“Tuberculosis Hypoxia”
Max Planck Institute for Infection Biology
MPIB-0202-10VSBL
Study Overview
Objective
To identify biochemicals that are altered in Mycobacterium tuberculosis
cultured under hypoxic conditions in a snow globe model. A secondary
objective is to identify biochemicals that are differentially released into the
culture media and/or consumed from the media.
2
Snow Globe Overview
Two time courses completed
• Both processed
• One set shipped
Sauton’s w/tyloxapol
Citric acid
Ferric ammonium citrate
Glycerol
Asparagine
Biofilm
7H9 w/tyloxapol?
3
Study Design
Lipidomics
Pellet
Pellet & Supernatant
Metabolomics
Proteomics/
Glycomics
2 Pellets & Supernatant
Pellet
Transcriptomics
Hypoxia
0
2 hours
1
2
3
Reaeration
4
5
6
7
+1
+6 hours
+2
Days
Each arrow indicates a set of quadruplicate biological
replicates of either pellet or supernatant + pellet as indicated
The primary purpose of this time course is to provide data
to help correlate lipidomics, transcriptomics, and proteomics
from the snow globe run
4
Metabolon Platform Technology
UHPLC-MS/MS (+ESI)
Instrumentation
Biochemical
Extraction
UHPLC-MS/MS (-ESI)
Biochemical Analysis
Metabolyzer™
Peak Detection
Peak Integration
Library Search
RT, Mass, MS/MS
QA/QC
GC-MS (+EI)
21-hydroxyprogesterone
3-hydroxyoctanoic acid
1-methylguanidine-hydrochloride
3-hydroxydecanoic acid
3-indoleacrylic acid
DL-3-phenyllactic acid
DL-alpha-hydroxyisocaproic acid
DL-hexanoyl-carnitine
O-acetyl-L-carnitine-hydrochloride
EDTA
l-aspartyl-l-phenylalanine
Library Search for Biochemical ID
Biochemical
Metabolyzer Software
cholesterol
3.17 min
Database
Of
Standards
Mass spectrum
4.01
cholesterol
Biochemical ID
14.43
5.84
4.38
10.66
8.46
10.18
4.55
6.52
6.73
4
5
11.76
9.34
6
7
11.03
9.47
7.50
5.34
3.17
7.74
8.01
8
9
Time (min)
11.79
11.21
10
11
13.05
12.89
12
13
13.30
14
147,926
289,530
58,939
281,085
177,587
3,281,189
231,486
118,902,022
29,443,151
992,513
6,520,826
Amount
143,789
UHPLC-MS/MS (+ESI)
Metabolyzer™
Peak Detection
Biochemical
Extraction
UHPLC-MS/MS (-ESI)
Peak Integration
Library Search
RT, Mass, MS/MS
GC-MS (+EI)
Metabolon Platform Technology
Global Biochemical
Pathway Changes
Disease Biomarkers
Mechanistic Toxicology
Drug MOA
Cellular Characteristics
QA/QC
Statistical Analysis
Biochemical
Interpretation
• Pathway analysis
• Literature
Heat Maps by Pathway
Quality Control Processes
1. Significant component is QC
30% of samples
dedicated to
quality control
2. Multiple embedded QC standards in every sample
+ Recovery
Standards
Sample
+ Internal
Standards
Extraction/recovery
Injection into Instrument
3. Matrix-specific technical replicates and QC injections across a study run-day
1st
Injection
Equal aliquot from ALL
experimental samples pooled
as “client matrix” (CMTRX)
CMTRX
CMTRX
Final
Injection
Process Blank
Experimental
samples
These processes allow for monitoring platform and process variability
Platform QC and Metabolite Summary
Data Quality and Precision
Median RSD
Quality Control
Sample (Matrix)
Cells
Media
Internal Standards
5%
5%
Endogenous
Biochemicals
9%
14%
Internal Standards: standards spiked into each of the study samples prior to injection into the MS instrument
Endogenous Biochemicals: from CMTRX samples – technical replicates created from a small portion of
experimental samples
These data are within Metabolon’s QC specifications.
Number of Biochemicals
Compound
Classification
Cells
Media
Total
281
61
Named / Identified
134
45
Unnamed
147
16
Statistical Analyses: T-tests
 Welch’s Two-Sample T-Test was used to determine whether the means of two
populations were different.
p-value: evidence that the means are different (smaller is better)
q-value: estimate of the false discovery rate (smaller is better)
p≤0.05 was taken as significant
Sample Statistics Table
Fold of Change - Snow Globe
SUPER
PATHWAY
SUB PATHWAY
Amino acid Alanine and aspartate metabolism
0.55
1.71
1.43
1.20
BIOCHEMICAL NAME
Day 1 vs. Day 0
Day 7 vs. Day 0
Day 8 vs. Day 0
Day 8 vs. Day 7
alanine
1.43
2.87
4.10
1.43
beta-alanine
0.95
0.91
1.76
1.92
cyano-alanine
1.47
0.72
0.40
0.55
aspartate
1.69
6.61
8.08
1.22
asparagine
0.79
0.36
0.28
0.76
Green: indicates significant difference (p≤0.05) between the groups shown; GREEN indicates a ratio < 1
Red: indicates significant difference (p≤0.05) between the groups shown; RED indicates a ratio > 1
Bold blue: narrowly missed cutoff for significance; p>0.05 , p<0.10
Non-colored text and cell: mean values are not significantly different for that comparison
The full t-test table is supplied as a separate excel file
10
Statistical Analyses: Summary
Welch's Tw o-Sam ple t -test
Snow Globe
Cells
Media
Fresh vs.
Day 0
Day 1 vs.
Day 0
Day 7 vs.
Day 0
Day 8 vs.
Day 0
Day 8 vs.
Day 7
Total number of biochemicals w ith p≤0.05
-
21
58
75
27
Biochemicals (↑↓)
-
10|11
23|35
51|24
25|2
q-value
-
0.17
0.07
0.08
0.24
Total number of biochemicals w ith p≤0.05
10
3
20
23
0
Biochemicals (↑↓)
4|6
2|1
15|5
18|5
0|0
q-value
0.09
0.27
0.02
0.01
-
Intensity
Scaled
Scaled Intensity
Visualization with Box/Line Plots
Metabolite
Name,
glycine,
C Matrix
7
6
5
“C” = cells; “M” = media
4
3
2
1
0
D0
D1
Scaled
ScaledIntensity
Intensity
Snow Globe (Box Plots)
2 .5
D7
Day
Experiment
D8
Snow Globe
Box and Whiskers Legend
+
Metabolite
Name,
glycine,
M Matrix
___
2
1 .5
1
0 .5
0
FM
Fermentor (Line Plots)
D0
D1
D3
D5
D7
Day
Experiment
Fermentor
D8
D9
Mean Value
Median Value
Extreme Data Points
Upper Quartile
Lower Quartile
“Max” of distribution
“Min” of distribution
Biochemical Data and Interpretation
13
M. tuberculosis and dormancy
• M. tuberculosis strains express a two component regulatory system (dosT/dosS)
that is regulated by O2 content.
• These kinases show differential sensitivity to oxygen.
• M. tuberculosis also express resuscitation-promoting factors
•Required for virulence and resuscitation from dormancy
•Dispensible for survival in vitro
• Factors that are regulated by hypoxia/starvation control cell envelope synthesis
virulence factors
Oxygen status affects the glycolytic pathway
glucose, C
3
2
Scaled Intensity
Scaled Intensity
2 .5
1 .5
glucose
1
0 .5
glucose 6-P
0
D0
D1
D7
D8
Snow Globe
fructose-6-phosphate, C
8
7
6
5
4
3
2
1
0
D0
D1
fructose 6-P
6
D8
glucose-6-phosphate (G6P), C
fructose 1,6-bisP
5
Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate, C
4
3 .5
3
glyceraldehyde-3-P
1
0
D1
D7
1,3-bisphosphoglycerate
D8
Snow Globe
2 .5
2
1 .5
1
0 .5
0
3-phosphoglycerate
D0
D1
D7
D8
Snow Globe
2-phosphoglycerate
pyruvate, C
2 .5
3-phosphoglycerate, C
6
5
phosphoenolpyruvate
2
1 .5
pyruvate
1
Scaled Intensity
D0
3
Scaled Intensity
Dihydroacetone phosphate
2
Scaled Intensity
Scaled Intensity
D7
Snow Globe
4
3
2
1
0
0 .5
Acetyl CoA
0
D0
D1
D7
Snow Globe
D8
D0
D1
D7
Snow Globe
D8
The TCA cycle and CO2 loss
glucose
pyruvate
lactate
acetyl-CoA
The generation of CO2 by the bacteria
would acidify the environment
citrate
oxaloacetate
malate
cis-aconitate
isocitrate
fumarate
succinate
CO2
α-ketoglutarate
succinyl-CoA
CO2
The loss of carbon atoms would decrease
the energy yield
Glyoxylate Cycle
glucose, M
3 .5
1 .2
Scaled Intensity
3
glucose
2 .5
2
1 .5
1
1
0 .8
0 .6
0 .4
0 .2
0 .5
0
0
FM
D0
D1
D7
D8
FM
pyruvate
Snow Globe
D0
D1
D7
D8
Snow Globe
lactate
Scaled Intensity
citrate
malate, M
10
cis-aconitate, M
2
acetyl-CoA
oxaloacetate
8
1 .5
1
0 .5
cis-aconitate
0
FM
6
D0
D1
D7
Snow Globe
malate
4
2
glyoxylate
isocitrate
acetyl-CoA
0
D0
D1
D7
D8
fumarate
Snow Globe
Scaled Intensity
FM
succinate
isocitrate, M
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
FM
D0
D1
Snow Globe
Scaled Intensity
Scaled Intensity
Scaled Intensity
citrate, M
1 .4
succinate, M
8
7
6
5
4
3
2
1
0
FM
D0
D1
Snow Globe
D7
D8
D7
D8
D8
Glyoxylate Cycle
Scaled Intensity
2 .5
lactate, C
2
1 .5
1
Scaled Intensity
0 .5
pyruvate
0
D0
D1
D7
D8
Snow Globe
lactate
acetyl-CoA
D1
D7
D8
oxaloacetate
malate, C
5
4
malate
glyoxylate
3
D0
cis-aconitate
isocitrate
1
fumarate
0
D1
D7
D1
D0
D1
Scaled Intensity
Scaled Intensity
3
2
isocitrate, C
2 .5
succinate
4
2
1 .5
1
0 .5
0
1
D0
D1
D7
Snow Globe
0
D0
D7
Snow Globe
D8
succinate, C
D1
D7
Snow Globe
D8
D8
cis-aconitate, C
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
Snow Globe
5
D7
Snow Globe
acetyl-CoA
2
D0
citrate, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
citrate
Snow Globe
Scaled Intensity
D0
Scaled Intensity
Scaled Intensity
glucose
4
3 .5
3
2 .5
2
1 .5
1
0 .5
0
acetyl CoA, C
3
D8
D8
Glyoxylate Cycle
pyruvate
2 .5
2
1 .5
1
1
0 .5
0
2
D -1 D 0
lactate
1 .5
1
D1 D2
D3 D5 D7
D8 D9
Fermentor
0 .8
0 .6
0 .4
0 .2
acetyl-CoA
0 .5
citrate, C
1 .2
Scaled Intensity
3
0
0
D -1 D 0
D1 D2
D3 D5 D7
D -1 D 0
citrate
D8 D9
D1 D2
D3 D5 D7
D8 D9
Fermentor
Fermentor
oxaloacetate
cis-aconitate
malate, C
malate
2
glyoxylate
isocitrate
Scaled Intensity
2 .5
acetyl-CoA
1 .5
1
fumarate
0 .5
0
D1 D2
D3 D5 D7
D -1 D 0
succinate
succinate, C
2
1 .5
1
0 .5
D1 D2
D3 D5 D7
Fermentor
D8 D9
D1 D2
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D1 D2
D3 D5 D7
Fermentor
D3 D5 D7
Fermentor
isocitrate, C
D -1 D 0
0
D -1 D 0
cis-aconitate, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D8 D9
Fermentor
Scaled Intensity
D -1 D 0
Scaled Intensity
Scaled Intensity
Scaled Intensity
Scaled Intensity
lactate, C
3 .5
acetyl CoA, C
2 .5
glucose
D8 D9
D8 D9
Glyoxylate Cycle
glucose
citrate, M
1 .4
Scaled Intensity
1 .2
pyruvate
lactate
1
0 .8
0 .6
0 .4
0 .2
acetyl-CoA
0
FM
oxaloacetate
2
malate
1
0 .5
glyoxylate
D3
D5
D7
D8
D9
Fermentor
cis-aconitate, M
2 .5
cis-aconitate
1 .5
isocitrate
acetyl-CoA
2
1 .5
1
0 .5
0
0
D0
D1
D3
D5
D7
D8
D9
D0
D1
D3
isocitrate, M
1 .2
1
4
3
2
0 .8
0 .6
0 .4
0 .2
1
0
FM
0
FM
D0
D1
D3
D5
Fermentor
D7
D8
D9
D5
Fermentor
succinate
succinate, M
5
FM
fumarate
Fermentor
Scaled Intensity
FM
Scaled Intensity
Scaled Intensity
D1
Scaled Intensity
malate, M
2 .5
D0
citrate
D0
D1
D3
D5
Fermentor
D7
D8
D9
D7
D8
D9
Methylcitrate cycle
acetyl CoA, C
3
pyruvate
1 .5
2
1 .5
1
0 .5
1
acetyl-CoA
0
D0
0 .5
D0
D1
D7
D8
propionyl CoA
oxaloacetate
Snow Globe
malate
methyl-isocitrate
acetyl-CoA
malate, C
5
cis-aconitate
glyoxylate
fumarate
4
3
pyruvate
2
succinate
1
0
D1
D7
D8
succinate, C
5
Snow Globe
Scaled Intensity
D0
4
3
2
1
0
D0
D1
D7
Snow Globe
D8
D1
D7
Snow Globe
methylcitrate
0
Scaled Intensity
Scaled Intensity
2
Scaled Intensity
2 .5
propionyl CoA, C
D8
Snow Globe: significant proteome changes
Relative Counts
Protein Description
Locus Tag
Day 0
Day 1
Day 7
Day 8
heat shock protein hspX [Mycobacterium tuberculosis H37Rv]
gi Number
15609168
Rv2031c
0.0455
0.4614
1
0.6198
serine protease PepA [Mycobacterium tuberculosis H37Rv]
15607267
Rv0125
0.095
0.85
0.975
1
isoniazid inductible gene protein INIB [Mycobacterium tuberculosis H37Rv]
15607482
Rv0341
0.1591
0.0833
0.6023
1
hypothetical protein Rv2744c [Mycobacterium tuberculosis H37Rv]
57117019
Rv2744c
0.1342
0.2081
1
0.9195
succinyl-CoA synthetase subunit beta [Mycobacterium tuberculosis H37Rv]
15608091
Rv0951
0.1034
0.0431
1
0.931
transcription antitermination protein NusG [Mycobacterium tuberculosis H37Rv]
15607779
Rv0639
0.1667
0.2604
0.9688
1
dihydrolipoamide acetyltransferase [Mycobacterium tuberculosis H37Rv]
15609352
Rv2215
0.1529
0.0941
1
0.7176
hypothetical protein Rv0569 [Mycobacterium tuberculosis H37Rv]
15607709
Rv0569
0.0602
0.6386
1
0.5422
D-3-phosphoglycerate dehydrogenase [Mycobacterium tuberculosis H37Rv]
57117042
Rv2996c
0.029
0.058
0.971
1
hypothetical protein Rv1738 [Mycobacterium tuberculosis H37Rv]
15608876
Rv1738
0
0.7302
1
0.6667
hypothetical protein Rv2626c [Mycobacterium tuberculosis H37Rv]
15609763
Rv2626c
0
0.2203
1
0.4576
transcriptional regulatory protein [Mycobacterium tuberculosis H37Rv]
15608542
Rv1404
0
0.3585
1
0.9623
malate dehydrogenase [Mycobacterium tuberculosis H37Rv]
15608380
Rv1240
0.1429
0.0238
0.9286
1
heat shock protein hsp (heat-stress-induced ribosome-binding protein A) [Mycobacterium tuberculosis H37Rv]
15607392
Rv0251c
0
0.0286
1
0.6857
dehydrogenase [Mycobacterium tuberculosis H37Rv]
15610525
Rv3389c
0.0769
0
1
0.6923
glyceraldehyde-3-phosphate dehydrogenase [Mycobacterium tuberculosis H37Rv]
15608574
Rv1436
0.0455
0.0455
0.5
1
hypothetical protein Rv2623 [Mycobacterium tuberculosis H37Rv]
15609760
Rv2623
0
0
0.8462
1
short chain dehydrogenase [Mycobacterium tuberculosis H37Rv]
15610360
Rv3224
0.1
0.4
1
0.5
isocitrate dehydrogenase [Mycobacterium tuberculosis H37Rv]
15607208
Rv0066c
0.1111
0.4444
1
0.8889
aspartate-semialdehyde dehydrogenase [Mycobacterium tuberculosis H37Rv]
15610844
Rv3708c
0
0
0.7
1
50S ribosomal protein L18 [Mycobacterium tuberculosis H37Rv]
15607860
Rv0720
1
0.9706
0.0882
0.2059
50S ribosomal protein L19 [Mycobacterium tuberculosis H37Rv]
15610041
Rv2904c
0.5556
1
0.0556
0.1389
Esat-6 like protein esxJ (Esat-6 like protein 2) [Mycobacterium tuberculosis H37Rv]
15608178
Rv1038c
0.9274
1
0.3347
0.3992
low molecular weight antigen CFP2 (low molecular weight protein antigen 2) (CFP-2) [Mycobacterium tuberculosis H37Rv]
15609513
Rv2376c
1
0.6577
0.2613
0.5315
Amino acid levels decreased during hypoxia
isoleucine, C
leucine, C
2 .5
1 .5
Scaled Intensity
Scaled Intensity
2
1
0 .5
0
2
1 .5
1
0 .5
0
D0
D1
D7
D8
D0
Snow Globe
D8
tyrosine, C
3
1 .2
2 .5
Scaled Intensity
Scaled Intensity
D7
Snow Globe
asparagine, C
1 .4
D1
1
0 .8
0 .6
0 .4
2
1 .5
1
0 .5
0 .2
0
0
D0
D1
D7
Snow Globe
D8
D0
D1
D7
D8
Snow Globe
• Amino acid levels decreased early in hypoxia and began to recover at Day 7
•Amino acids possibly were shuttled into the TCA/glyoxylate cycle for the
biosynthesis energy (anapleurotic reactions)
• Aeration of the culture resulted in increased levels of amino acids.
TCA cycle intermediates accumulate in media
glucose
glucose, M
3 .5
1 .2
Scaled Intensity
3
2 .5
2
1 .5
pyruvate
1
0
FM
D0
D1
D7
0 .6
0 .4
0
lactate
D8
1
0 .8
0 .2
0 .5
FM
D0
D1
D7
D8
Snow Globe
acetyl-CoA
Snow Globe
citrate
Scaled Intensity
cis-aconitate
malate, M
10
cis-aconitate, M
2
oxaloacetate
1 .5
1
0 .5
8
0
FM
6
glyoxylate
malate
4
D0
D1
D7
Snow Globe
isocitrate
2
0
D0
D1
D7
D8
Snow Globe
fumarate
succinate
succinate, M
8
7
6
5
4
3
2
1
0
Scaled Intensity
FM
Scaled Intensity
Scaled Intensity
Scaled Intensity
citrate, M
1 .4
isocitrate, M
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
FM
D0
D1
Snow Globe
FM
D0
D1
Snow Globe
D7
D8
D7
D8
D8
Aeration induces an increase in pentose phosphate pathway
intermediates
Glucose
3
2 .5
Scaled Intensity
Scaled Intensity
Gluconate
nicotinamide adenine dinucleotide phosphate (NADP+), C
NADPH
ribitol, C
4
3 .5
3
2 .5
2
1 .5
1
0 .5
0
6-Phosphogluconate
2
1 .5
1
0 .5
NADPH
0
D0
D1
Ribulose-5-P
D0
D1
D7
D7
D8
Snow Globe
D8
Snow Globe
Ribulose-5-P
sedoheptulose-7-phosphate, C
5
Glyceraldehyde-3-P + Sedoheptulose-7-P
Scaled Intensity
Xylulose-5-P
6
4
3
2
1
0
D0
D1
D7
Snow Globe
fructose, C
3
Fructose-6-P + Erythrose-4-P
Xylulose-5-P
Scaled Intensity
2 .5
2
1 .5
1
Glyceraldehyde-3-P + Fructose-6-P
0 .5
0
D0
D1
D7
Snow Globe
D8
D8
Glucose utilization in bacteria
Fuhrer et al., J Bacteriol. 187(5): 1581-1590
nicotinate, C
7
Salvage Pathway
Scaled Intensity
6
5
4
NAD(P) breakdown
3
Scaled Intensity
NAD+ synthesis tightly regulated in M. tb
nicotinamide, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D0
2
D1
0
D1
D7
D8
Snow Globe
Nicotinic Acid
3
Nicotinamide
Nicotinamide
Riboside
nicotinic acid mononucleotide (NaMN), C
Nicotinic Acid
Mononucleotide
Nicotinamide
Mononucleotide
nicotinate ribonucleoside*, C
2
1 .5
1
0 .5
0
2 .5
D0
D1
D7
Snow Globe
2
Nicotinic Acid
Dinucleotide
1 .5
1
0
D1
D7
Snow Globe
D8
3 .5
3
0 .5
D0
nicotinamide adenine dinucleotide (NAD+), C
NAD
NADP
Scaled Intensity
Scaled Intensity
D8
2 .5
Scaled Intensity
D0
3
D7
Snow Globe
1
2 .5
2
1 .5
1
0 .5
0
NAD+
D0
D1
D7
•
starvation is a cidal event in tubercle bacilli
• NAD+ production is tightly regulated
• Enzymes common to the de novo and salvage pathways are hypothesized to
be good drug targets
Snow Globe
D8
D8
NAD+ synthesis tightly regulated in M. tb
nicotinamide, C
1
1 .2
Scaled Intensity
1
0 .8
Salvage Pathway
0 .6
Scaled Intensity
nicotinate, C
0 .8
0 .6
0 .4
0 .2
0 .4
NAD(P) breakdown
0 .2
0
D -1 D 0
D1 D2
D -1 D 0
D1 D2
D3 D5 D7
D8 D9
Fermentor
Nicotinamide
Nicotinamide
Riboside
Nicotinic Acid
Mononucleotide
Nicotinamide
Mononucleotide
Scaled Intensity
Nicotinic Acid
3
D3 D5 D7
D8 D9
Fermentor
0
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
nicotinic acid mononucleotide (NaMN), C
nicotinate ribonucleoside*, C
D -1 D 0
D1 D2
D3 D5 D7
D8 D9
Fermentor
Nicotinic Acid
Dinucleotide
2
1 .5
NAD
nicotinamide adenine dinucleotide (NAD+), C
1
0 .5
0
D -1 D 0
D1 D2
D3 D5 D7
Fermentor
D8 D9
NADP
Scaled Intensity
Scaled Intensity
2 .5
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
• NAD+ starvation is a cidal event in tubercle bacilli
• NAD+ production is tightly regulated
• Enzymes common to the de novo and salvage pathways are hypothesized to
be good drug targets
D -1 D 0
D1 D2
D3 D5 D7
Fermentor
D8 D9
β-oxidation and hypoxia
Scaled Intensity
Scaled Intensity
3
2 .5
2
1 .5
1
0 .5
2
1 .5
1
0 .5
1 .4
0
1 .2
0
D0
D1
D7
caprylate (8:0), C
2 .5
D8
D0
D1
heptanoate (7:0), C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D7
D8
Snow Globe
glycerol, C
1
0 .8
0 .6
0 .4
0 .2
pelargonate (9:0), C
6
0
D0
5
Scaled Intensity
Scaled Intensity
Snow Globe
Scaled Intensity
caproate (6:0), C
3 .5
D1
D7
D8
Snow Globe
4
3
2
1
0
D0
D1
D7
Snow Globe
D8
D0
D1
D7
D8
Snow Globe
• In vivo, M. tuberculosis preferentially oxidizes fatty acids as their primary energy
source
• Medium chain fatty acids decreased during hypoxia but rebounded after aeration
of the culture
The methylcitrate cycle regulates propionyl CoA levels
pyruvate
acetyl-CoA
Scaled Intensity
methylcitrate
propionyl CoA, C
2
Propionyl-CoA
1 .5
oxaloacetate
1
cis-aconitate
0 .5
0
D0
D1
D7
D8
malate
Snow Globe
methyl-isocitrate
fumarate
pyruvate
succinate
• Propionyl CoA is generated during the β-oxidation of odd-chain length fatty acids
• Propionyl CoA is toxic at high concentrations.
• The methylcitrate cycle consumes propionyl CoA in order to maintain homeostasis
Long chain fatty acids accumulate during hypoxia
stearate (18:0), C
arachidate (20:0), C
2 .5
1 .5
1
0 .5
2
1 .5
1
0 .5
0
2
1 .5
1
0 .5
0
D0
D1
D7
D8
0
D0
Snow Globe
D1
D7
D8
D0
Snow Globe
Scaled Intensity
2
1 .5
1
0 .5
0
D0
D1
D7
Snow Globe
D8
D1
D7
D8
Snow Globe
lignocerate (24:0), C
2 .5
Scaled Intensity
behenate (22:0), C
2 .5
Scaled Intensity
2
Scaled Intensity
Scaled Intensity
2 .5
hexacosanoate (26:0), C
4
3 .5
3
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
D8
Snow Globe
• Long chain fatty acids are liberated from the cell envelope and then
undergo β-oxidation
• M. tuberculosis remodels its cellular envelope in order to form granulomas.
It is possibly synthesizing new lipids for this process
Urea cycle: production of arginine
citrulline, C
3 .5
Scaled Intensity
3
CO2 + NH4+ + ATP
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
D8
Snow Globe
aspartate
citrulline
1 .5
aspartate, C
3 .5
3
1
Scaled Intensity
Scaled Intensity
carbamoyl
phosphate
ornithine, C
2
0 .5
0
D0
D1
D7
D8
Urea
Cycle
ornithine
Snow Globe
argininosuccinate
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
Snow Globe
urea
fumarate
H2O
3
arginine, C
3
2 .5
2
Scaled Intensity
Scaled Intensity
arginine
urea, C
4
1
0
D0
D1
D7
Snow Globe
D8
2
1 .5
1
0 .5
0
D0
D1
D7
Snow Globe
D8
to Krebs
Cycle
D8
Metabolite biosynthesis during hypoxia
Glucose
serine, C
Hexose-P
aspartate, C
3 .5
Glycerate-P
Scaled Intensity
3
Serine
Scaled Intensity
2 .5
2
1 .5
1
0 .5
0
D0
2 .5
PEP
1 .5
1
0
D0
D1
D7
D8
Snow Globe
Aspartate
Scaled Intensity
OAA
malate, C
Pyruvate
Acetyl-CoA
Alanine
Scaled Intensity
0 .5
5
D1
D7
D8
Snow Globe
2
alanine, C
4
3 .5
3
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
D8
Snow Globe
Malate
4
TCA/Glyoxylate
Cycle
3
2
1
0
D0
D1
D7
D8
Snow Globe
• Alanine and aspartate levels increased during hypoxia possibly from the increased
levels of their precursor metabolites
cAMP and Hypoxia
16
14
12
10
8
6
4
2
0
N6-acetyllysine, C
3 .5
3
Scaled Intensity
Scaled Intensity
adenosine 3',5'-cyclic monophosphate (cAMP), C
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
Snow Globe
D8
D0
D1
D7
D8
Snow Globe
• cAMP is an important signaling molecule in M. tuberculosis pathogenesis
• cAMP binding acetyltransferases are crucial for virulence.
Lysine is n-acetylated on stress proteins which causes their activation
cAMP and Hypoxia
16
14
12
10
8
6
4
2
0
adenosine 3',5'-cyclic monophosphate (cAMP), C
Scaled Intensity
Scaled Intensity
adenosine 3',5'-cyclic monophosphate (cAMP), C
D0
D1
D7
Snow Globe
D8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D -1 D 0
D1 D2
D3 D5 D7
D8 D9
Fermentor
• cAMP is an important signaling molecule in M. tuberculosis pathogenesis
• cAMP binding acetyltransferases are crucial for virulence.
Lysine is n-acetylated on stress proteins which causes their activation
Unknown Compounds that Decrease with Aeration
X - 11461, C
4
Scaled Intensity
Scaled Intensity
5
3
2
1
0
D0
D1
D7
X - 11877, C
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D8
D0
D1
X - 11404, C
8
7
6
5
4
3
2
1
0
D7
D8
Snow Globe
X - 14955, C
6
5
Scaled Intensity
Scaled Intensity
Snow Globe
4
3
2
1
0
D0
D1
D7
Snow Globe
D8
D0
D1
D7
Snow Globe
D8
Unknown Compounds that Increase with Aeration
X - 15657, C
Scaled Intensity
2 .5
2
1 .5
1
0 .5
0
D1
D7
D8
X - 11687_200, C
2 .5
4
3
2
1
0
D0
2
1 .5
1
0 .5
0
D0
Snow Globe
D1
D7
D8
Snow Globe
6
5
4
3
2
1
0
D0
D1
D7
Snow Globe
D0
D1
D7
Snow Globe
X - 16050, C
7
Scaled Intensity
Scaled Intensity
X - 15489, C
5
Scaled Intensity
3
D8
D8
Scaled Intensity
D1
D7
D8
D0
Snow Globe
D1
D7
4
3
2
1
0
D0
2
1 .5
1
0 .5
D0
D8
D1
D1
D7
Snow Globe
D8
D7
Snow Globe
Snow Globe
X - 15177, C
5
X - 11542, C
2 .5
0
Scaled Intensity
D0
X - 11407_200, C
9
8
7
6
5
4
3
2
1
0
Scaled Intensity
X - 16039, C
9
8
7
6
5
4
3
2
1
0
Scaled Intensity
Scaled Intensity
Unknown Compounds that Increase with Aeration
X - 11533, C
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D0
D1
D7
Snow Globe
D8
D8
Unknown Compounds that Increase with Aeration
X - 16038, C
4
Scaled Intensity
Scaled Intensity
5
3
2
1
0
D0
D1
D7
Snow Globe
D8
X - 14359, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D0
D1
D7
Snow Globe
D8
Media Components Throughout the Time course
citrate, M
Scaled Intensity
1 .2
Scaled Intensity
glycerol, M
2 .5
1
0 .8
0 .6
0 .4
0 .2
0
1
2
1 .5
1
0 .5
0 .6
0 .4
0 .2
D0
D1
D7
0
FM
D8
D0
Snow Globe
D1
D7
FM
D8
0 .6
0 .4
Scaled Intensity
Scaled Intensity
0 .8
0 .8
0 .6
0 .4
0 .2
0 .2
0
0
D0
D1
D3
D5
Fermentor
D7
D8
D9
D7
D8
asparagine, M
2 .5
1
1
D1
Snow Globe
glycerol, M
1 .2
1 .2
FM
D0
Snow Globe
citrate, M
1 .4
Scaled Intensity
0 .8
0
FM
asparagine, M
1 .2
Scaled Intensity
1 .4
2
1 .5
1
0 .5
0
FM
D0
D1
D3
D5
D7
D8
D9
FM
D0
D1
Fermentor
D3
D5
Fermentor
• Citrate and glycerol levels are maintain for the entire 8 day period.
• Asparagine levels decrease significantly from day 0 to day 8.
This may serve as a limiting nitrogen source for the cell culture reaction.
D7
D8
D9
Amino Acids Accumulate in the Media: Possible Cell Death
Scaled Intensity
Scaled Intensity
5
4
3
2
1
0
FM
D0
D1
D7
lysine, M
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D8
Scaled Intensity
glycine, M
6
FM
D0
Snow Globe
FM
2
1
2
1 .5
1
D7
D8
D0
D1
D7
FM
3
2
0
4
3
2
D8
D8
D7
D8
8
6
4
2
0
D7
D7
10
1
1
D1
tyrosine, M
12
Scaled Intensity
Scaled Intensity
4
Snow Globe
D0
Snow Globe
5
5
D1
1
D8
phenylalanine, M
6
6
D0
1 .5
Snow Globe
tryptophan, M
FM
2
0
FM
Snow Globe
7
D8
0 .5
0
D1
D7
2 .5
2 .5
0 .5
D0
D1
proline, M
3
Scaled Intensity
3
FM
D0
Snow Globe
3
4
Scaled Intensity
Scaled Intensity
D8
glutamate, M
3 .5
0
Scaled Intensity
D7
Snow Globe
aspartate, M
5
D1
valine, M
8
7
6
5
4
3
2
1
0
0
FM
D0
D1
Snow Globe
D7
D8
FM
D0
D1
Snow Globe
X - 16027, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
Scaled Intensity
Scaled Intensity
Unknown Compounds Found in Media and Cells
D0
D1
D7
X - 16027, M
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D8
FM
D0
Snow Globe
X - 16063, C
3 .5
Scaled Intensity
Scaled Intensity
3
2 .5
2
1 .5
1
0 .5
0
D0
D1
D7
Snow Globe
D1
D7
D8
Snow Globe
D8
X - 16063, M
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
FM
D0
D1
Snow Globe
D7
D8
Unknown Compounds Found in Media and Cells (Fermentor)
X - 16027, C
X - 16027, M
2
8
Scaled Intensity
Scaled Intensity
10
6
4
2
0
1 .5
1
0 .5
0
D -1 D 0
D1 D2
D3
D5 D7
D8
D9
FM
D0
D1
D3
Fermentor
X - 16063, C
3
Scaled Intensity
Scaled Intensity
2 .5
2
1 .5
1
0 .5
0
D -1 D 0
D1 D2
D3 D5 D7
Fermentor
D5
D7
D8
D9
D8
D9
Fermentor
D8 D9
X - 16063, M
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
FM
D0
D1
D3
D5
Fermentor
D7
Conclusion & Path Forward
Main biochemical
findings:
• Glycolytic intermediates are decreased during the
hypoxic phase and then increase upon addition of
oxygen
• TCA/glyoxylate intermediate, with the exception of
citrate are increased suggesting an increased utilization
of amino acids for energy production
• Acetyl CoA is increased during hypoxia suggesting
increased β-oxidation of fatty acids.
• Urea cycle intermediates were altered suggesting a
need to synthesize arginine
Possible path forward:
• Increase sample size in order to strengthen the
confidence of the statistical analysis
• Include longer timepoints post aeration to determine
metabolic effects due to the resupply of oxygen
• Include more timepoints during the hypoxic phase to
gain more resolution of the effects of hypoxia in this in
vitro model
46
lactate, C
glucose
Scaled Intensity
2 .5
pyruvate
D0
D1
D7
acetyl CoA, C
3
2
1 .5
1
0 .5
0
D0
D8
D1
lactate
Snow Globe
Scaled Intensity
4
3 .5
3
2 .5
2
1 .5
1
0 .5
0
D7
D8
Snow Globe
acetyl-CoA
citrate, C
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D0
D1
Scaled Intensity
cis-aconitate
oxaloacetate
malate, C
5
4
3
glyoxylate
malate
2
D7
D8
Snow Globe
citrate
cis-aconitate, C
1 .8
1 .6
1 .4
1 .2
1
0 .8
0 .6
0 .4
0 .2
0
D0
isocitrate
D1
D7
D8
Snow Globe
1
0
D1
D7
D8
isocitrate, C
2 .5
Snow Globe
fumarate
succinate
succinate, C
5
4
Scaled Intensity
D0
Scaled Intensity
Scaled Intensity
Scaled Intensity
The glyoxylate cycle reduces CO2 loss
2
1 .5
1
0 .5
0
3
D0
D1
D7
Snow Globe
2
1
0
D0
D1
D7
Snow Globe
D8
D8