Bridgette M. Cumming1, Kelvin Addicott1, John H. Adamson1, Adrie
J.C. Steyn1,21Africa Health Research Institute, Durban, South
Africa;
2Department of Microbiology, UAB Center for AIDS Research,
University of Alabama at Birmingham, USA
Mycobacterium tuberculosis infection decelerates bioenergetic
metabolism and alters mitochondrial substrate preferences in the
macrophage host
SUPPORTING COMPANY LOGOS
• About 1/3 of the world’s population is latently infected with
Mycobacterium tuberculosis (Mtb)1.
• Infection with Mtb does not confer immunity against future
infections demonstrating Mtb hasdefinitive strategies to manipulate
the immune response and prevent complete eradication.
• At the onset of tuberculosis infection, macrophages
phagocytose Mtb as the first line of defence.Several studies have
indicated that Mtb infection of murine bone marrow derived
macrophagesresults in a shift to aerobic glycolysis that induces
the production of pro-inflammatory cytokines2,3.
• To further our understanding of Mtb modulation of the
macrophage to consider host-directedadjunctive therapy as a novel
intervention against TB, we investigated how Mtb tempers
thebioenergetic metabolism of human monocyte derived macrophages
using extracellular flux andmetabolite analyses.
Introduction
• Human monocyte derived macrophages (hMDMs) were infected with
Mtb H37Rv, M. bovis BCG(BCG) or heat killed Mtb (Dead Mtb) at
multiplicities of infection (MOI) of 1, 2.5 or 5 (as indicated)for
24 hrs.
• The infected macrophages were analysed for their:• Overall
bioenergetic status using extracellular flux technology (Agilent
Seahorse XF96).• Metabolite levels and isotopomers after incubation
with [U-13C]glucose using LC-MS/MS and
13C-tracing.
Methods
Mtb infection modulates the bioenergetic phenotype of
macrophages distinct to that of BCG and heat killed Mtb
infections
0 2 0 4 0 6 0 8 0 1 0 0
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M O I 1
T im e (m in )
gly
co
PE
R (
pm
ol/
min
) h M D M
M tb M O I 1
B C G M O I 1
D e a d M tb M O I 1
R ot& A n tiA 2 -D G
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M O I 2 .5
T im e (m in )
gly
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R (
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min
) h M D M
M tb M O I 2 .5
B C G M O I 2 .5
D e a d M tb M O I 2 .5
R ot& A n tiA 2 -D G
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0
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M O I 5
T im e (m in )
gly
PE
R (
pm
ol/
min
)
h M D M
M tb M O I 5
B C G M O I 5
D e a d M tb M O I 5
R ot& A n tiA 2 -D G
B a s a l C o m p e n s a to ry
0
5 0
1 0 0
1 5 0
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2 5 0M O I 1
gly
co
PE
R (
pm
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min
) h M D M
M tb M O I 1
B C G M O I 1
D e a d M tb M O I 1
+
B a s a l C o m p e n s a to ry
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
M O I 2 .5
gly
co
PE
R (
pm
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min
) h M D M
M tb M O I 2 .5
B C G M O I 2 .5
D e a d M tb M O I 2 .5
*
B a s a l C o m p e n s a to ry
0
5 0
1 0 0
1 5 0
2 0 0
2 5 0
M O I 5
gly
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R (
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) h M D M
M tb M O I 5
B C G M O I 5
D e a d M tb M O I 5
#
#
A B C
D E F
Figure 1. Increasing MOI of Mtb depresses mitochondrial
respiration in hMDM in contrast to BCG infection,
which increases the spare respiratory capacity of the hMDMs.
(A-C) Cell Mito Stress Test (CMST) of hMDM
infected with increasing MOI of Mtb, BCG and heat-killed Mtb for
24 hrs. (D-F) Respiratory parameters of the
macrophages calculated from the CMST. (G-I) Phenograms
demonstrating a shift in Mtb infected hMDMs towards an
energy phenotype reminiscent of quiescence at an MOI of 5.
Figure 2. Unlike BCG infection, Mtb does not increase the rate
of glycolysis in hMDMs. (A-C) Glycolytic Rate
Assay of hMDM infected with increasing MOI of Mtb, BCG and
heat-killed Mtb for 24 hrs. (D-F) Basal Glycolysis and
compensatory glycolysis of the macrophages expressed as proton
efflux rate (PER, pmol/min) as determined from the
Glycolytic Rate Assay on the XF96. The glycolytic proton efflux
rate gives a direct measure of the acidification rate due
to glycolysis without the acidification contribution from
mitochondrial respiration.
Figure 3. Mtb infection decelerates bioenergetic metabolism in
hMDMs.13C-tracing of metabolites
extracted from hMDM infected with Mtb, BCG or heat-killed Mtb at
an MOI of 4 for 24 hours. Less 13C
incorporation is observed in the metabolites of glycolysis, TCA
cycle and the pentose phosphate pathway
of the Mtb infected hMDMs in comparison to the uninfected hMDMs
and those that are infected with BCG
and heat-killed Mtb. Although the levels of the glycolytic
intermediates are decreased in all of the infected
hMDMs, the levels of the TCA cycle intermediates from Mtb
infected hMDMs are higher possibly due to
lack of cycling through the TCA cycle.
Glc
UI
Glc
Mtb
Glc
BC
G
Glc
D
ead
Mtb
Gln
UI
Gln
Mtb
Gln
BC
G
Gln
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ead
Mtb
FA
UI
FA
Mtb
FA
BC
G
FA
D
ead
Mtb
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Fu
el
ox
ida
tio
n
(% o
f G
lc+
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+F
A o
xid
ati
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)
% D e p e n d e n c y
% F le x ib ility
+ +
+
+*
+
+
*
#
*
#
0 2 0 4 0 6 0
2 5
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7 5
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O x a m a te
E C A R (m p H /m in / g p ro te in )
OC
R (
pm
ole
s/m
in/
g p
ro
tein
)
U I
U I+ 2 0 m M
M tb 1
M tb 1 + 2 0 m M
M tb 5
M tb 5 + 2 0 m M
0 1 0 2 0 3 0 4 0 5 0
2 5
5 0
7 5
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1 2 5
2 -D e o x y g lu c o s e
E C A R (m p H /m in / g p ro te in )
OC
R (
pm
ole
s/m
in/
g p
ro
tein
)
U I
U I+ 2 0 m M
M tb 1
M tb 1 + 2 0 m M
M tb 5
M tb 5 + 2 0 m M
M tb 5 + 3 0 m M
1 5 2 0 2 5 3 0
0
5 0
1 0 0
1 5 0
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2 5 0
D ic h lo r o a c e tic a c id
E C A R (m p H /m in / g p ro te in )
OC
R (
pm
ole
s/m
in/
g p
ro
tein
)
U I
U I+ 1 0 m M
U I+ 2 0 m M
U I+ 3 0 m M
M tb
M tb + 1 0 m M
M tb + 2 0 m M
M tb + 3 0 m M
D e a d M tb
Figure. 4. Inhibition of glycolysis (2-deoxyglucose) or lactate
production (oxamate and
dichloroacetic acid) do not redirect the bioenergetic metabolism
of Mtb infected macrophages
towards OXPHOS. Phenograms of uninfected and Mtb (1 or 5: MOI)
hMDM cells treated with (A)
oxamate (B) 2-deoxyglucose (C) and RAW264.7 cells (Mtb, MOI 5)
treated with dichloroacetic acid.
Circles: uninfected macrophages, Squares: Mtb infected
macrophages.
Inhibition of glycolysis in Mtb infected macrophages does not
redirect energy metabolism to OXPHOS
Mtb infection alters mitochondrial substrate preference of the
macrophage
Mtb infection decelerates bioenergetic metabolism of the
macrophage
Figure 5. Mtb infection decreases the mitochondrial
dependency on glucose and increases the dependency on
glutamine and fatty acid oxidation. hMDM were infected with
Mtb, BCG and heat-killed Mtb (MOI 4) for 24 hrs prior to
conducting the Mitochondrial Fuel Flex Test on the XF96.
Glc,
glucose; Gln, glutamine; FA, fatty acids
• Basal respiration, ATP-linked OCR and maximalrespiration are
reduced in Mtb infected hMDMs withincreasing MOI in contrast to
infection with BCG andheat-killed Mtb that do not affect
respiration at lowMOIs.
• Furthermore, BCG infection increases the maximalrespiration
and the spare respiratory capacity ofhMDMs above that of uninfected
hMDMs.
• Unlike BCG or heat killed Mtb infection, Mtb infectiondoes not
increase the rate of basal glycolysis orcompensatory glycolysis in
hMDMs.
• Mtb infection decelerates flux through glycolysis andthe TCA
cycle of the hMDMs, confirming the observedshift to a quiescent
energy phenotype.
• ADP/ATP and AMP/ATP ratios increase in Mtb infectedhMDMs
supporting the overserved reduced rates ofOXPHOS and
glycolysis.
• Inhibition of glycolysis or lactate production did notredirect
the bioenergetic metabolism of the macrophageto OXPHOS suggesting
that Mtb has an irreversibledepressive effect on mitochondrial
respiration.
• Mtb infection of hMDMs changes the substratepreference of
mitochondria from glucose to glutamineand fatty acids, whereas
heat-killed Mtb increases thedependency of the macrophages on
glucose with noflexibility.
• Mtb infection of hMDMs slows down energy metabolismof the host
cell, including both OXPHOS and glycolysis.This is probably part of
Mtb’s strategy to “diminish” theinnate immune functions of the
macrophage and delayapoptosis of the macrophage to sustain bacilli
growth.
• Future work includes investigating the “apoptotic status”of
Mtb infected hMDMs displaying the quiescent energyphenotype.
Conclusions
This work was supported by the National Institutes ofHealth
grants R01 AI 111 940 and R21 127182 and theUAB Center for AIDS
Research.
Acknowledgements
References1. Global TB Report, 2017, WHO.
2. Gleeson et al. (2016) J. Immun. Vol. 196, pp. 2444;
3. Stanley et al. (2016) Vol. 197, pp. 1287
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AT
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Resp
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+
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over
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M O I 5
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OC
R (
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U I
M tbB C G
M tb
Q u ie s c e n t G ly c o ly t ic
A e ro b ic E n e rg e t ic
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OC
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ol/
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/ g
pro
tein
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U I
M tb
M tb
B C G
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M O I 5
E C A R (m p H /m in / g p ro te in )
OC
R (
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ol/
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/ g
pro
tein
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U I
M tb
B C G
M tb
A B C
D E F
G H I
A B C
