“ “tuberculosis hypoxia” max planck institute for infection biology mpib-0202-10vsbl

“Tuberculosis Hypoxia”Max Planck Institute for Infection Biology

MPIB-0202-10VSBL

Study Overview

Objective

2

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.

Snow Globe Overview

3

Two time courses completed• Both processed• One set shipped

Sauton’s w/tyloxapol

Citric acid

Ferric ammonium citrate

Glycerol

Asparagine

Biofilm

7H9 w/tyloxapol?

Study Design

4

0 Days1 2 3 4 5 6 7 +1 +2

Hypoxia Reaeration

Transcriptomics

Lipidomics Pellet

Pellet

Proteomics/ Glycomics

Metabolomics Pellet & Supernatant

2 Pellets & Supernatant

+6 hours2 hours

The primary purpose of this time course is to provide data to help correlate lipidomics, transcriptomics,

and proteomics from the snow globe run

Each arrow indicates a set of quadruplicate biological replicates of either pellet or supernatant + pellet as

indicated

BiochemicalExtraction

Metabolyzer™ UHPLC-MS/MS (+ESI)

GC-MS (+EI)

UHPLC-MS/MS (-ESI)

Instrumentation

Biochemical Analysis

Metabolon Platform Technology

Peak Detection

Peak Integration

Library SearchRT, Mass, MS/MS

QA/QC

tryptophan 984,812glutamic acid 23,387histidine 32,597leucine 117,683,785cholesterol 197,866asparagine 1,245,744creatinine 127,611cytidine 721,266lactate 2,111,697alpha-keto-glutarate 14,105,6544-hydroxyphenylacetate 444,8173-hydroxybutanoic acid 1,639,392cytosine 106,032arabinose 778,911fructose 2,519,658X-3370 1,101,581mannose 2,833,397pyruvate 250,670uridine 135,807Cholic acid 26,394,344allantoin 208,938isocitrate 5,825,474heptadecanoic acid 913,112inosine 1,115,331isoleucine 452,549alanine 462,648threonine 208,938tyrosine 67,989lysine 296,740methionine 49,879malic acid 156,499X-3697 46,230n-hexadecanoic acid 298,590octadecanoic acid 240,570trans-4-hydroxyproline 132,151X-9043 13,989,1722-deoxyguanosine 5,913,5193-hydroxyphenylacetate 7,214,4063-phospho-d-glycerate 8,649,644Kynurenic acid 468,6635-s-methyl-5-thioadenosine 14,569,292(p-Hydroxyphenyl)lactic acid 1,303,643alphahydroxybenzeneacetic acid 450,807ornithine 326,9495-oxoproline 890,753orotic acid 97,674palmitoleic acid 4,036,188pantothenic acid 94,772,385salicylic acid 379,417alpha-tocopherol 766,717citric acid 4,245,654X-6270 228,209glyceric acid 8,267,905histamine 912,667N-acetyl-L-leucine 45,949N-acetylneuraminic acid 2,178,243uric acid 312,025arginine 364,344ascorbic acid 584,247fumaric acid 4,459,712n-dodecanoate 370,805glutamine 109,597serine 321,078valine 2,241,354pyridoxal 78,842urea 437,094riboflavine 2,520,194proline 901,738pyrophosphate 1,045,231taurine 101,103,133X-3030 5,726,735citrulline 1,243,836biliverdin 420,511pyridoxamine 218,468serotonin 5,105,113gamma-L-glutamyl-L-glutamine 174,652gamma-L-glutamyl-L-tyrosine 8,854,738guanosine-5-monophosphate 82,6823-indoxyl-sulfate 369,207X-3100 135,999phosphate 35,095glycine 6,209,638nonanate 1,936,241DL-homocysteine 418,561L-kynurenine 3,451,095tartaric acid 631,615sn-Glycerol-3-phosphate 282,554carnitine 76,3072-methylhippuric acid 462,4873-methyl-2-oxovaleric acid 395,8394-Guanidinobutanoic acid 73,352,9894-methyl-2-oxopentanoate 272,564,647beta-hydroxypyruvic acid 431,358X-1656 118,271hippuric acid 7,724,959ethylmalonic acid 182,320D-lyxose 267,189maltose 27,024S-5-adenosyl-L-methionine 216,4052-deoxycytidine 1,151,739L-alpha-glycerophosphorylcholine 992,513aspartate 6,520,826p-hydroxybenzaldehyde 106,124X-1962 147,926D-sphingosine 289,530cortodoxone 58,939DL-indole-3-lactic acid 281,085gamma-glu-leu 177,587glycocholic acid 725,542taurocholic acid 3,281,189X-10381 231,486phenylalanine 118,902,022taurodeoxycholic acid 29,443,151inositol 146,593X-10419 554,814D-glucose 65,293,8461,5-anhydro-D-glucitol 5,183,545meso-erythritol 1,151,739X-3026 992,5132-hydroxybutyric acid 6,520,826digalacturonic acid 106,1243-methyl-2-oxobutyric 147,926monopalmitin 289,5301-stearoyl-rac-glycerol 58,93921-hydroxyprogesterone 147,9263-hydroxyoctanoic acid 289,5301-methylguanidine-hydrochloride 58,9393-hydroxydecanoic acid 281,0853-indoleacrylic acid 177,587DL-3-phenyllactic acid 3,281,189DL-alpha-hydroxyisocaproic acid 231,486DL-hexanoyl-carnitine 118,902,022O-acetyl-L-carnitine-hydrochloride 29,443,151EDTA 992,513l-aspartyl-l-phenylalanine 6,520,826

Metabolyzer Software

4 5 6 7 8 9 10 11 12 13 14Time (min)

4.01 14.435.84

4.3810.66

8.46

10.18

11.764.55 6.526.73 7.74

9.3411.7911.03

13.059.477.5011.215.34

12.893.17 13.30

8.01

Mass spectrum

3.17 min

Biochemical Amountcholesterol 143,789

DatabaseOf

Standards

cholesterol

Biochemical ID

Library Search for Biochemical ID

Global Biochemical Pathway Changes

Disease BiomarkersMechanistic Toxicology

Drug MOACellular Characteristics

BiochemicalInterpretation• Pathway analysis

• Literature

Heat Maps by Pathway

Metabolon Platform Technology Statistical Analysis

BiochemicalExtraction

Metabolyzer™ UHPLC-MS/MS (+ESI)

GC-MS (+EI)

UHPLC-MS/MS (-ESI)

Peak Detection

Peak Integration

Library SearchRT, Mass, MS/MS

QA/QC

Quality Control Processes

Sample

+ Recovery Standards

Extraction/recoveryInjection into Instrument

+ Internal Standards

Equal aliquot from ALL experimental samples pooledas “client matrix” (CMTRX)

1st

InjectionFinal

Injection

CMTRX Process Blank

Experimentalsamples

30% of samples dedicated to quality control

CMTRX

1. Significant component is QC 2. Multiple embedded QC standards in every sample

3. Matrix-specific technical replicates and QC injections across a study run-day

These processes allow for monitoring platform and process variability

Platform QC and Metabolite Summary

Internal Standards: standards spiked into each of the study samples prior to injection into the MS instrumentEndogenous Biochemicals: from CMTRX samples – technical replicates created from a small portion of experimental samples

Data Quality and Precision

These data are within Metabolon’s QC specifications.

Number of Biochemicals

Cells Media

Internal Standards 5% 5%

14%

Median RSDQuality Control Sample (Matrix)

Endogenous Biochemicals

9%

Compound Classification

Cells Media

Total 281 61

Named / Identified 134 45

Unnamed 147 16

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

Statistical Analyses: T-tests

10

Sample Statistics Table

The full t-test table is supplied as a separate excel file

0.55 Green: indicates significant difference (p≤0.05) between the groups shown; GREEN indicates a ratio < 11.71 Red: indicates significant difference (p≤0.05) between the groups shown; RED indicates a ratio > 11.431.20

Bold blue: narrowly missed cutoff for significance; p>0.05 , p<0.10Non-colored text and cell: mean values are not significantly different for that comparison

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

Amino acid

Fold of Change - Snow Globe

BIOCHEMICAL NAMESUB PATHWAYSUPER

PATHWAY Day 1 vs. Day 0 Day 7 vs. Day 0 Day 8 vs. Day 0 Day 8 vs. Day 7

Alanine and aspartate metabolism

Statistical Analyses: Summary

Total number of biochemicals with 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 with 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 -

Fresh vs. Day 0

Cells

Media

Day 8 vs. Day 0

Day 8 vs. Day 7

Day 1 vs. Day 0

Day 7 vs. Day 0

Welch's Two-Sample t-test

Snow Globe

Visualization with Box/Line Plots

“C” = cells; “M” = media

Box and Whiskers Legend

“Max” of distribution“Min” of distribution

Median Value___

Extreme Data PointsUpper QuartileLower Quartile

Mean Value+

glycine, C

D0 D1 D7 D8Snow Globe

0

1

2

3

4

5

6

7

Sca

led Inte

nsi

ty

Metabolite Name, Matrix

Scale

d

Inte

nsit

y

DayExperimentSnow Globe (Box Plots)

glycine, M

FM D0 D1 D3 D5 D7 D8 D9Fermentor

0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

Metabolite Name, Matrix

Scale

d

Inte

nsit

y

DayExperimentFermentor (Line

Plots)

13

Biochemical Data and Interpretation

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

fructose 6-P

glucose 6-P

fructose 1,6-bisP

1,3-bisphosphoglycerate

glyceraldehyde-3-P

3-phosphoglycerate

2-phosphoglycerate

phosphoenolpyruvate

pyruvate

Acetyl CoA

glucose

Dihydroacetone phosphate

glucose-6-phosphate (G6P), C


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

glucose, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

pyruvate, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

3-phosphoglycerate, C


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

fructose-6-phosphate, C


0

1

2

34

5

6

7

8

Sca

led Inte

nsi

ty

Isobar: fructose 1,6-diphosphate, glucose 1,6-diphosphate, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

The TCA cycle and CO2 loss

pyruvate

acetyl-CoA

citrate

cis-aconitate

isocitrate

succinyl-CoAsuccinate

fumarate

malate

oxaloacetate

glucose

lactate

α-ketoglutarateCO2

CO2

The generation of CO2 by the bacteriawould acidify the environment

The loss of carbon atoms would decreasethe energy yield

Glyoxylate Cycle

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA isocitrate, M

FM D0 D1 D7 D8Snow Globe

0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

citrate, M


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

cis-aconitate, M


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

succinate, M


0

1

2

34

5

6

7

8

Sca

led Inte

nsi

ty

malate, M


0

2

4

6

8

10

Sca

led Inte

nsi

ty

glucose, M


0

0.5

1

1.5

2

2.5

3

3.5S

cale

d Inte

nsi

ty

Glyoxylate Cycleacetyl CoA, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

succinate, C


0

1

2

3

4

5

Sca

led Inte

nsi

typyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

citrate, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

lactate, C


0

0.5

1

1.52

2.5

3

3.5

4

Sca

led Inte

nsi

ty

malate, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

cis-aconitate, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

isocitrate, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

Glyoxylate Cycle

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

citrate, C

D-1 D0 D1 D2 D3 D5 D7 D8 D9Fermentor

0

0.2

0.4

0.6

0.8

1

1.2

Sca

led Inte

nsi

ty

cis-aconitate, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

isocitrate, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

succinate, C


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

malate, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

lactate, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

acetyl CoA, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

Glyoxylate Cycle

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

citrate, M


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

cis-aconitate, M


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

isocitrate, M


0

0.2

0.4

0.6

0.8

1

1.2

Sca

led Inte

nsi

ty

succinate, M


0

1

2

3

4

5

Sca

led Inte

nsi

ty

malate, M


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

Methylcitrate cycle

pyruvate

acetyl-CoA

methylcitrate

cis-aconitate

methyl-isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

pyruvate

propionyl CoA

propionyl CoA, C


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

acetyl CoA, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

malate, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

succinate, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

Snow Globe: significant proteome changes

Relative Counts

Protein Description gi Number Locus Tag Day 0 Day 1 Day 7 Day 8

heat shock protein hspX [Mycobacterium tuberculosis H37Rv] 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

• 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.

isoleucine, C


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

leucine, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

tyrosine, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

asparagine, C


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

TCA cycle intermediates accumulate in media

isocitrate, M


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

citrate, M


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

cis-aconitate, M


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

pyruvate

acetyl-CoA

citrate

cis-aconitate

isocitrate

succinatefumarate

malate

oxaloacetate

glucose

lactate

glyoxylate

succinate, M


0

1

2

34

5

6

7

8

Sca

led Inte

nsi

ty

malate, M


0

2

4

6

8

10

Sca

led Inte

nsi

tyglucose, M


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

Aeration induces an increase in pentose phosphate pathway intermediates

ribitol, C


0

0.5

1

1.52

2.5

3

3.5

4

Sca

led Inte

nsi

ty

sedoheptulose-7-phosphate, C


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

fructose, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

nicotinamide adenine dinucleotide phosphate (NADP+), C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

6-Phosphogluconate

Ribulose-5-P

Ribulose-5-P

Xylulose-5-P

Gluconate

Glucose

Glyceraldehyde-3-P + Sedoheptulose-7-P

NADPH

NADPH

Fructose-6-P + Erythrose-4-PXylulose-5-P

Glyceraldehyde-3-P + Fructose-6-P

Glucose utilization in bacteria

Fuhrer et al., J Bacteriol. 187(5): 1581-1590

NAD+ synthesis tightly regulated in M. tb

• 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

Nicotinic AcidMononucleotide

Nicotinic AcidDinucleotide

NicotinamideMononucleotide

NAD

Nicotinic Acid Nicotinamide

NAD(P) breakdown

NADP

Salvage Pathway

NicotinamideRiboside

nicotinate, C


0

1

2

3

4

5

6

7

Sca

led Inte

nsi

ty

nicotinate ribonucleoside*, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

nicotinamide adenine dinucleotide (NAD+), C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

nicotinic acid mononucleotide (NaMN), C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

nicotinamide, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

NAD+ synthesis tightly regulated in M. tb

• 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

Nicotinic AcidMononucleotide

Nicotinic AcidDinucleotide

NicotinamideMononucleotide

NAD

Nicotinic Acid Nicotinamide

NAD(P) breakdown

NADP

Salvage Pathway

NicotinamideRiboside

nicotinamide, C


0

0.2

0.4

0.6

0.8

1

Sca

led Inte

nsi

ty

nicotinamide adenine dinucleotide (NAD+), C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

nicotinate, C


0

0.2

0.4

0.6

0.8

1

1.2

Sca

led Inte

nsi

ty

nicotinic acid mononucleotide (NaMN), C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

nicotinate ribonucleoside*, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

β-oxidation and hypoxia

caproate (6:0), C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

heptanoate (7:0), C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

caprylate (8:0), C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

pelargonate (9:0), C


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

• 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

glycerol, C


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

The methylcitrate cycle regulates propionyl CoA levels

pyruvate

acetyl-CoA

methylcitrate

cis-aconitate

methyl-isocitrate

succinate

fumarate

malate

oxaloacetate

Propionyl-CoApropionyl CoA, C


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

• 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

pyruvate

Long chain fatty acids accumulate during hypoxia

stearate (18:0), C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

arachidate (20:0), C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

behenate (22:0), C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

lignocerate (24:0), C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

hexacosanoate (26:0), C


0

0.5

1

1.52

2.5

3

3.5

4

Sca

led Inte

nsi

ty

• 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

aspartate, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

ornithine, C


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

urea, C


0

1

2

3

4

Sca

led Inte

nsi

ty

arginine, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

citrulline, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

citrulline

argininosuccinate

arginine

ornithine

carbamoyl phosphate

CO2 + NH4+ + ATP

aspartate

fumarateto Krebs

Cycle

UreaCycle

H2O

urea

Metabolite biosynthesis during hypoxia

• Alanine and aspartate levels increased during hypoxia possibly from the increased levels of their precursor metabolites

Glycerate-P

PEP

Pyruvate

Hexose-P

Glucose

Serine

Acetyl-CoA

Alanine

TCA/Glyoxylate Cycle

Malate

OAAAspartate

alanine, C


0

0.5

1

1.52

2.5

3

3.5

4

Sca

led Inte

nsi

ty

serine, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

aspartate, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

malate, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

cAMP and Hypoxia

• 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

adenosine 3',5'-cyclic monophosphate (cAMP), C


0

2

4

68

10

12

14

16

Sca

led Inte

nsi

ty

N6-acetyllysine, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

cAMP and Hypoxia

• 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



0

2

4

68

10

12

14

16

Sca

led Inte

nsi

ty



0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

Unknown Compounds that Decrease with Aeration

X - 11461, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

X - 11877, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

X - 11404, C


0

1

2

34

5

6

7

8

Sca

led Inte

nsi

ty

X - 14955, C


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

Unknown Compounds that Increase with Aeration

X - 15657, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

X - 15489, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

X - 11687_200, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

X - 16050, C


0

1

2

3

4

5

6

7

Sca

led Inte

nsi

ty


X - 16039, C


0123456789

Sca

led Inte

nsi

ty

X - 15177, C


0

1

2

3

4

5

Sca

led Inte

nsi

tyX - 11407_200, C


0123456789

Sca

led Inte

nsi

ty

X - 11542, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

X - 11533, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty


X - 16038, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

X - 14359, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

Media Components Throughout the Time course

citrate, M


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

glycerol, M


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

asparagine, M


0

0.2

0.4

0.6

0.8

1

1.2

Sca

led Inte

nsi

ty

• 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.

asparagine, M


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

citrate, M


0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sca

led Inte

nsi

ty

glycerol, M


0

0.2

0.4

0.6

0.8

1

1.2

Sca

led Inte

nsi

ty

Amino Acids Accumulate in the Media: Possible Cell Death

glycine, M


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

aspartate, M


0

1

2

3

4

5

Sca

led Inte

nsi

ty

glutamate, M


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

lysine, M


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

phenylalanine, M


0

1

2

3

4

5

6

Sca

led Inte

nsi

ty

tyrosine, M


0

2

4

6

8

10

12

Sca

led Inte

nsi

ty

tryptophan, M


0

1

2

3

4

5

6

7

Sca

led Inte

nsi

ty

valine, M


0

1

2

34

5

6

7

8

Sca

led Inte

nsi

ty

proline, M


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

Unknown Compounds Found in Media and Cells

X - 16027, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6S

cale

d Inte

nsi

ty

X - 16027, M


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

X - 16063, M


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

X - 16063, C


0

0.5

1

1.5

2

2.5

3

3.5

Sca

led Inte

nsi

ty

Unknown Compounds Found in Media and Cells (Fermentor)

X - 16063, M


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

X - 16063, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

tyX - 16027, C


0

2

4

6

8

10S

cale

d Inte

nsi

tyX - 16027, M


0

0.5

1

1.5

2

Sca

led Inte

nsi

ty

Conclusion & Path Forward

•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

Main biochemical findings:

•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

Possible path forward:

The glyoxylate cycle reduces CO2 loss

citrate, C


0

0.2

0.4

0.60.8

1

1.2

1.4

1.6

Sca

led Inte

nsi

ty

acetyl CoA, C


0

0.5

1

1.5

2

2.5

3

Sca

led Inte

nsi

ty

lactate, C


0

0.5

1

1.52

2.5

3

3.5

4

Sca

led Inte

nsi

ty

malate, C


0

1

2

3

4

5

Sca

led Inte

nsi

ty

succinate, C


0

1

2

3

4

5

Sca

led Inte

nsi

tycis-aconitate, C


00.20.40.60.8

11.21.41.61.8

Sca

led Inte

nsi

ty

isocitrate, C


0

0.5

1

1.5

2

2.5

Sca

led Inte

nsi

ty

pyruvate

acetyl-CoA

citrate

cis-aconitate

isocitrate

succinatefumarate

malate

oxaloacetate

glucose

lactate

glyoxylate

“ “tuberculosis hypoxia” max planck institute for infection biology mpib-0202-10vsbl

Documents

qc injections

snow globe overview3

snow globe model

distributionmedian value

study samples

number of biochemicals

multiple embedded qc

better qvalue