consumption may be the generation of ROS, which was observed in the INH/Cys-treated Mtb cultures, and which might contribute to the enhanced bactericidal activity of the INH/Cys combination (Fig. S7). We have previously demonstrated that respiration can be controlled by the poise of the menaquinone/menaquinol pool and that respiration is inhibited when the pool is in an oxidized state (22). Here, we show that the addition of cysteine to INH-treated Mtb cultures shifts the pool toward a reduced state and allows Mtb cells that were not respiring to resume respiration. This finding is in accordance with our transcriptional data, which showed up-regulation of the DosR regulon, which enhances the emergence of Mtb from nonrespiring dormancy (31). This effect on the DosR regulon might also be a consequence of the reduction of copper by cysteine, which can lead to the generation of nitric oxide that activates the sensor histidine kinases DosS and DosT (32, 33). In conclusion, we suggest that the control of respiration via the menaquinol pool is a key step in fostering drug tolerance and drug resistance in Mtb. Moreover, considering the increasing incidence of MDR- and XDR-TB, new strategies to treat resistant infections and prevent the emergence of resistance are urgently needed; the addition of nontoxic, small molecules, such as NAC to TB chemotherapy, presents a promising approach to these chemotherapeutic challenges in the treatment of TB, warranting further study.

Fig. S7.

Proposed model for the mechanism of action of the INH/Cys combination. The oxidation of cysteine into cysteine in the presence of ferric ions generates ROS production via the Fenton and Harber–Weiss reactions followed by DNA damage. In parallel, this oxidation reaction shifts the balance of menaquinone toward menaquinol, which was shown to increase Mtb respiration resulting in actively replicating Mtb cells. Mtb cells, which do not shut down their metabolism and are kept in an active replication state, are consistently susceptible to INH bactericidal activity. The combination of INH inhibitory activity and oxidative damage causes Mtb cell death.

Materials and Methods

Bacterial Strains and Reagents.

The Mtb strains used in this study and the phage Φ²DRM9 were obtained from laboratory stocks. The Mtb strains were grow at 37 °C while shaking in Middlebrook 7H9 medium (Difco) supplemented with 10% (vol/vol) OADC enrichment (oleic acid-albumin-dextrose-catalase, Difco), 0.2% (vol/vol) glycerol, and 0.05% (vol/vol) tyloxapol. The solid media used was Middlebrook 7H10 medium (Difco) supplemented with 10% (vol/vol) OADC enrichment (Difco) and 0.2% (vol/vol) glycerol. Plates were incubated at 37 °C for up to 6 wk. For safety purposes, specific experiments were performed with Mtb mc²6230, a nonpathogenic deletion mutant (ΔRD1 ΔpanCD) of strain H37Rv that can safely be used in a Biosafety Level 2 laboratory. To culture mc²6230, D-pantothenic acid hemicalcium salt (25 mg/L) was added to liquid and solid media. All chemicals were obtained from Sigma unless otherwise stated. All solvents used were LC-MS grade (Fisher).

Growth of Mtb in Vitro Cultures.

Mtb cultures were grown at 37 °C to an optical density at 600 nm (OD_600nm) of 0.7–1.0. The cultures were diluted 1/50, treated with the appropriate chemicals, and incubated at 37 °C with shaking for the duration of the experiment. The addition of 4 mM of cysteine to Mtb cultures did not change the pH of the cultures. The OD_600nm was recorded, and CFUs were obtained by plating serial dilutions (see media described above).

Determination of Total Thiol Concentration.

An exponentially growing Mtb mc²6230 culture was diluted to an OD_600nm ∼ 0.15, treated with the appropriate chemicals, and incubated at 37 °C while shaking. The cultures (10 mL) were centrifuged, and the cell pellets were washed once with PBS and resuspended in 50 mM Tris (pH 8.0; 0.5 mL) containing 5 mM EDTA. Glass beads (0.1 mm diameter, 0.1 mL) were added, and the cell pellets were lysed using the MP Bio FastPrep machine (45 s, speed 6 M/s, three times). After cooling, the samples were centrifuged and the supernatants were filter-sterilized before analysis. The total thiol concentration was obtained by measuring spectrophotometrically at 412 nm a 1-mL solution containing 50 mM Tris (pH 8.0), 5 mM 5,5′-dithiobis(2-nitrobenzoic acid) (10 μL), and the sample lysate using the following extinction coefficient: ε₄₁₂ _nm 2-nitro-5-thiobenzoate anion 14,150 M⁻¹⋅cm⁻¹.

ROS and DNA Breaks Analyses.

Mtb mc²6230 was grown to OD_600nm 1.0 and diluted to an OD_600nm of 0.2 in fresh media containing the appropriate chemicals. For ROS analysis, aliquots were taken at the indicated time points, and cells were stained with dihydroethidium (5 μM) for 30 min at 37 °C and immediately analyzed on a BD FACS Calibur (BD Biosciences) as described previously (25). Aliquots from the same mycobacterial cells used for ROS analysis were removed at indicated time points, fixed, treated with TUNEL reaction mix from the In Situ Cell Death Detection Kit (Roche Molecular Biochemicals), and analyzed by flow cytometry, as described previously (25).

Determination of Oxygen Consumption.

The oxygen consumption rate was determined using a Clark-type oxygen electrode (Rank Brothers) linked to an ADC-24 data logger (Pico Technology). Mtb mc²6230 was grown to stationary phase and 5 mL were added to the incubation chamber to measure basal level oxygen consumption. After 100 s, cysteine, INH, or INH/Cys was added at the indicated concentration, and oxygen consumption was recorded for another 300 s, after which maximal uncoupled respiration was measured with the addition of carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 200 nmol) for 100 s.

J774 Murine Macrophage Experiments.

J774A.1 macrophages (ATCC) were subcultured twice a week according to the supplier’s recommendations in DMEM (Invitrogen), supplemented with 10% FBS (Invitrogen). Cell viability was assessed using the MTT assay (34). For the infection assays, the macrophages (∼100,000 cells) were seeded into each chamber of a 24-well tissue culture plate and cultured for 3 d. At the time of the infection, cell density was ∼3.5 × 10⁵ cells per well. For the infection, a Mtb H37Rv culture was grown to midlog (OD_600nm ∼ 0.6–0.8), washed twice in PBS supplemented with 0.05% tyloxapol, resuspended in PBS, and sonicated twice for 10 s. The culture was then diluted in DMEM supplemented with 10% FBS and used to infect the J774 cells for 4 h at 37 °C in 5% CO₂ at an approximate multiplicity of infection (MOI) of 1 to allow for bacterial uptake. Cell monolayers were then washed twice with PBS, and cultured in DMEM supplemented with 10% FBS and the appropriate test compounds, while incubating at 37 °C in 5% CO₂. The media with compounds was replenished at days 2 and 4. At each time point, the media was removed from the wells; the wells were washed once with PBS and then treated for 5 min with 0.05% aqueous SDS solution to lyse the macrophages. The lysates were serially diluted and plated for CFU determination.

Additional methods are available in SI Materials and Methods.

SI Materials and Methods

Microarray Analysis.

Cultures of Mtb H37Rv (10 mL, OD_600nm of 0.2) were treated with INH (7.3 μM), with and without cysteine (4.0 mM), incubated for 4 h at 37 °C, and harvested. The cell pellets were resuspended in Qiagen RNA protect reagent (1 mL; Qiagen) and incubated for 4 h at room temperature. The samples were frozen at −80 °C until ready to use. The cells were spun down, resuspended in buffer RLT (1 mL, Qiagen RNeasy kit), and lysed using a Fast-Prep apparatus (MP Bio). Upon cooling, the samples were spun down and the supernatants were treated with absolute ethanol (0.5 mL) and applied to RNeasy columns in two applications. RNA was purified following Qiagen protocol. RNA yield, purity, and integrity were measured on a Nanodrop spectrophotometer (Nanodrop Products) and an Agilent Bioanalyzer 2100 microfluidic system (Agilent Technologies). Ambion Turbo DNA-free (Ambion) was used to remove contaminating DNA. cDNA probes were prepared as described in ref. 10. Total RNA (2 μg) was used as template for reverse transcription with random hexamer primers and aminoallyl-dUTP in a 2:3 ratio to dTTP in the nucleotide mix. After overnight incubation at 42 °C, the Qiagen MinElute kit was used to stop the reaction, hydrolyze RNA, and remove unincorporated nucleotides and free amines. cDNA concentration and purity were measured by Nanodrop. Cy3 and Cy5 dyes were coupled to the aminoallyl-cDNA, and then purified with a MinElute kit. Probe concentration, purity, and dye incorporation were measured by Nanodrop. Probes were then combined, dried, dissolved in hybridization buffer, and hybridized according to the PFGRC protocol SOP#M008 to 70-mer oligo DNA microarrays representing the complete Mycobacterium tuberculosis genome with four in-slide replicates (JCVI PFGRC M. tuberculosis v. 4), which had been prehybridized. One of the replicates was dye-flipped. Slides were washed, dried, and then scanned on a GenePix 4000A scanner (Molecular Devices). Images were processed with the TM4 software suite (www.TM4.org) and TIGR Spotfinder was used to grid and quantitate spots. TIGR MIDAS was used for Lowess normalization, SD regularization, and in-slide replicate analysis, with all quality control flags on and one bad channel tolerance policy set to generous. Results were analyzed in megaelectronvolts with Significance Analysis of Microarrays and hierarchical clustering algorithms. The array data have been deposited in the Gene Expression Omnibus at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/geo) with accession no. GSE86184.

Determination of Cystine Levels by UPLC-MS.

An exponentially growing Mtb mc²6230 culture was diluted to an OD_600nm ∼ 0.3, treated with the appropriate chemicals, and incubated at 37 °C while shaking. The cultures (5 mL) were quenched with 10 mL of cold methanol and centrifuged at 0 °C for 10 min. The cell pellets were resuspended in 1 mL acetonitrile/methanol/water (40/40/20; vol/vol/v) containing 5 mM EDTA. Glass beads (0.1 mL) were added and the cells were lysed using the MP Bio FastPrep machine (45 s, speed 6 M/s, three times). After cooling, the samples were centrifuged for 1 min, and the supernatants were filter-sterilized before analyzing on an Acquity UPLC (Waters) coupled with a quadrupole time-of-flight mass spectrometer (Synapt G2, Waters). The UPLC column was an Acquity amide column 1.7 μm, the flow rate was 0.5 mL/min, and the column temperature was set at 55 °C. The mobile phases were: A, acetonitrile with 0.1% formic acid; B, water with 0.1% formic acid. The conditions used for the UPLC were: 0 min, 99% A; 1 min, 99% A; 14 min, 65% A; 17 min, 40% A; 18 min, 40% A; 19 min, 99% A; 20 min, 99% A. The MS conditions were: acquisition mode, MS^E; ionization mode, ESI (+); capillary voltage, 2 kV; cone voltage, 30 V; desolvation gas flow, 800 L/h; desolvation temperature, 550 °C; source temperature, 120 °C; acquisition range, 50–1200 m/z with a scan time of 0.5 s. Leucine enkephalin (2 ng/μL) was used as lock mass. Data generated from the experiment were processed and analyzed with Waters MarkerLynx software, which integrates, normalizes, and aligns MS datapoints and converts them into exact mass retention time pairs to build a matrix composed of retention time, exact mass m/z, and intensity pairs. Cysteine and cystine standards were run to determine retention time and m/z.

Viability Under Anaerobic Conditions.

Mtb mc²6230 was grown under a normal aerobic atmosphere to OD_600nm 0.5–0.8 and diluted 1/50 before being shifted to an anaerobic chamber (<0.0001% O₂, 5% CO₂, 10% H₂, 85% N₂). After 24 h of anaerobic incubation, the culture was divided and treated with RIF, cysteine, or both. At each time point, aliquots were taken and removed from the chamber to be serially diluted and plated. The plates were incubated at 37 °C under aerobic conditions for 4 wk.

Determination of Ferrous and Ferric Ion Concentrations.

An exponentially growing culture of Mtb mc²6230 was grown to an OD_600nm ∼ 0.15, split and treated with cysteine (4 mM), INH (7.3 μM) or the combination INH/Cys using the same concentrations as the individual treatment. At each time point, samples (10 mL) were taken and spun down. The supernatants were kept on ice while the cell pellets were washed twice with cold phosphate buffer solution PBS (Corning). The cell pellets were resuspended in 1 mL 50 mM NaOH with 0.1 mL of glass beads and lysed using a Fast Prep machine. The cell pellet lysate (0.1 mL) or a sample of the supernatant (0.1 mL) was mixed with 10 mM HCl (0.1 mL) and incubated at 60 °C for 2 h. After cooling, 0.03 mL iron detection reagent (6.5 mM ferrozine + 6.5 mM neocuproine + 2.5 M ammonium acetate + 1 M ascorbic acid in water) was added and the sample absorbance was read at 550 nm to quantify ferrous and ferric ion concentrations. Ferrous ion concentrations were obtained as described above, omitting ascorbic acid in the iron detection reagent. The iron concentrations were determined based on a standard curve obtained with increasing concentrations of ferric chloride standard and normalized to protein content.

Determination of Hydrogen Peroxide Concentration.

An exponentially growing Mtb mc²6230 culture was diluted to an OD_600nm ∼ 0.15, treated with cysteine, INH, or INH/Cys at the indicated concentrations, and incubated at 37 °C while shaking. At each time point, the cultures (10 mL) were centrifuged, and the cell pellets were washed once with PBS and resuspended in 0.5 mL PBS. Glass beads (0.1 mL) were added and the cells lysed using the MP Bio FastPrep machine (45 s, speed 6 M/s, three times). After cooling, the samples were centrifuged, and the supernatants were filter-sterilized before analysis. H₂O₂ concentrations were measured in the lysed cells and supernatants using Amplex Red reagent, according to the manufacturer’s instructions (Molecular Probes).

Determination of NADH and NAD⁺ Concentrations.

An exponentially growing culture of Mtb mc²6230 was grown to an OD_600nm ∼ 0.15, split, and treated with cysteine (4 mM), INH (7.3 μM), or the combination INH/Cys using the same concentrations as the individual treatment. At each time point, samples (10 mL) were taken, spun down, and treated with an aqueous solution of 0.2 N hydrochloric acid (1 mL, for NAD⁺ determination) or an aqueous solution of 0.2 N sodium hydroxide (1 mL, for NADH determination). The suspension were heated at 55 °C for 10 min, cooled down and neutralized with an aqueous solution of 0.1 N hydrochloric acid (1 mL, for NADH determination) or an aqueous solution of 0.1 N sodium hydroxide (1 mL, for NAD⁺ determination). The suspensions were kept at 0 °C for 10 min and centrifuged. The supernatant were filtered through a 0.2-μm filter twice before analysis. The reagent solution (an equal part of 1 M bicine, 40 mM EDTA, 16.6 mM phenazine ethosulfate, 4.2 mM ethylthiazolyldiphenyl-tetrazolium bromide, and 100% ethanol; 0.1 mL), the supernatant (0.09 mL) and alcohol dehydrogenase (5 units, 0.01 mL) were added to a 96-well plate and the plate was read at 570 nm for 10 min. The concentrations were determined based on a standard curve obtained with increasing concentrations of NAD⁺ standard.

Determination of the Menaquinol:Menaquinone Ratio.

An exponentially growing Mtb mc²6230 culture was diluted to an OD_600nm ∼ 0.15, treated with cysteine, INH, or INH/Cys at the indicated concentrations, and incubated at 37 °C while shaking. At each time point, a sample (2 mL) of each culture was removed and mixed with 6 mL of cold methanolic 0.2M perchloric acid solution. The menaquinol/menaquinone pools were extracted with petroleum ether (40–60 °C; 4 mL, × 2) and dried under nitrogen. The samples were resuspended in 2-propanol (0.2 mL) and immediately analyzed on an Acquity UPLC (Waters) coupled with a quadrupole time-of-flight mass spectrometer (Synapt G2, Waters). The UPLC column was an Acquity BEH C18 Column, the flow rate was 0.4 mL/min, and the column temperature was set at 55 °C. The mobile phases were: A, acetonitrile/water 60/40 with 0.1% formic acid and 10 mM aqueous ammonium formate solution; B, acetonitrile/isopropanol (10/90) with 0.1% formic acid and 10 mM aqueous ammonium formate solution. The conditions used for the UPLC were: 0 min, 60% A; 2 min, 57% A (curve 6); 2.1 min, 50% A (curve 1); 12 min, 46% A (curve 6); 12.1 min, 30% A (curve 1); 18 min, 1% A (curve 6); 18.1 min, 60% A (curve 6); 20 min, 60% A (curve 1). The MS conditions were: acquisition mode, MS^E; ionization mode, ESI (^+/−), acquisition range: 100–5,000 m/z with a scan time of 0.5 s, capillary 2 kV (positive), cone voltage 30 V; desolvation gas flow, 900 L/h; desolvation temperature, 550 °C; source temperature, 120 °C; acquisition range, 50–1,200 m/z with a scan time of 0.5 s. Leucine enkephalin (2 ng/μL) was used as lock mass. Data generated from the experiment were processed and analyzed with Waters MarkerLynx software, which integrates, normalizes, and aligns MS data points and converts them into exact mass retention time pairs to build a matrix composed of retention time, exact mass m/z, and intensity pairs. Menaquinone-9 (Santa Cruz Biotechnology) was run to determine retention time and m/z.

Detection of the Mtb Persister Population Using Φ²DRMs.

Flow cytometry.

An exponentially growing Mtb mc²6230 culture was washed twice with mycobacteriophage (MP) buffer (50 mM Tris, 150 mM NaCl, 10 mM MgCl₂, 2 mM CaCl₂) and diluted to an OD_600nm ∼ 0.1 in 7H9 media without tyloxapol in 96-well plates. Each well contained 0.1 mL of culture, which was treated with cysteine (4 mM), INH (7.3 μM), or INH/Cys at the indicated concentrations, at 37 °C for up to 4 d. The samples, along with the untreated control, were infected with 0.1 mL of the phage Φ²DRM9 at a MOI of 10 for 16 h at 37 °C. The phage Φ²DRM9 expresses both the L5 promoter driving GFP (mVenus) expression and the INH persister-specific dnaK promoter fused to the gene encoding the red fluorescent protein (RFP, tdTomato). Next, 10,000 events were acquired for each sample, along with the uninfected control, on S3e cell sorter (Bio-Rad) after gating the singlets on FSC-A and SSC-A on log₁₀ scale. The data were analyzed using the FlowJo software package (v10.0.7, TreeStar) by gating for GFP⁺ and GFP⁺tdTomato⁺ cells in comparison with the uninfected and the single color-positive Mtb mc²6230 controls. The RFP population was back-gated to determine GFP expression distribution in these cells.

Microscopy.

In parallel, the samples were also analyzed by microscopy using a Nikon Eclipse Ti microscope at >10 fields for each sample used for flow-cytometry. Images were captured with bright-field, FITC and Texas-Red filters. The exposure time for each filter is kept constant throughout the experiment.

Acknowledgments

W.R.J. acknowledges generous support from the NIH Centers for AIDS Research Grant (CFAR) AI-051519 at the Albert Einstein College of Medicine. This work was supported by the National Institutes of Health Grants AI26170 and U19AI111276 (to W.R.J.), Potts Memorial Foundation research grant (to P.J.), and Stony Wold-Herbert foundation (P.J.).

Footnotes

↵¹To whom correspondence should be addressed. Email: jacobsw{at}hhmi.org.

Author contributions: C.V. and W.R.J. designed research; C.V., T.H., B.W., P.J., T.R.W., and L.W.L., performed research; J.S.F. contributed new reagents/analytic tools; C.V., T.H., B.W., P.J., T.R.W., L.W.L., and W.R.J. analyzed data; and C.V. and W.R.J. wrote the paper.
Reviewers: C.C.A., University of Minnesota; M.H.L., Medical Research Council; and A.S., National Institute of Allergy and Infectious Diseases.
The authors declare no conflict of interest.
Data deposition: The data reported in this paper have been deposited in the Gene Expression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. GSE86184).
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1704376114/-/DCSupplemental.

References

↵
1. Raviglione M,
2. Sulis G
(2016) Tuberculosis 2015: Burden, challenges and strategy for control and elimination. Infect Dis Rep 8:6570.
.
OpenUrl
↵
1. Zhang Y,
2. Heym B,
3. Allen B,
4. Young D,
5. Cole S
(1992) The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358:591–593.
.
OpenUrl CrossRef PubMed
↵
1. Vilchèze C,
2. Jacobs WR Jr
(2014) Resistance to isoniazid and ethionamide in Mycobacterium tuberculosis: Genes, mutations, and causalities. Microbiol Spectr 2:MGM2-0014-2013.
.
OpenUrl
↵
1. Ung KS,
2. Av-Gay Y
(2006) Mycothiol-dependent mycobacterial response to oxidative stress. FEBS Lett 580:2712–2716.
.
OpenUrl CrossRef PubMed
↵
1. Buchmeier NA,
2. Newton GL,
3. Koledin T,
4. Fahey RC
(2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 47:1723–1732.
.
OpenUrl CrossRef PubMed
↵
1. Newton GL, et al.
(1999) Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255:239–244.
.
OpenUrl CrossRef PubMed
↵
1. Rawat M, et al.
(2002) Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob Agents Chemother 46:3348–3355.
.
OpenUrl Abstract/FREE Full Text
↵
1. Vilchèze C, et al.
(2011) Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis. Antimicrob Agents Chemother 55:4422–4423.
.
OpenUrl Abstract/FREE Full Text
↵
1. Vilchèze C, et al.
(2008) Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis. Mol Microbiol 69:1316–1329.
.
OpenUrl CrossRef PubMed
↵
1. Jain P, et al.
(2016) Dual-reporter mycobacteriophages (Φ²DRMs) reveal preexisting Mycobacterium tuberculosis persistent cells in human sputum. MBio 7:e01023-16.
.
OpenUrl Abstract/FREE Full Text
↵
1. Maciag A, et al.
(2007) Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J Bacteriol 189:730–740.
.
OpenUrl Abstract/FREE Full Text
↵
1. Boshoff HI, et al.
(2004) The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: Novel insights into drug mechanisms of action. J Biol Chem 279:40174–40184.
.
OpenUrl Abstract/FREE Full Text
↵
1. Voskuil MI, et al.
(2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198:705–713.
.
OpenUrl Abstract/FREE Full Text
↵
1. Schnappinger D, et al.
(2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment. J Exp Med 198:693–704.
.
OpenUrl Abstract/FREE Full Text
↵
1. Park S,
2. Imlay JA
(2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction. J Bacteriol 185:1942–1950.
.
OpenUrl Abstract/FREE Full Text
↵
1. Kachur AV,
2. Koch CJ,
3. Biaglow JE
(1999) Mechanism of copper-catalyzed autoxidation of cysteine. Free Radic Res 31:23–34.
.
OpenUrl CrossRef PubMed
↵
1. Demple B,
2. Harrison L
(1994) Repair of oxidative damage to DNA: Enzymology and biology. Annu Rev Biochem 63:915–948.
.
OpenUrl CrossRef PubMed
↵
1. Imlay JA,
2. Chin SM,
3. Linn S
(1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240:640–642.
.
OpenUrl Abstract/FREE Full Text
↵
1. Piccaro G,
2. Giannoni F,
3. Filippini P,
4. Mustazzolu A,
5. Fattorini L
(2013) Activities of drug combinations against Mycobacterium tuberculosis grown in aerobic and hypoxic acidic conditions. Antimicrob Agents Chemother 57:1428–1433.
.
OpenUrl Abstract/FREE Full Text
↵
1. Nath KA,
2. Salahudeen AK
(1993) Autoxidation of cysteine generates hydrogen peroxide: Cytotoxicity and attenuation by pyruvate. Am J Physiol 264:F306–F314.
.
OpenUrl
↵
1. Lobritz MA, et al.
(2015) Antibiotic efficacy is linked to bacterial cellular respiration. Proc Natl Acad Sci USA 112:8173–8180.
.
OpenUrl Abstract/FREE Full Text
↵
1. Hartman T, et al.
(2014) Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis. PLoS Pathog 10:e1004510.
.
OpenUrl CrossRef
↵
1. Wakamoto Y, et al.
(2013) Dynamic persistence of antibiotic-stressed mycobacteria. Science 339:91–95.
.
OpenUrl Abstract/FREE Full Text
↵
1. Grant SS,
2. Kaufmann BB,
3. Chand NS,
4. Haseley N,
5. Hung DT
(2012) Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci USA 109:12147–12152.
.
OpenUrl Abstract/FREE Full Text
↵
1. Vilchèze C,
2. Hartman T,
3. Weinrick B,
4. Jacobs WR Jr
(2013) Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Nat Commun 4:1881.
.
OpenUrl CrossRef PubMed
↵
1. Lamprecht DA, et al.
(2016) Turning the respiratory flexibility of Mycobacterium tuberculosis against itself. Nat Commun 7:12393.
.
OpenUrl CrossRef PubMed
↵
1. Amaral EP, et al.
(2016) N-acetyl-cysteine exhibits potent anti-mycobacterial activity in addition to its known anti-oxidative functions. BMC Microbiol 16:251.
.
OpenUrl
↵
1. Baniasadi S, et al.
(2010) Protective effect of N-acetylcysteine on antituberculosis drug-induced hepatotoxicity. Eur J Gastroenterol Hepatol 22:1235–1238.
.
OpenUrl CrossRef PubMed
↵
1. Kranzer K, et al.
(2015) A systematic review and meta-analysis of the efficacy and safety of N-acetylcysteine in preventing aminoglycoside-induced ototoxicity: Implications for the treatment of multidrug-resistant TB. Thorax 70:1070–1077.
.
OpenUrl Abstract/FREE Full Text
↵
1. Dinesh N,
2. Sunil M,
3. Sanjay G
(2013) Supplementation of N-acetylcysteine as an adjuvant in treatment of newly diagnosed pulmonary tuberculosis patients: A prospective, randomized double blind, placebo controlled study. Eur Respir J 42:P2833.
.
OpenUrl
↵
1. Leistikow RL, et al.
(2010) The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy. J Bacteriol 192:1662–1670.
.
OpenUrl Abstract/FREE Full Text
↵
1. Marcus SA,
2. Sidiropoulos SW,
3. Steinberg H,
4. Talaat AM
(2016) CsoR is essential for maintaining copper homeostasis in Mycobacterium tuberculosis. PLoS One 11:e0151816.
.
OpenUrl
↵
1. Dicks AP,
2. Williams DL
(1996) Generation of nitric oxide from S-nitrosothiols using protein-bound Cu2+ sources. Chem Biol 3:655–659.
.
OpenUrl CrossRef PubMed
↵
1. Mosmann T
(1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.
.
OpenUrl CrossRef PubMed
↵
1. Park HD, et al.
(2003) Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48:833–843.
.
OpenUrl CrossRef PubMed

Article Alerts

Email Article

Citation Tools

Request Permissions

Article Classifications

Biological Sciences
Microbiology

Proceedings of the National Academy of Sciences: 114 (17)

Table of Contents

Submit

[1] ↵
Raviglione M,
Sulis G
(2016) Tuberculosis 2015: Burden, challenges and strategy for control and elimination. Infect Dis Rep 8:6570.
.
OpenUrl

[2] Raviglione M,

[3] Sulis G

[4] ↵
Zhang Y,
Heym B,
Allen B,
Young D,
Cole S
(1992) The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature 358:591–593.
.
OpenUrl CrossRef PubMed

[5] Zhang Y,

[6] Heym B,

[7] Allen B,

[8] Young D,

[9] Cole S

[10] ↵
Vilchèze C,
Jacobs WR Jr
(2014) Resistance to isoniazid and ethionamide in Mycobacterium tuberculosis: Genes, mutations, and causalities. Microbiol Spectr 2:MGM2-0014-2013.
.
OpenUrl

[11] Vilchèze C,

[12] Jacobs WR Jr

[13] ↵
Ung KS,
Av-Gay Y
(2006) Mycothiol-dependent mycobacterial response to oxidative stress. FEBS Lett 580:2712–2716.
.
OpenUrl CrossRef PubMed

[14] Ung KS,

[15] Av-Gay Y

[16] ↵
Buchmeier NA,
Newton GL,
Koledin T,
Fahey RC
(2003) Association of mycothiol with protection of Mycobacterium tuberculosis from toxic oxidants and antibiotics. Mol Microbiol 47:1723–1732.
.
OpenUrl CrossRef PubMed

[17] Buchmeier NA,

[18] Newton GL,

[19] Koledin T,

[20] Fahey RC

[21] ↵
Newton GL, et al.
(1999) Characterization of Mycobacterium smegmatis mutants defective in 1-d-myo-inosityl-2-amino-2-deoxy-alpha-d-glucopyranoside and mycothiol biosynthesis. Biochem Biophys Res Commun 255:239–244.
.
OpenUrl CrossRef PubMed

[22] Newton GL, et al.

[23] ↵
Rawat M, et al.
(2002) Mycothiol-deficient Mycobacterium smegmatis mutants are hypersensitive to alkylating agents, free radicals, and antibiotics. Antimicrob Agents Chemother 46:3348–3355.
.
OpenUrl Abstract/FREE Full Text

[24] Rawat M, et al.

[25] ↵
Vilchèze C, et al.
(2011) Coresistance to isoniazid and ethionamide maps to mycothiol biosynthetic genes in Mycobacterium bovis. Antimicrob Agents Chemother 55:4422–4423.
.
OpenUrl Abstract/FREE Full Text

[26] Vilchèze C, et al.

[27] ↵
Vilchèze C, et al.
(2008) Mycothiol biosynthesis is essential for ethionamide susceptibility in Mycobacterium tuberculosis. Mol Microbiol 69:1316–1329.
.
OpenUrl CrossRef PubMed

[28] Vilchèze C, et al.

[29] ↵
Jain P, et al.
(2016) Dual-reporter mycobacteriophages (Φ²DRMs) reveal preexisting Mycobacterium tuberculosis persistent cells in human sputum. MBio 7:e01023-16.
.
OpenUrl Abstract/FREE Full Text

[30] Jain P, et al.

[31] ↵
Maciag A, et al.
(2007) Global analysis of the Mycobacterium tuberculosis Zur (FurB) regulon. J Bacteriol 189:730–740.
.
OpenUrl Abstract/FREE Full Text

[32] Maciag A, et al.

[33] ↵
Boshoff HI, et al.
(2004) The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: Novel insights into drug mechanisms of action. J Biol Chem 279:40174–40184.
.
OpenUrl Abstract/FREE Full Text

[34] Boshoff HI, et al.

[35] ↵
Voskuil MI, et al.
(2003) Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198:705–713.
.
OpenUrl Abstract/FREE Full Text

[36] Voskuil MI, et al.

[37] ↵
Schnappinger D, et al.
(2003) Transcriptional adaptation of Mycobacterium tuberculosis within macrophages: Insights into the phagosomal environment. J Exp Med 198:693–704.
.
OpenUrl Abstract/FREE Full Text

[38] Schnappinger D, et al.

[39] ↵
Park S,
Imlay JA
(2003) High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction. J Bacteriol 185:1942–1950.
.
OpenUrl Abstract/FREE Full Text

[40] Park S,

[41] Imlay JA

[42] ↵
Kachur AV,
Koch CJ,
Biaglow JE
(1999) Mechanism of copper-catalyzed autoxidation of cysteine. Free Radic Res 31:23–34.
.
OpenUrl CrossRef PubMed

[43] Kachur AV,

[44] Koch CJ,

[45] Biaglow JE

[46] ↵
Demple B,
Harrison L
(1994) Repair of oxidative damage to DNA: Enzymology and biology. Annu Rev Biochem 63:915–948.
.
OpenUrl CrossRef PubMed

[47] Demple B,

[48] Harrison L

[49] ↵
Imlay JA,
Chin SM,
Linn S
(1988) Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro. Science 240:640–642.
.
OpenUrl Abstract/FREE Full Text

[50] Imlay JA,

[51] Chin SM,

[52] Linn S

[53] ↵
Piccaro G,
Giannoni F,
Filippini P,
Mustazzolu A,
Fattorini L
(2013) Activities of drug combinations against Mycobacterium tuberculosis grown in aerobic and hypoxic acidic conditions. Antimicrob Agents Chemother 57:1428–1433.
.
OpenUrl Abstract/FREE Full Text

[54] Piccaro G,

[55] Giannoni F,

[56] Filippini P,

[57] Mustazzolu A,

[58] Fattorini L

[59] ↵
Nath KA,
Salahudeen AK
(1993) Autoxidation of cysteine generates hydrogen peroxide: Cytotoxicity and attenuation by pyruvate. Am J Physiol 264:F306–F314.
.
OpenUrl

[60] Nath KA,

[61] Salahudeen AK

[62] ↵
Lobritz MA, et al.
(2015) Antibiotic efficacy is linked to bacterial cellular respiration. Proc Natl Acad Sci USA 112:8173–8180.
.
OpenUrl Abstract/FREE Full Text

[63] Lobritz MA, et al.

[64] ↵
Hartman T, et al.
(2014) Succinate dehydrogenase is the regulator of respiration in Mycobacterium tuberculosis. PLoS Pathog 10:e1004510.
.
OpenUrl CrossRef

[65] Hartman T, et al.

[66] ↵
Wakamoto Y, et al.
(2013) Dynamic persistence of antibiotic-stressed mycobacteria. Science 339:91–95.
.
OpenUrl Abstract/FREE Full Text

[67] Wakamoto Y, et al.

[68] ↵
Grant SS,
Kaufmann BB,
Chand NS,
Haseley N,
Hung DT
(2012) Eradication of bacterial persisters with antibiotic-generated hydroxyl radicals. Proc Natl Acad Sci USA 109:12147–12152.
.
OpenUrl Abstract/FREE Full Text

[69] Grant SS,

[70] Kaufmann BB,

[71] Chand NS,

[72] Haseley N,

[73] Hung DT

[74] ↵
Vilchèze C,
Hartman T,
Weinrick B,
Jacobs WR Jr
(2013) Mycobacterium tuberculosis is extraordinarily sensitive to killing by a vitamin C-induced Fenton reaction. Nat Commun 4:1881.
.
OpenUrl CrossRef PubMed

[75] Vilchèze C,

[76] Hartman T,

[77] Weinrick B,

[78] Jacobs WR Jr

[79] ↵
Lamprecht DA, et al.
(2016) Turning the respiratory flexibility of Mycobacterium tuberculosis against itself. Nat Commun 7:12393.
.
OpenUrl CrossRef PubMed

[80] Lamprecht DA, et al.

[81] ↵
Amaral EP, et al.
(2016) N-acetyl-cysteine exhibits potent anti-mycobacterial activity in addition to its known anti-oxidative functions. BMC Microbiol 16:251.
.
OpenUrl

[82] Amaral EP, et al.

[83] ↵
Baniasadi S, et al.
(2010) Protective effect of N-acetylcysteine on antituberculosis drug-induced hepatotoxicity. Eur J Gastroenterol Hepatol 22:1235–1238.
.
OpenUrl CrossRef PubMed

[84] Baniasadi S, et al.

[85] ↵
Kranzer K, et al.
(2015) A systematic review and meta-analysis of the efficacy and safety of N-acetylcysteine in preventing aminoglycoside-induced ototoxicity: Implications for the treatment of multidrug-resistant TB. Thorax 70:1070–1077.
.
OpenUrl Abstract/FREE Full Text

[86] Kranzer K, et al.

[87] ↵
Dinesh N,
Sunil M,
Sanjay G
(2013) Supplementation of N-acetylcysteine as an adjuvant in treatment of newly diagnosed pulmonary tuberculosis patients: A prospective, randomized double blind, placebo controlled study. Eur Respir J 42:P2833.
.
OpenUrl

[88] Dinesh N,

[89] Sunil M,

[90] Sanjay G

[91] ↵
Leistikow RL, et al.
(2010) The Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy. J Bacteriol 192:1662–1670.
.
OpenUrl Abstract/FREE Full Text

[92] Leistikow RL, et al.

[93] ↵
Marcus SA,
Sidiropoulos SW,
Steinberg H,
Talaat AM
(2016) CsoR is essential for maintaining copper homeostasis in Mycobacterium tuberculosis. PLoS One 11:e0151816.
.
OpenUrl

[94] Marcus SA,

[95] Sidiropoulos SW,

[96] Steinberg H,

[97] Talaat AM

[98] ↵
Dicks AP,
Williams DL
(1996) Generation of nitric oxide from S-nitrosothiols using protein-bound Cu2+ sources. Chem Biol 3:655–659.
.
OpenUrl CrossRef PubMed

[99] Dicks AP,

[100] Williams DL

[101] ↵
Mosmann T
(1983) Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63.
.
OpenUrl CrossRef PubMed

[102] Mosmann T

[103] ↵
Park HD, et al.
(2003) Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48:833–843.
.
OpenUrl CrossRef PubMed

[104] Park HD, et al.

Main menu

User menu

Search

Significance

Abstract

Results

Addition of Cysteine to INH-Treated, Exponentially Growing Cultures of Mtb Results in Sterilization of Drug-Susceptible and Drug-Resistant Mtb.

A Free Thiol Group Is Required in Combination with INH for Sterilizing Activity.

The Combination of Cysteine and RIF Prevents the Emergence of RIF-Resistant Mutants.

Transcriptional Profiling Reveals That INH/Cys Triggers an Oxidative Process.

Cysteine Is Rapidly Oxidized into Cystine Leading to ROS Production.

The INH/Cys Combination Generates a Transient Increase in H2O2 Concentration.

The INH/Cys Combination Increases Mtb Cellular Respiration and Prevents the Emergence of INH Persisters.

The INH/Cys Combination Potentiates INH Activity in Macrophages.

Discussion

Materials and Methods

Bacterial Strains and Reagents.

Growth of Mtb in Vitro Cultures.

Determination of Total Thiol Concentration.

ROS and DNA Breaks Analyses.

Determination of Oxygen Consumption.

J774 Murine Macrophage Experiments.

SI Materials and Methods

Microarray Analysis.

Determination of Cystine Levels by UPLC-MS.

Viability Under Anaerobic Conditions.

Determination of Ferrous and Ferric Ion Concentrations.

Determination of Hydrogen Peroxide Concentration.

Determination of NADH and NAD+ Concentrations.

Determination of the Menaquinol:Menaquinone Ratio.

Detection of the Mtb Persister Population Using Φ2DRMs.

Flow cytometry.

Microscopy.

Acknowledgments

Footnotes

References

Citation Manager Formats

Article Classifications

Sign up for Article Alerts

Jump to section

You May Also be Interested in

Similar Articles

Articles

PNAS Portals

Information

The INH/Cys Combination Generates a Transient Increase in H₂O₂ Concentration.

Determination of NADH and NAD⁺ Concentrations.

Detection of the Mtb Persister Population Using Φ²DRMs.