The antibiotic bedaquiline activates host macrophage innate immune resistance to bacterial infection
- Unit for Integrated Mycobacterial Pathogenomics, CNRS UMR 3525, Institut Pasteur, France
- Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, France
- Mycobacterial Genetics Unit, Institut Pasteur, France
- Bioinformatics and Biostatistics, Department of Computational Biology, USR 3756 CNRS, Institut Pasteur, France
- MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Faculty of Natural Sciences, Imperial College London, United Kingdom
- Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, United Kingdom
- Department of Tuberculosis Control and Prevention, Shenzhen Nanshan Center for Chronic Disease Control, China
Figures
Figure 1 with 2 supplements
BDQ modulates the response of human macrophages.
Cells from four individual donors were treated with BDQ (5 μg/mL) for 18 hr. Differentially expressed genes were identified by mRNAseq. (A) Gene ontology enrichment analysis of genes whose …
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Figure 1—source data 1
Genes whose expression is upregulated in naive macrophages upon BDQ treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data1-v1.xlsx
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Figure 1—source data 2
Genes whose expression is downregulated in naive macrophages upon BDQ treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data2-v1.xlsx
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Figure 1—source data 3
Genes whose expression is upregulated in BDQr-MTB-infected macrophages upon BDQ treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data3-v1.xlsx
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Figure 1—source data 4
Genes whose expression is downregulated in BDQr-MTB-infected macrophages upon BDQ treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data4-v1.xlsx
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Figure 1—source data 5
Genes differentially expressed in BDQr-MTB infected macrophages by BDQ.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data5-v1.xlsx
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Figure 1—source data 6
Genes differentially expressed in naive macrophages by BDQ.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data6-v1.xlsx
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Figure 1—source data 7
Differentially expressed genes both in naive and in BDQr-MTB infected macrophages upon BDQ treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig1-data7-v1.xlsx
Figure 1—figure supplement 1
Cell viability assay of macrophages incubated with BDQ.
(A) Cells were treated with various concentrations of BDQ. After 4 hr, 2 and 7 days, cell viability was evaluated with the MTT assay (Trevigen) according to the manufacturer's instructions. (B) …
Figure 1—figure supplement 2
Generation of BDQ resistant MTB strain (BDQr-MTB) and evaluation of its virulence.
(A) Amino acid sequence alignment of the ATP synthase c-subunit gene in wild-type (WT) and BDQ-resistant H37Rv strain. The mutation was indicated in red, at position 63. (B) Optical density (OD) …
Figure 2 with 1 supplement
Modulation of host metabolism by BDQ.
(A–B) The Glycolytic Rate Assay was performed in heat killed-MTB stimulated macrophages treated with BDQ, in the presence of rotenone/antimycin A (Rot/AA) and 2-deoxy-D-glycose (2-DG), inhibitors of …
Figure 2—figure supplement 1
BDQ modulates the metabolism of naïve macrophages.
(A–B) The Glycolytic Rate Assay was performed in macrophages, in the presence of rotenone/antimycin A (Rot/AA) and 2-deoxy-D-glycose (2-DG), respectively inhibitors of mitochondrial electron …
Figure 3 with 2 supplements
BDQ activates the lysosomal pathway in human MTB-infected macrophages.
(A) Heatmap showing differential expression of genes included in the Lysosome KEGG category (p-value<0.05). Each column corresponds to one donor. Data were normalized to determine the log ratio with …
Figure 3—figure supplement 1
BDQ activates the lysosomal pathway in human naive macrophages.
(A) Macrophages were incubated with BDQ (5 μg/mL) for 3 hr, 18 hr and 48 hr. Acid organelles were then labeled with 100 nM LysoTracker DND-99 for 1 hr. The fluorescence intensity was quantified by …
Figure 3—figure supplement 2
BDQ activates the lysosomal pathway, regardless of infection with live MTB.
(A) Briefly, cells were untreated, infected with drug-susceptible MTB or BDQr-MTB, or stimulated with LPS (TLR4 agonist), Pam3CSK4 (TLR1/2 agonist) or heat-killed bacteria, and then treated with …
Figure 4 with 1 supplement
BDQ potentiates PZA antimycobacterial activity.
(A) Macrophages were infected with BDQr-MTB and treated with BDQ (1 μg/mL) and PZA. After 7 of days treatment, cells were lysed and bacteria were enumerated by CFU (counted in triplicate). (B) …
Figure 4—figure supplement 1
BDQ did not potentiate the activity of the other first-line anti-TB drugs, in liquid culture or in BDQr-MTB infected macrophages.
(A) Cells were infected with BDQr-MTB and treated with BDQ (1 μg/mL) and RIF, INH or EMB. After 5 days treatment, cells were lysed and bacteria were enumerated by CFU (counted in triplicate). (B) …
Figure 5 with 1 supplement
Classical anti-TB drugs did not activate the lysosomal pathway in human macrophages.
(A) Number of differentially-expressed genes upon treatment with commonly used anti-TB drugs relative to untreated control. Briefly, naïve- and hk-MTB-stimulated macrophages were treated with AMK …
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Figure 5—source data 1
Differentially expressed genes in hk-MTB stimulated macrophages upon EMB treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data1-v1.xlsx
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Figure 5—source data 2
Differentially expressed genes in hk-MTB stimulated macrophages upon RIF treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data2-v1.xlsx
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Figure 5—source data 3
Differentially expressed genes in naive macrophages upon RIF treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data3-v1.xlsx
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Figure 5—source data 4
Differentially expressed genes in hk-MTB stimulated macrophages upon PZA treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data4-v1.xlsx
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Figure 5—source data 5
Regulated genes naive macrophages upon PZA treatment.
FDR < 0.05.
- https://cdn.elifesciences.org/articles/55692/elife-55692-fig5-data5-v1.xlsx
Figure 5—figure supplement 1
Scatterplot depicting the correlation between the log2 fold changes (log2FC) in gene expression levels using live MTB and heat-killed bacteria.
Figure 6
BDQ induced autophagy in MTB-infected macrophages.
(A) BDQr-MTB-infected macrophages were incubated with BDQ (5 μg/mL) and different inhibitors of autophagy; bafilomycin (BAF, 100 nM), chloroquine (CQ, 40 μM) and 3-methyladenine (3-MA, 5 mM). After …
Figure 7
BDQ increases macrophage bactericidal functions.
(A) Growth of S. Typhimurium and S. aureus in liquid medium in the presence of BDQ (20 μg/mL). (B) Macrophages were incubated with BDQ and then infected with S. Typhimurium or S. aureus. The number …
Figure 8 with 2 supplements
BDQ does not affect mitochondrial functions.
(A) Macrophages were treated with BDQ for 6, 24 and 48 hr and incubated with TMRM. Fluorescence was quantified by flow cytometry. Oligomycin (OM) is a positive control. (B) Cells were treated as in …
Figure 8—figure supplement 1
The hERG gene is not expressed in human monocyte-derived macrophages.
RT-qPCR was performed in order to detect hERG mRNA expression in macrophages, in hERG-transfected and non-transfected HEK293 cells (kind gift from Craig T. January, University of Wisconsin–Madison). …
Figure 8—figure supplement 2
Oxygen consumption rate (OCR) measured by Seahorse extracellular flux assay of cells incubated with BDQ for 48 hr.
Basal respiration, ATP production, maximal respiration, respiratory reserve and nonmitochondrial respiration were followed by sequential additions of oligomycin (OM), FCCP, and the inhibitors of …
Figure 9 with 1 supplement
Activation of TFEB by BDQ.
(A) Representative fluorescence microscopy images of macrophages treated with BDQ for 6 hr and 18 hr, or incubated in HBSS for 1 hr (starvation). Cells were stained with antibody against TFEB (red). …
Figure 9—figure supplement 1
BDQ-induced lysosome activation is dependent on TFEB.
(A) TFEB expression was inhibited in macrophages using siRNA-mediated gene silencing. After 48 hr, relative gene expression of TFEB was measured by RT-qPCR. Relative expression levels were …
Tables
Table 1
Gene Ontology (GO) functional annotation of genes differentially expressed by BDQ only in naïve- and BDQr-MTB-infected macrophages.
| Specific NAIVE BDQ genes | ||||||
|---|---|---|---|---|---|---|
| GO category | avg. LogFC | p-value | ||||
| Cell division | -0.51 | 8.34E-05 | ||||
| Sphingolipid metabolic process | 0.33 | 1.42E-04 | ||||
| Angiogenesis | 0.67 | 5.16E-04 | ||||
| Spindle | -0.63 | 5.46E-04 | ||||
| Lysosomal lumen | 0.35 | 1.21E-04 | ||||
| Glycosphingolipid metabolic process | 0.35 | 1.21E-03 | ||||
| Response to oxidative stress | 0.49 | 1.26E-03 | ||||
| Mitotic cell cycle | -0.58 | 1.29E-03 | ||||
| Specific INFECTED BDQ genes | ||||||
| GO category | avg. LogFC | p-value | ||||
| Endoplasmatic reticulum-Golgi intermediate compartment | -0.38 | 2.90E-07 | ||||
| Membrane raft | -0.34 | 4.93E-05 | ||||
| Cellular protein metabolic process | -0.35 | 3.32E-04 | ||||
| Lipid binding | -0.36 | 4.88E-04 | ||||
| Ribonucleoprotein complex binding | -0.37 | 5.17E-04 | ||||
| Protein dephosphorylation | -0.34 | 5.43E-04 | ||||
| Lysosomal membrane | -0.34 | 6.31E-04 | ||||
| Ubiquitin-dependent protein catabolic process | 0.74 | 6.48E-04 | ||||
Table 2
Gene Ontology (GO) functional annotation of differentially expressed genes in naïve- or hk-MTB-stimulated cells treated with PZA or RIF.
| hk-MTB + PZA | ||||||
|---|---|---|---|---|---|---|
| GO category | avg. LogFC | p-value | ||||
| Integral to lumenal side of endoplasmic reticulum membrane | 0.16 | 2.79E-04 | ||||
| Cytokine-mediated signaling pathway | 0.27 | 3.28E-04 | ||||
| Interferon-gamma-mediated signaling pathway | 0.21 | 3.56E-04 | ||||
| MHC class I receptor activity | 0.16 | 7.82E-04 | ||||
| Cytosolic small ribosomal subunit | 0.26 | 7.98E-03 | ||||
| MHC class I protein complex | 0.16 | 1.04E-03 | ||||
| Regulation of immune response | 0.22 | 1.33E-03 | ||||
| Negative regulation of MAPK cascade | 0.20 | 1.34E-03 | ||||
| Naïve + RIF | ||||||
| GO category | avg. LogFC | p-value | ||||
| Mitotic cell cycle | -0.37 | 1.53E-17 | ||||
| DNA replication | -0.34 | 2.59E-13 | ||||
| Cell cycle checkpoint | -0.36 | 4.98E-10 | ||||
| S phase of mitotic cell cycle | -0.32 | 6.29E-10 | ||||
| DNA strand elongation involved in DNA replication | -0.33 | 1.88E-09 | ||||
| G1/S transition of mitotic cell cycle | -0.36 | 2.02E-09 | ||||
| Cell division | -0.34 | 3.96E-09 | ||||
| Cell cycle | -0.32 | 2.26E-07 | ||||
| hk-MTB + RIF | ||||||
| GO category | avg. LogFC | p-value | ||||
| Melanosome | 0.26 | 4.09E-07 | ||||
| Endoplasmic reticulum unfolded protein response | 0.29 | 7.95E-07 | ||||
| Electron carrier activity | 0.23 | 9.11E-07 | ||||
| Tissue regeneration | -0.23 | 1.63E-05 | ||||
| Response to drug | -0.29 | 2.32E-05 | ||||
| NADP binding | 0.22 | 3.07E-05 | ||||
| Cellular lipid metabolic process | -0.19 | 4.05E-05 | ||||
| Lipid metabolic process | -0.23 | 4.98E-05 | ||||
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Buffy coats (Homo sapiens) | PBMC | Etablissement Français du sang (EFS) | ||
| Cell line (Homo sapiens) | HEK 293 cell line | Obtained from Dr. Craig T. January, University of Wisconsin–Madison | ||
| Cell line (Homo sapiens) | HEK 293 cell line stably expressing hERG | Obtained from Dr. Craig T. January, University of Wisconsin–Madison | ||
| Strain, strain background (Mycobacterium tuberculosis) | H37Rv | PMID:12574362 | ||
| Strain, strain background (Salmonella Typhimurium) | S. Typhimurium | PMID:25793259 | ||
| Strain, strain background (Staphylococcus aureus) | S. aureus | PMID:25793259 | ||
| Antibody | Donkey anti- Rabbit Secondary Antibody, Alexa Fluor 555 | Thermo Fisher | Cat. #: A-31572 RRID:AB_162543 | IF (1:500) |
| Antibody | Goat anti-Mouse Secondary Antibody, Alexa Fluor 555 | Thermo Fisher | Cat. #: A-21424 RRID:AB_141780 | IF (1:500) |
| Antibody | Mouse monoclonal anti-LC3 | MBL | Cat. #: M152-3 RRID:AB_1953013 | IF (1:100) |
| Antibody | Rabbit IgG HRP Linked Whole Ab | GE Healthcare | Cat. #: NA934 RRID:AB_2722659 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti- α/β-Tubulin | Cell Signaling Technology | Cat. #: 2148S RRID:AB_2288042 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-LC3B | Abcam | Cat. #: Ab48394 RRID:AB_881433 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-SQSTM1/p62 | Cell Signaling Technology | Cat. #: 5114S RRID:AB_10624872 | WB (1:1000) |
| Antibody | Rabbit polyclonal anti-TFEB | Thermo Fisher | Cat. #: PA5-65566 RRID:AB_2662642 | IF (1:100) |
| Commercial assay or kit | Griess Reaction Assay | Promega | Cat. #: G2930 RRID:SCR_006724 | |
| Commercial assay or kit | Illumina TruSeq RNA Sample Preparation kit v2 | Illumina | Cat. #: RS-122-2001/2002 RRID:SCR_010233 | |
| Commercial assay or kit | MTT Cell Proliferation Assay kit | Trevigen | Cat. #: 4890–25K RRID:SCR_012449 | |
| Commercial assay or kit | PierceTM BCA Protein Assay kit | Thermo Fisher | Cat. #: 23227 | |
| Commercial assay or kit | RNeasy Mini kit | Qiagen | Cat. #: 74104 RRID:SCR_008539 | |
| Commercial assay or kit | Seahorse XF Cell Mito Stress Test Kit | Agilent Technologies | Cat. #: 103708–100 | |
| Commercial assay or kit | Seahorse XF Glycolytic rate Assay kit | Agilent Technologies | Cat. #: 103710–100 | |
| Chemical compound, drug | Bedaquiline | Combi-Blocks | Cat. #: QV-7478 | |
| Chemical compound, drug | BAPTA-AM | Sigma-Aldrich | Cat. #: A1076 | |
| Commercial assay or kit | Image-IT TMRM | Thermo Fisher | Cat. #: I34361 | |
| Commercial assay or kit | LysoTracker DND-99 | Thermo Fisher | Cat. #: L7528 | |
| Commercial assay or kit | MitoSOX Red | Thermo Fisher | Cat. #: M36008 | |
| Commercial assay or kit | MitoTrackerTM Deep Red FM | Thermo Fisher | Cat. #: M22426 | |
| Commercial assay or kit | Fluo-8 AM | Abcam | Cat. #: Ab142773 | |
| Commercial assay or kit | DQ Green BSA | Thermo Fisher | Cat. #: D12050 | |
| Commercial assay or kit | SuperScript III Reverse Transcriptase | Thermo Fisher | Cat. #: 18080093 | |
| Commercial assay or kit | Power SYBR Green PCR Master Mix | Thermo Fisher | Cat. #: 4367659 | |
| Sequenced-based reagent | RT-qPCR primers | This paper | See Supplementary file 1 | |
| Sequenced- based reagent | siRNA: nontargeting control | Dharmacon | Cat. #: D-001810–10- 05 | |
| Sequenced-based reagent | siRNA: ON-TARGETplus Human TFEB (7942) siRNA - SMARTpool | Dharmacon | Cat. #: L-009798–00- 0005 | |
| Software, algorithm | GraphPad Prism | GraphPad Prism (https://graphpad.com) | RRID:SCR_002798 | Version 7 |
| Software, algorithm | ClueGO (Cytospace plug-in) | http://apps.cytoscape.org/apps/cluego | RRID:SCR_005748 | |
| Software, algorithm | DESeq2 | https://bioconductor.org/packages/release/ bioc/html/DESeq2.html | RRID:SCR_015687 | Version 1.18.1 |
| Software, algorithm | Icy | http://icy.bioimageanalysis.org/download/ | RRID:SCR_010587 | Version 1.0 |
| Software, algorithm | Seahorse Wave | http://www.agilent.com/en-us/products/ cell-analysis-(seahorse)/software-download-for- wave-desktop | RRID:SCR_014526 | |
| Software, algorithm | ImageJ | http://imagej.nih.gov/ij | RRID:SCR_003070 |
Additional files
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Supplementary file 1
Supplementary materials and methods.
- https://cdn.elifesciences.org/articles/55692/elife-55692-supp1-v1.docx
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Supplementary file 2
Oligonucleotide sequences.
- https://cdn.elifesciences.org/articles/55692/elife-55692-supp2-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/55692/elife-55692-transrepform-v1.pdf
