Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages
- Africa Health Research Institute, South Africa
- University of Alabama at Birmingham, United States
Figures
Figure 1
Schematic illustration of cellular metabolism pathways and XF assays used to analyze metabolic pathways.
(A) The XF measures oxygen consumption rate (OCR) of the cell, which is mostly consumed at complex IV of the electron transport chain (ETC) in the mitochondria, and extracellular acidification rate …
Figure 2 with 3 supplements
Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI.
Respiratory profiles (OCR) and respiratory parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and ∆Dead Mtb (heat-killed Mtb) at MOIs of 5 for 24 h. …
Figure 2—figure supplement 1
Respiratory profiles and parameters of infected macrophages are dependent on cell type, mycobacterial strain and MOI.
Respiratory profiles (OCR) and respiratory parameters of (A–D) PMA differentiated THP-1 macrophages, and (E–H) hMDMs infected with Mtb, BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and …
Figure 2—figure supplement 2
Contribution of extracellular mycobacteria to OCR and ECAR of infected macrophages and percentage of Mtb-infected macrophages.
OCR (A, B) and ECAR (C, D) of the remaining extracellular Mtb (A, C) and BCG (B,D) in the final wash of infected THP-1 macrophages that were adhered to wells of an XF96 culture plate using Cell-TakTM…
Figure 2—figure supplement 3
Mitochondrial respiration assays without FCCP.
Mitochondrial respiratory profiles of THP-1 cells (A–D) and hMDMs (E, F) without the addition of FCCP were generated to measure the non-mitochondrial respiration. The non-mitochondrial respiration …
Figure 3 with 1 supplement
Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI.
ECAR profiles and glycolytic parameters of (A–B) PMA differentiated THP-1 macrophages, and (C–D) hMDMs infected with Mtb, BCG and dead Mtb at MOI of 5 for 24 h. Refer to Figure 3—figure supplement 1 …
Figure 3—figure supplement 1
Extracellular acidification profiles and glycolytic parameters of THP-1 and hMDMs are affected by macrophage type, mycobacterial strain and MOI.
ECAR profiles and glycolytic parameters of (A–D) PMA differentiated THP-1 macrophages, and (E–H) hMDMs infected with Mtb, BCG and dead Mtb at MOIs of 1 or 2.5 for 24 h. Profiles and glycolytic …
Figure 4
Phenograms demonstrate that increasing MOI of Mtb shifts macrophages towards quiescent energy phenotypes.
Basal OCR and ECAR measurements from the respiratory assay (Figure 2) before addition of oligomycin were plotted to generate phenograms of (A–C) PMA-differentiated THP-1 cells and (D–F) hMDMs …
Figure 5 with 1 supplement
Mtb infection reduces the glycolytic proton efflux rate of macrophages.
(A) Extracellular acidification can be caused by both lactate and protons produced from pyruvate, the final product of glycolysis, in addition to carbonic acid generated from CO2 from pyruvate …
Figure 5—figure supplement 1
Mtb infection reduces the glycolytic proton efflux rate of macrophages.
(A–D) Profiles of total proton efflux rate (PER) in red and the glycolytic proton efflux rate (glycoPER) in blue of hMDMs infected with Mtb, BCG and dead Mtb at MOI of 1 for 18 h. The difference …
Figure 6 with 1 supplement
Mtb infection decelerates flux through glycolysis and the TCA cycle of the macrophage.
(A–C) 13C-tracing of metabolites extracted from hMDM cells infected with Mtb, BCG and ∆Dead Mtb at MOI 5 (hMDM) for 10 h followed by incubation with [U-13C]glucose for 8 h. The stacked mass …
Figure 6—figure supplement 1
Mtb at a MOI of 2.5 decelerates flux through glycolysis in THP-1 cells and induces breaks in the TCA cycle at citrate and succinate.
13C-tracing of extracted metabolites of THP-1 cells infected with Mtb, BCG and ∆Dead Mtb at a MOI of 2.5 for 10 h followed by incubation with [U-13C] glucose for 8 h. The stacked mass isotopomer …
Figure 7
Mtb infection alters the mitochondrial substrate preference of macrophages.
(A–C) UK5099, etomoxir and BPTES were used to assess the mitochondrial flexibility and dependency on glucose (Glc), fatty acids (FA) and glutamine (Gln) in (B) THP-1 and (C) hMDM cells infected with …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mycobacterium Tuberculosis) | Mtb | BEI resources | NR-123 | |
| Strain, strain background (Mycobacterium bovis) | BCG | Stratens Serum Institut | Danish BCG vaccine strain 1331 | |
| Strain, strain background (Mycobacterium Tuberculosis) H37Rv AYs330 | Mtb-GFP | Steyn Laboratory | GFP Reporter strain | |
| Cell line (Homo sapiens) | THP-1 | ATCC Cat# TIB-202 | RRID:CVCL_0006 | |
| Biological sample (Homo sapiens) | Buffy coats | South African National Blood Service | ||
| Biological sample (Homo sapiens) | Human plasma | South African National Blood Service | ||
| Antibody | CD14 microbeads, Human | Miltenyi Biotec | Cat# 130- 050-201 | MACS (1:25) |
| Commercial assay or kit | Seahorse XF Mito Fuel Flex Test Kit | Agilent | Cat# 103260–100 | |
| Commercial assay or kit | Lookout Mycoplasma PCR detection kit | Sigma | Cat# MO0035 | |
| Chemical compound, drug | Seahorse XF Palmitate- BSA FAO Substrate | Agilent | Cat# 102720–100 | |
| Chemical compound, drug | Oligomycin (from Streptomyces diastatochromogenes) | Sigma | Cat# O4876 | |
| Chemical compound, drug | Carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) | Sigma | Cat# C2920 | |
| Chemical compound, drug | Antimycin A | Sigma | Cat# A8674 | |
| Chemical compound, drug | Rotenone | Sigma | Cat# R8875 | |
| Chemical compound, drug | D-(+)- Glucose | Sigma | G8270 | |
| Chemical compound, drug | GlutaMAXTM | Gibco | Cat# 35050–038 | |
| Chemical compound, drug | DMEM | Lonza | Cat# BE12-604F | |
| Chemical compound, drug | L-Carnitine hydrochloride | Sigma | Cat# C0283 | |
| Chemical compound, drug | NaCl | Sigma | Cat# S3014 | |
| Chemical compound, drug | KCl | Sigma | Cat# SAAR5042020EM | |
| Chemical compound, drug | CaCl2 | Sigma | Cat# 1023780500 | |
| Chemical compound, drug | HEPES | Sigma | Cat# H0887 | |
| Chemical compound, drug | Sodium pyruvate | Sigma | Cat# S8636 | |
| Chemical compound, drug | XF Base medium | Agilent | Cat# 102353–100 | |
| Chemical compound, drug | Etomoxir | Sigma | Cat# E1905 | |
| Chemical compound, drug | Formalin Buffered, Neutral | Sigma | Cat# SAAR2436021EL | |
| Chemical compound, drug | Methanol | Sigma | Cat# 34860 | |
| Chemical compound, drug | Acetonitrile | Sigma | Cat# 34851 | |
| Chemical compound, drug | Human GM-CSF | Celtic Diagnostics | Cat# 300-03-100 | |
| Chemical compound, drug | Histopaque 1077 | Sigma | Cat# 10771 | |
| Chemical compound, drug | DPBS | Lonza | Cat# BE17-512F | |
| Chemical compound, drug | DMSO | Sigma | Cat# 41639 | |
| Chemical compound, drug | Bradford Dye | BIO-RAD | Cat# 500–0205 | |
| Chemical compound, drug | Middlebrook 7H11 Agar | BD | Cat# 283810 | |
| Chemical compound, drug | Polymyxin B | Sigma | Cat# P1004 | |
| Chemical compound, drug | Amphotericin B | Sigma | Cat# A4888 | |
| Chemical compound, drug | Carbenicillin | Sigma | Cat# C1389 | |
| Chemical compound, drug | Trimethoprim | Sigma | Cat# T7883 | |
| Chemical compound, drug | Cell-TakTM | Corning | Cat# 354241 | |
| Chemical compound, drug | Phorbol 12 -myristate 13-acetate | Sigma | Cat# P8139 | |
| Chemical compound, drug | Agarose | Sigma | Cat# A9539 | |
| Chemical compound, drug | SyBr Safe | Invitrogen | Cat# 533102 | |
| Chemical compound, drug | Middlebrook OADC | BD | Cat# 212240 | |
| Chemical compound, drug | RPMI1640 | Lonza | Cat# BE12-167F | |
| Chemical compound, drug | 2-Mercapt oethanol | Gibco | Cat# 21985023 | |
| Chemical compound, drug | 2-Deoxy-D-Glucose | Sigma | Cat# D6134 | |
| Chemical compound, drug | D-Glucose 13C6 | LC Scientific | Cat# GG601L | |
| Chemical compound, drug | Middlebrook 7H9 Broth | BD | Cat# 271310 | |
| Chemical compound, drug | MgSO4 | Sigma | Cat# SAAR4123920EM | |
| Chemical compound, drug | NaH2PO4 | Sigma | Cat# S9638 | |
| Software, algorithm | Wave desktop software | Agilent | Version 2.6 | |
| Software, algorithm | GraphPad Prism | Graphpad Prism | Version 7.04 | |
| Software, algorithm | CorelDRAW | Corel | Version X8 | |
| Software, algorithm | XF Cell Mito Stress Test Report Generator | Agilent | https://www.agilent.com/en/ products/cell-analysis/xf-cell-mito -stress-test-report-generator | |
| Software, algorithm | XF Glycolysis Stress Test Report Generator | Agilent | https://www.agilent.com/en/products/cell-analysis/xf-glycolysis-stress-test-report-generator | |
| Software, algorithm | XF Glycolytic Rate Assay Report Generator | Agilent | https://www.agilent.com/en/products/cell-analysis/xf-glycolytic-rate-assay-report-generator | |
| Software, algorithm | XF Mito Fuel Flex Test Report Generator | Agilent | https://www.agilent.com/en/products/cell-analysis/report-generator-for-the-xf-mito-fuel-flex-test | |
| Software, algorithm | XF Real-Time ATP Rate Assay Report Generator | Agilent | https://www.agilent.com/en/products/cell-analysis/xf-real-time-atp-rate-assay-report- generator |
Additional files
-
Transparent reporting form
- https://doi.org/10.7554/eLife.39169.015
