1. Biochemistry and Chemical Biology
  2. Microbiology and Infectious Disease
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Mycobacterium tuberculosis induces decelerated bioenergetic metabolism in human macrophages

  1. Bridgette M Cumming
  2. Kelvin W Addicott
  3. John H Adamson
  4. Adrie JC Steyn  Is a corresponding author
  1. Africa Health Research Institute, South Africa
  2. University of Alabama at Birmingham, United States
Tools and Resources
Cite this article as: eLife 2018;7:e39169 doi: 10.7554/eLife.39169
7 figures, 1 table and 1 additional file

Figures

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 (ECAR), which is generated from lactic acid produced from pyruvate, the end-product of glycolysis, and carbonic acid produced from CO2 released during the TCA cycle. Assays performed on the XF include: (B) mitochondrial respiration test, (C) extracellular acidification test, (D) glycolytic rate assay, (E) mitochondrial fuel test, (F) fatty acid oxidation assay and (G) real-time ATP rate assay. Oligo, oligomycin; FCCP, cyanide-4-[trifluoromethoxy]phenylhydrazone; AntiA and Rot, antimycin A and rotenone; 2-DG, 2-Deoxyglucose; G-6-P, glucose-6-phosphate; G-3-P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; α-KG, α-ketoglutarate; OAA, oxaloacetate.

https://doi.org/10.7554/eLife.39169.002
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. Refer to Figure 2—figure supplement 1 for profiles of lower MOIs. After obtaining basal respiration, cells were subjected to oligomycin (Oligo, 1.5 µM), which inhibits ATP synthase and demonstrates the mitochondrial ATP-linked OCR, followed by FCCP (cyanide-4-[trifluoromethoxy]phenylhydrazone), which uncouples mitochondrial respiration and maximizes OCR (1 µM for THP-1 and hMDMs), and finally antimycin A and rotenone (AntiA and Rot), which inhibit complex III and I in the ETC, respectively, and shut down respiration (0.5 µM of each for THP-1; 2.5 µM of each for hMDMs). Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.003
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 respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.004
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 prior to a mitochondrial respiration assay. The percentage of THP-1 (E) and hMDM cells (F) that were infected with Mtb GFP-reporter strain at MOI 1, 2.5 or 5 after 16 h. Data shown are the mean ± SD (n = 5 biological replicates) Student’s t test, ϕ, p < 0.001; *p < 0.005.

https://doi.org/10.7554/eLife.39169.005
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 was then used to calculate the basal respiration and proton leak in scenarios when the addition of FCCP induces oxidative bursts in infected cells at a high MOI. Profiles and respiratory parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.006
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 for profiles at lower MOIs. After obtaining non-glycolytic acidification, glucose (Glc, 10 mM) was added to the cells, followed by oligomycin (1.5 µM), which inhibits ATP synthase inducing maximal glycolysis to compensate for loss of mitochondrial generated ATP, and finally 2-deoxyglucose (2-DG, 100 mM) to inhibit glycolysis and demonstrate that the prior acidification was generated by glycolysis. Profiles and glycolytic parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.007
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 parameters are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.008
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 infected with Mtb, BCG and ∆Dead Mtb at MOIs of 1, 2.5 and 5. Data are representative of three independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.009
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 oxidation in the mitochondria. Calculating proton efflux rate (PER) enables the glycolytic PER to be elucidated separately from the mitochondrial PER (Figure 5—figure supplement 1A–D). (B–E) Basal and compensatory glycolytic PER of THP-1 cells (B–C) and hMDMs (D–E) infected with Mtb, BCG and ∆Dead Mtb at MOI of 5 for 18 h. Refer to Figure 5—figure supplement 1E–L for profiles at lower MOIs. Following basal measurement of ECAR and OCR, to determine basal glycolytic PER, rotenone and antimycin A were added to determine compensatory PER. This was followed by addition of 2-DG to ensure that the PER observed was caused by glycolysis. Profiles and PER are representative of two independent experiments. Data shown are the mean ± SD (n = 6). Student’s t test relative to the uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. (F–G) Total rate of ATP production was calculated as the sum of glycolytic ATP rate formation (equivalent to glycoPER) and mitochondrial-derived ATP rate formation that was estimated from the ATP-linked OCR, assuming a P/O ratio of 2.79. Rate of ATP formation in (F) THP-1 cells and (G) hMDM cells infected with Mtb, BCG or ∆Dead Mtb at indicated MOI for 18 h. Refer to Figure 5—figure supplement 1M–N for % contribution of glycolysis and OXPHOS to the total rate of ATP production. Error bars are SD (n = 6 biological replicates). Student’s t test; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.010
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 between the total PER and glycoPER will give the PER caused by mitochondrial respiration. (E–L) Basal and compensatory glycolytic PER of THP-1 cells (E–H) and hMDMs (I–L) infected with Mtb, BCG and dead-Mtb at MOIs of 1 and 2.5 for 18 h. (M, N) % Contribution of glycolysis and OXPHOS to the total rate of ATP production in (M) THP-1 cells and (N) hMDM cells infected with Mtb, BCG or dead Mtb at indicated MOI for 18 h. Profiles, PER and % contribution of glycolysis and OXPHOS to total ATP production are representative of two independent experiments. Data shown are the mean ± SD (n = 6 biological replicates). Student’s t test relative to the uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.011
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 isotopomer distributions of the intracellular metabolites depict the contribution of glucose to (A) glycolysis, (B) the tricarboxylic acid (TCA) cycle and (C) the pentose phosphate pathway (PPP). Figure 6—figure supplement 1 demonstrates the isotopomer distributions of the intracellular metabolites of THP-1 cells infected at a MOI of 2.5. (D–E) 13C enrichment of pyruvate and lactate, including total peak areas of lactate in the supernatant of the (D) hMDM cells and (E) THP-1 cells used for metabolite analysis. Data are representative of two independent experiments and shown as the mean ± SD (n = 6 biological replicates). Student’s t test relative to uninfected cells; #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05.

https://doi.org/10.7554/eLife.39169.012
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 distributions of the intracellular metabolites depict the contribution of glucose to the TCA cycle and the PPP and some amino acids associated with the TCA cycle.

https://doi.org/10.7554/eLife.39169.013
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 Mtb, BCG and ∆Dead Mtb at MOIs of 1 and 5, respectively, for 18 h. Data shown are the mean ±SEM of five independent experiments. Student’s t test relative to uninfected cells (UI); #, p < 0.0001; χ, p < 0.0005; ϕ, p < 0.001; *p < 0.005; +, p < 0.05. (D–I) The oxidation of endogenous or exogenous fatty acids in (D–F) uninfected hMDM cells in addition to (G–I) Mtb-infected macrophages at a MOI of 5 were assessed by adding a palmitate-BSA (palm-BSA) conjugate and BSA controls before analysis on the XF with the respiration test. Etomoxir was used to assess inhibition of the transport of long-chain fatty acids into the mitochondria. BR, basal respiration; MR, maximal respiration (which gives a measure of respiration under conditions of stress); FAO, fatty acid oxidation; FA, fatty acids. (J–L) Pie charts illustrating the mitochondrial fatty acid preferences of (J) uninfected, (K) Mtb- and (L) BCG-infected hMDMs under basal and stressful conditions. Profiles and pie charts are representative of two independent experiments (n = 6 biological replicates).

https://doi.org/10.7554/eLife.39169.014

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource
or reference
IdentifiersAdditional
information
Strain, strain
background
(Mycobacterium
Tuberculosis)
MtbBEI
resources
NR-123
Strain, strain
background
(Mycobacterium
bovis)
BCGStratens
Serum
Institut
Danish BCG
vaccine strain
1331
Strain, strain
background
(Mycobacterium
Tuberculosis)
H37Rv AYs330
Mtb-GFPSteyn
Laboratory
GFP Reporter
strain
Cell line
(Homo sapiens)
THP-1ATCC 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
AntibodyCD14
microbeads,
Human
Miltenyi
Biotec
Cat# 130-
050-201
MACS
(1:25)
Commercial
assay or kit
Seahorse XF
Mito Fuel Flex
Test Kit
AgilentCat#
103260–100
Commercial
assay or kit
Lookout
Mycoplasma PCR
detection kit
SigmaCat#
MO0035
Chemical
 compound,
drug
Seahorse XF
Palmitate-
BSA FAO
Substrate
AgilentCat#
102720–100
Chemical
compound,
drug
Oligomycin
(from
Streptomyces
diastatochromogenes)
SigmaCat# O4876
Chemical
compound,
drug
Carbonilcyanide
p-triflouromethoxyphenylhydrazone (FCCP)
SigmaCat# C2920
Chemical
compound,
drug
Antimycin ASigmaCat# A8674
Chemical
compound,
drug
RotenoneSigmaCat# R8875
Chemical
compound,
drug
D-(+)-
Glucose
SigmaG8270
Chemical
compound,
drug
GlutaMAXTMGibcoCat#
35050–038
Chemical
compound,
drug
DMEMLonzaCat#
BE12-604F
Chemical
compound,
drug
L-Carnitine
hydrochloride
SigmaCat# C0283
Chemical
compound,
drug
NaClSigmaCat# S3014
Chemical
compound,
drug
KClSigmaCat#
SAAR5042020EM
Chemical
compound,
drug
CaCl2SigmaCat#
1023780500
Chemical
compound,
drug
HEPESSigmaCat#
H0887
Chemical
compound,
drug
Sodium
pyruvate
SigmaCat#
S8636
Chemical
compound,
drug
XF Base
medium
AgilentCat#
102353–100
Chemical
compound,
drug
EtomoxirSigmaCat# E1905
Chemical
compound,
drug
Formalin Buffered,
Neutral
SigmaCat#
SAAR2436021EL
Chemical
compound,
drug
MethanolSigmaCat# 34860
Chemical
compound,
drug
AcetonitrileSigmaCat#
34851
Chemical
compound,
drug
Human
GM-CSF
Celtic
Diagnostics
Cat#
300-03-100
Chemical
compound,
drug
Histopaque
1077
SigmaCat#
10771
Chemical
compound,
drug
DPBSLonzaCat#
BE17-512F
Chemical
compound,
drug
DMSOSigmaCat#
41639
Chemical
compound,
 drug
Bradford
Dye
BIO-RADCat#
500–0205
Chemical
compound,
drug
Middlebrook
7H11 Agar
BDCat#
283810
Chemical
compound,
drug
Polymyxin BSigmaCat#
P1004
Chemical
compound,
drug
Amphotericin BSigmaCat#
A4888
Chemical
compound,
drug
CarbenicillinSigmaCat#
C1389
Chemical
compound,
drug
TrimethoprimSigmaCat#
T7883
Chemical
compound,
drug
Cell-TakTMCorningCat#
354241
Chemical
compound,
drug
Phorbol 12
-myristate
13-acetate
SigmaCat#
P8139
Chemical
compound,
drug
AgaroseSigmaCat#
A9539
Chemical
compound,
drug
SyBr SafeInvitrogenCat#
533102
Chemical
compound,
drug
Middlebrook
OADC
BDCat#
212240
Chemical
compound,
drug
RPMI1640LonzaCat#
BE12-167F
Chemical
compound,
drug
2-Mercapt
oethanol
GibcoCat#
21985023
Chemical
compound,
drug
2-Deoxy-D-GlucoseSigmaCat#
D6134
Chemical
compound,
drug
D-Glucose
13C6
LC ScientificCat#
GG601L
Chemical
compound,
drug
Middlebrook
7H9 Broth
BDCat#
271310
Chemical
compound,
drug
MgSO4SigmaCat#
SAAR4123920EM
Chemical
compound,
drug
NaH2PO4SigmaCat#
S9638
Software,
algorithm
Wave desktop
software
AgilentVersion 2.6
Software,
algorithm
GraphPad
Prism
Graphpad
Prism
Version 7.04
Software,
algorithm
CorelDRAWCorelVersion X8
Software,
algorithm
XF Cell Mito
Stress
Test Report
Generator
Agilenthttps://www.agilent.com/en/
products/cell-analysis/xf-cell-mito
-stress-test-report-generator
Software,
algorithm
XF Glycolysis
Stress
Test Report
Generator
Agilenthttps://www.agilent.com/en/products/cell-analysis/xf-glycolysis-stress-test-report-generator
Software,
algorithm
XF Glycolytic
Rate Assay
Report Generator
Agilenthttps://www.agilent.com/en/products/cell-analysis/xf-glycolytic-rate-assay-report-generator
Software,
algorithm
XF Mito Fuel
Flex Test
Report Generator
Agilenthttps://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
Agilenthttps://www.agilent.com/en/products/cell-analysis/xf-real-time-atp-rate-assay-report-
generator

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Additional files

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