Research ArticleTuberculosis

Targeting redox heterogeneity to counteract drug tolerance in replicating Mycobacterium tuberculosis

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Science Translational Medicine  13 Nov 2019:
Vol. 11, Issue 518, eaaw6635
DOI: 10.1126/scitranslmed.aaw6635
  • Fig. 1 RNA-seq of intraphagosomal Mtb derived from EMSH-reduced and EMSH-basal fractions.

    (A) Schematic depiction of flow sorting–coupled RNA-seq of intraphagosomal bacteria present in EMSH-basal and EMSH-reduced fractions of THP-1 macrophages infected with Mtb/Mrx1-roGFP2. Mtb cells (optical density at 600 nm, 0.4) harvested and resuspended in RPMI for 24 hours were used as an in vitro control. FACS, fluorescence-activated cell sorting; GTC, guanidinium thiocyanate. (B) Scatter plot indicates relative distribution of differentially expressed genes (DEGs) from the EMSH-reduced and EMSH-basal fractions on the basis of log2 fold changes (FC) (blue, DEGs specific to EMSH-reduced; red, DEGs unique to EMSH-basal; black, DEGs common to both; gray, nonsignificant genes). (C) The table summarizes the transcriptional overlap between this study and the response of Mtb under intramacrophage and pH stress conditions. Fisher’s exact test with P < 0.05 as a cutoff for significance. ns, no significant difference. (D) Heat maps indicate log2 fold changes of DEGs belonging to various functional categories (obtained from Mycobrowser, École polytechnique fédérale de Lausanne) in EMSH-reduced and EMSH-basal fractions. Genes were considered differentially expressed on the basis of the false discovery rate (FDR) of ≤0.05 and absolute fold change of ≥1.5 (tables S1 and S3).

  • Fig. 2 Cysteine utilization pathways promote redox heterogeneity and drug tolerance in Mtb.

    (A) Various cysteine (CySH) utilization pathways in Mtb. Expression of genes (blue) coordinating CySH flux into pathways for mycothiol (MSH), Fe-S cluster, and H2S biogenesis is induced in the EMSH-reduced fraction. (B) THP-1 macrophages were infected for 24 hours with the indicated strains of Mtb expressing Mrx1-roGFP2, and the percent distribution of redox-diverse fractions was measured at 24 hours p.i. and depicted as a stacked bar plot. **P < 0.01, by Mann-Whitney test compares EMSH-reduced fraction in various strains of Mtb with WT Mtb. (C) THP-1 macrophages infected for 24 hours with the indicated strains of Mtb were exposed for an additional 48 hours to Inh (2.18 μM, 3× of in vitro MIC) or left untreated. Bacillary load was determined by CFU enumeration, and percent survival was quantified by normalizing the CFU in Inh-treated samples at 48 hours against untreated samples (UT) at 0 hours. *P < 0.05, **P < 0.01, #P < 0.05, and ##P < 0.01, by Mann-Whitney test. Number signs (#) and asterisks (*) compare survival between WT Mtb and other strains under UT and Inh-treated conditions, respectively. (D and E) Indicated strains of Mtb grown in 7H9-tyloxapol broth acidified to pH 6.2 or pH 4.5 were exposed to Inh (7.25 μM, 10× of in vitro MIC) or kept unexposed. Bacterial load was quantified after 5 days of treatment by CFU enumeration, and percent survival was quantified strain-wise by normalizing the bacterial load in Inh-treated samples at day 5 against untreated samples. *P < 0.05, **P < 0.001, by Mann-Whitney test. Asterisks compare survival between WT Mtb and other strains after 5 days of Inh treatment. Data shown in each panel are the results of three independent experiments performed in triplicate (means ± SD). ns, no significant difference (P > 0.05).

  • Fig. 3 Phagosomal pH is required for the redox-dependent multidrug tolerance of Mtb.

    (A) THP-1 macrophages—untreated or pretreated with 10 nM BafA1, 10 mM NH4Cl, or 10 μM CQ—were infected with Mtb/Mrx1-roGFP2, and percent distribution of redox-diverse fractions was measured at 24 hours p.i. **P < 0.01, by Mann-Whitney test to compare the EMSH-reduced fraction with untreated sample. (B and C) THP-1 macrophages, untreated or pretreated with 10 nM BafA1 or 10 μM CQ, were infected with WT Mtb for 24 hours and exposed to Inh (2.18 μM) or Rif (1 μM) or left unexposed for an additional 48 hours. Percent survival was quantified by normalizing the CFU in drug-treated samples at 48 hours against untreated samples at 0 hours. *P < 0.05, **P < 0.01, by Mann-Whitney test. (D) THP-1 macrophages were infected with Mtb/Mrx1-roGFP2, and EMSH was measured at 24 hours p.i. After this, intraphagosomal bacteria were released and incubated in 7H9-albumen-dextrose-sodium chloride for 2 hours, and EMSH was determined. The 7H9-ADS–adapted Mtb was used to reinfect fresh THP-1 macrophages, with or without pretreatment with 10 μM CQ, and EMSH was measured at 24 hours p.i. **P < 0.01, ###P < 0.001, by Mann-Whitney test. Number signs (#) compare EMSH-reduced fractions between intramacrophage and 7H9-ADS–adapted Mtb. Asterisks (*) compare EMSH-reduced fractions between untreated and CQ-treated samples. (E) THP-1 macrophages harboring EMSH-reduced and EMSH-basal bacteria were flow-sorted at 24 hours p.i. (cycle 1 infection), and bacteria were released into 7H9-ADS. At 24-hour incubation, 7H9-ADS–adapted Mtb were used to infect THP-1 macrophages, with or without pretreatment with 10 μM CQ, for 24 hours (cycle 2 infection), and Inh tolerance was determined as mentioned earlier. *P < 0.05, **P < 0.01, by Mann-Whitney test. Data shown in each panel are the results of three independent experiments performed in triplicate (means ± SD). ns, no significant difference (P > 0.05).

  • Fig. 4 Phagosomal pH and redox heterogeneity drive drug tolerance during HIV-TB coinfection.

    (A) The course of HIV-1 replication upon stimulation of the U1 promonocytic cell line with PMA (5 ng/ml). Viral load was monitored by gag qRT-PCR. **P < 0.01, by Mann-Whitney test comparing gag expression with 0 hours. U937 (uninfected HIV-1 control) (B) and U1 macrophages (C) were stimulated with PMA and infected with Mtb/Mrx1-roGFP2, and percent distribution of redox-diverse fractions was measured over time. *P < 0.05, by Mann-Whitney test. Asterisks (*) compare EMSH-reduced fraction at various time points with 0 hours. (D) U1 macrophages—untreated or pretreated with 10 nM BafA1, 10 mM NH4Cl, and 10 μM CQ—were infected with Mtb/Mrx1-roGFP2, and percent distribution of redox-diverse fractions was measured at 12 hours p.i. *P < 0.05, **P < 0.01, by Mann-Whitney test. Asterisks (*) compare EMSH-reduced fractions between untreated and BafA1/NH4Cl/CQ-treated samples. U937 (E) and U1 macrophages (F), untreated or pretreated with 10 μM CQ or 10 nM BafA1, were infected with WT Mtb for 12 hours and exposed to Inh (2.18 μM) or left unexposed for an additional 48 hours. Bacillary load was determined by CFU enumeration, and percent survival was quantified by normalizing the CFU in drug-treated samples at 48 hours against untreated samples at 0 hours. *P < 0.05, **P < 0.01, by Mann-Whitney test. Data shown in each panel are the results of three independent experiments performed in triplicate (means ± SD).

  • Fig. 5 The drug-tolerant EMSH-reduced population is replicative and has high efflux pump activity.

    (A) Graphical depiction of Mrx1-roGFP2–coupled flow-sorting strategy to determine replication dynamics, metabolic state, and drug efflux activity in intramacrophage EMSH-reduced and EMSH-basal populations. (B and C) THP-1 macrophages were infected with pBP10-containing Mtb/Mrx1-roGFP2. At indicated time points, macrophages harboring EMSH-reduced and EMSH-basal bacteria were flow-sorted, and bacteria were released and plated in the presence or absence of kanamycin (Kan). The frequency of pBP10 loss and increase in cumulative bacterial burden (CBB) were calculated. *P < 0.05, **P < 0.01, #P < 0.05, ##P < 0.01, by Kruskal-Wallis test with Dunn’s correction over time p.i. Asterisks (*) and number signs (#) compare CFU per milliliter and percentage of pBP10 + ve bacteria over time p.i., respectively. (D) THP-1 macrophages were infected with Mtb/Mrx1-roGFP2, and at 24 hours p.i., the redox state of intraphagosomal Mtb/Mrx1-roGFP2 thiols was fixed using N-ethylmaleimide. Bacteria were released from macrophages and stained with calcein violet–AM (CV-AM). The CV-AM staining and EMSH status of Mtb cells were determined using multiparameter flow cytometric analysis. As a control, we performed CV-AM staining of Mtb grown in 7H9 broth for 24 hours at 4° and 37°C. *P < 0.05, by Mann-Whitney test. (E) THP-1 macrophages harboring EMSH-reduced and EMSH-basal bacteria were flow-sorted at 24 hours p.i., bacterial RNA was isolated, and expression of efflux pumps was measured by qRT-PCR. Expression was compared with in vitro control Mtb, and fold change was quantified after normalizing by 16S ribosomal RNA. *P < 0.05, **P < 0.01, by Mann-Whitney test for comparison with in vitro control Mtb. (F) THP-1 macrophages were infected with Mtb/Mrx1-roGFP2 bacteria preloaded with [14C]-Inh. At 24 hours p.i., macrophages harboring EMSH-reduced (Red) and EMSH-basal (Bas) bacteria were sorted and bacteria released. The relative distribution of radioactive [14C]-Inh was measured in bacterial and macrophage (MΦ) fractions. *P < 0.05, **P < 0.01, by Mann-Whitney test. Data shown in each panel are the results of two independent experiments performed in triplicate (means ± SD). ns, no significant difference (P > 0.05).

  • Fig. 6 CQ counteracts drug tolerance and reduces relapse in vivo.

    (A) Strategy to investigate the efficacy of CQ in reducing tolerance against Inh and Rif and posttherapeutic relapse in vivo. BALB/c mice (n = 6) were given an aerosol challenge with WT Mtb. From 4 weeks p.i. onward, groups of mice were left untreated or treated with anti-TB drugs (Inh/Rif) alone or in combination with CQ (CQ + Inh/ CQ + Rif). (B and C) Bacterial CFUs were measured in the lungs at the indicated time points. **P < 0.01, ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction across experimental groups at 12 weeks p.i. (D) Gross pathology of the lungs of WT Mtb–infected mice at 8 weeks of treatment across experimental groups. (E) Hematoxylin and eosin–stained lung sections (8 weeks of treatment) from mice infected with WT Mtb across experimental groups. The pathology sections show granuloma (G), alveolar space (AS), and bronchiole lumen (BL). All images were taken at ×40 magnification. Scale bars, 200 μm. (F) Outbred Hartley guinea pigs (n = 6) were given aerosol challenge with WT Mtb, and efficacy of CQ in reducing Inh tolerance was assessed as described in (B) and (C). **P < 0.01, ****P < 0.0001, by Kruskal-Wallis test with Dunn’s correction across experimental groups at 12 weeks p.i. (G) Hematoxylin and eosin–stained lung sections (8 weeks of treatment) from guinea pigs infected with WT Mtb across experimental groups. The pathology sections show granuloma (G), alveolar space (AS), and necrotic core (N). All images were taken at ×40 magnification. Scale bars, 200 μm. (H) Dexamethasone-induced reactivation of Mtb from the lungs of BALB/c mice (n = 5) after treatment with Inh alone or a combination of CQ plus Inh. Mann-Whitney test was used to compare the relapse frequency (Inh alone versus CQ + Inh combination) for effectiveness of CQ therapy (P = 0.0069). Data shown in each panel are the results of two independent experiments (means ± SD). ns, no significant difference (P > 0.05).

  • Fig. 7 CQ exhibits no adverse interactions with anti-TB drugs.

    (A) The table indicates three groups of treatment in BALB/c mice used in the pharmacokinetic study: CQ alone, front line anti-TB combination therapy (HREZ), and combination (CQ + HREZ). (B to F) Line plots indicate pharmacokinetic profiles of CQ and individual drugs of the anti-TB therapy regimen analyzed individually and in the presence of each other in plasma of animals over 24 hours. No significant difference was observed between groups at each time point indicated in each panel by Mann-Whitney test (P > 0.05). (G) The table depicts ratios of Cmax and AUClast of individual drugs alone or in combination to analyze drug-drug interaction. Doses used are the following: CQ, 10 mg/kg body weight, i.p.; Inh/H, 25 mg/kg body weight, p.o.; Rif/R, 10 mg/kg body weight, p.o.; Emb/E, 200 mg/kg body weight, p.o.; Pza/Z, 150 mg/kg body weight, p.o. p.o., per os consumption; BDL, below detection limit. All data are means ± SD of concentrations at each time point of samples in triplicates (n = 3 animals per group).

Supplementary Materials

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

    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 MtbmetB and MtbsufR.

    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.

    Table S1. List of differentially expressed genes from DESeq2 for EMSH-reduced, EMSH-basal, and in vitro control samples.

    Table S2. List of differentially expressed genes from DESeq2 for WT Mtb, MtbwhiB3, and whiB3-Comp strains at pH 6.6 and pH 4.5 used to specify the low pH–inducible WhiB3 regulon.

    Table S3. List of EMSH-reduced and EMSH-basal differentially expressed genes used to generate custom heat maps.

    Table S4. List of strains and primers used in this study.

    References (94112)

  • 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 MtbmetB and MtbsufR.
    • 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 (94112)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • 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, MtbwhiB3, 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.

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