“Biochemical Profiles of Mycobacterium

tuberculosis Grown Under Hypoxic Conditions (Snow Globe Model)”

Max Planck Institute for Infection Biology

MPIB-0206-09VSBL

Study OverviewObjective

Study Design

2

To identify biochemicals that are altered in Mycobacterium tuberculosis cultured under hypoxic conditions in the snow globe model and to identify biochemicals that are differentially released into the cell culture media and/or differentially consumed from the culture media.

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

Metabolomics Pellet & Supernatant

+6 hours2 hours

+3 +4 +5

Hypoxia Reaeration

Time Point / Condition Cell Pellet (n; Rep) Supernatant (n; Rep)

T=0 (log phase; hypox start) n=4; Reps A-D n=4; Reps A-D

T=1d (1d hypoxia) n=4; Reps A-D n=4; Reps A-D

T=7d (7d hypoxia) n=4; Reps A-D n=4; Reps A-D

T=8d (1d re-aeration) n=4; Reps A-D n=4; Reps A-D

T=12d (5d re-aeration) n=4; Reps A-D n=4; Reps A-Dn=1; Sauton's media no Tween (SnoT)

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

4

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)

4.0114.43

5.84

4.3810.66

8.46

10.18

11.764.55 6.526.73 7.74

9.3411.79

11.03

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

Automated Biochemical Identification

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

Compound Classification Cells Media

Named / Identified 133 70

Cells Media

Internal Standards 8% 6%

Endogenous Biochemicals

14% 15%

Quality Control Sample (Matrix)

Median RSD

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, q≤0.10 was taken as significant

Statistical Analyses: T-tests

8

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

Fold of Change

Welch's Two Sample t-Tests

SUB PATHWAY BIOCHEMICAL NAME PLATFORM COMP IDSG7-0SG7-1

SG7-0SG7-7

SG7-0SG7-8

SG7-0SG7-12

SG7-7SG7-8

SG7-7SG7-12

Glutamate metabolism

glutamate LC/MS pos 57 2.42 4.15 1.53 0.98 0.37 0.24

glutamine LC/MS pos 53 3.78 15.23 2.63 1.12 0.17 0.07

gamma-aminobutyrate (GABA)

LC/MS pos 1416 7.37 11.63 6.34 0.67 0.55 0.06

N-acetylglutamate LC/MS pos 15720 14.33 13.38 3.58 1.64 0.27 0.12

Statistical Analyses: Summary

Total number of biochemicals with p?0.05

Biochemicals (? ? )

p?0.05

Total number of biochemicals with

0.05<p <0.10

Biochemicals (? ? )

0.05<p<0.10

Total number of biochemicals with p?0.05

Biochemicals (? ? )

p?0.05

Total number of biochemicals with

0.05<p <0.10

Biochemicals (? ? )

0.05<p<0.10SG7-0SG7-1

40 31|9 15 10|5 10 1|9 7 1|6

SG7-0SG7-7

69 52|17 11 8|3 24 1|23 2 0|2

SG7-0SG7-8

54 40|14 14 9|5 24 1|23 6 0|6

SG7-0SG7-12

33 14|19 18 7|11 28 1|27 8 0|8

SG7-7SG7-8

42 17|25 19 5|14 2 1|1 2 0|2

SG7-7SG7-12

65 15|50 14 6|8 11 1|10 7 4|3

Statistical ComparisonsCells Media

Welch's Two Sample t-Tests

Visualization with Box Plots

Scale

d

Inte

nsit

y

Timepoint

Metabolite Name , Snow Globe

Box and Whiskers Legend

“Max” of distribution“Min” of distribution

Median Value___

Extreme Data PointsUpper QuartileLower Quartile

Mean Value+

Scale

d

Inte

nsit

y

Timepoint

Metabolite Name , Snow GlobeCells Media

alanine - SG7

S Ctrl 0 1 7 8 120

0.5

1

1.5

2

2.5alanine - SG7

0 1 7 8 120

0.5

1

1.5

2

Summary of Biochemical Findings

Key Observations

• The results for Snow Globe 7, and Snow Globe 6, show very good reproducibility compared with earlier snow globe analyses (MPIB-0202-09VSBL).

• During hypoxia, metabolic profiles of carbon sources suggest that M. tb is potentially relying on amino acids and lipids as sources of energy.

• Nucleotide profiles suggest that in an oxygen rich environment M. tb may synthesize nucleotides for cell division.

• NAD metabolites also increased during reaeration of the culture suggesting more oxidative metabolism during this time. This may also be linked to nucleotide metabolism.

Hypoxia and the Glyoxylate Cycle

Several glyoxylate intermediates accumulate during hypoxia

Succinate and acetyl-CoA are major entry points for anaplerotic reactions.

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

cis-aconitate - SG7

0 1 7 8 120

0.20.40.60.8

11.21.41.6

Cis-aconitate – SG7

succinate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3 Succinate – SG7

fumarate - SG7

0 1 7 8 120

0.5

1

1.5

2 Fumarate – SG7

citrate - SG7

0 1 7 8 120

0.4

0.8

1.2

1.6

Citrate – SG7malate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5Malate – SG7

isocitrate - SG7

0 1 7 8 120

0.5

1

1.5

2 Isocitrate – SG7

Anaplerosis and the Glyoxylate Cycle

The TCA cycle/glyoxylate cycle has several sites where amino acids, fatty acids and other molecules feed into in order to help produce energy in cells.

The increases in succinate and isocitrate may be indicative of anapleurotic reactions feeding into this pathway during hypoxia.

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

cis-aconitate - SG7

0 1 7 8 120

0.20.40.60.8

11.21.41.6

Cis-aconitate – SG7

succinate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3 Succinate – SG7

isocitrate - SG7

0 1 7 8 120

0.5

1

1.5

2Isocitrate – SG7

Hypoxia and Anaplerotic Reactions: A Summary

Cellular energetics plays a critical role in M. tb during hypoxia.

The bacterium has at least three possible stores of metabolites for utilization in energy production if carbon sources are depleted.

tyrosine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Tyrosine – SG7 adenosine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Adenosine – SG7

Amino Acid Levels Change with Oxygen Status

Hypoxia decreased the levels of amino acids suggesting that amino acids were possibly utilized for energy production.

Several amino acids, including those above decrease with hypoxia and increase with reaeration of the culture.

serine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5Serine – SG7

glutamate - SG7

0 1 7 8 120

0.5

1

1.5

2Glutamate – SG7 tryptophan - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5 Tryptophan – SG7

glutamine - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

Glutamine – SG7

Amino Acid Levels Change with Oxygen Status

Lysine and Tryptophan are metabolized to 2-aminoadipate and 5-methoxytryptamine, respectively.

The metabolites increase on day 7 whereas the amino acid molecules decrease.

This suggests that the amino acids are metabolized, possibly for energetic purposes.

lysine - SG6

0 1 7 8 11 140

0.5

1

1.5

2

2.5

3Lysine – SG6 2-aminoadipate - SG6

0 1 7 8 11 140

2

4

6

8

2-aminoadipate – SG6

tryptophan - SG6

0 1 7 8 11 140

1

2

3

4

5Tryptophan – SG6 5-methoxytryptamine - SG6

0 1 7 8 11 140

0.5

1

1.5

2

2.55-methoxytryptamine – SG6

Glyoxylate Intermediates Accumulate in Spent Media

Glyoxylate pathway intermediates accumulate in spent media.

These intermediates reach highest levels during hypoxia and may result from active excretion of metabolites

pyruvate

acetyl-CoA

glucose

lactate

citrate

cis-aconitate

isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

isocitrate - SG7

S Ctrl 0 1 7 8 120

0.2

0.4

0.6

0.8

1

1.2

1.4 Isocitrate – SG7succinate - SG7

S Ctrl 0 1 7 8 120

0.4

0.8

1.2

1.6

Succinate – SG7

citrate - SG7

S Ctrl 0 1 7 8 120

0.2

0.4

0.6

0.8

1

1.2Citrate– SG7

cis-aconitate - SG7

S Ctrl 0 1 7 8 120

0.20.40.60.8

11.21.41.6

Cis-aconitate – SG7malate - SG7

S Ctrl 0 1 7 8 120

0.5

1

1.5

2

2.5

3Malate – SG7

Malate and Aspartate Metabolism During the Snow Globe Culture Period

Glycerate-P

PEP

Pyruvate

Hexose-P

Glucose

Serine

Acetyl-CoA

Alanine

TCA/Glyoxylate Cycle

Malate

OAAAspartate

aspartate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5Aspartate – SG7

malate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5 Malate – SG7

Malate levels decrease during hypoxia and may suggest shuttling of malate from the glyoxylate pathway.

Glycolysis and Hypoxia in M. tb

6 carbon glycolytic intermediates decrease during hypoxia. Given the decrease in oxygen and possible lower metabolism, glycolysis may be slowing during hypoxia.

Reaeration of the culture increases this intermediates and may be providing glucose-6-phosphate to the pentose phosphate pathway

fructose 6-P

glucose 6-P

fructose 1,6-bisP

Dihydroacetone phosphate

1,3-bisphosphoglycerate

glyceraldehyde-3-P

3-phosphoglycerate

2-phosphoglycerate

phosphoenolpyruvate

pyruvate

Acetyl CoA

glucose

glucose - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Glucose – SG7

fructose-6-phosphate - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

Fructose-6-P – SG7

glucose-6-phosphate (G6P) - SG7

0 1 7 8 120

1

2

3

4

5

6 Glucose-6-P – SG7

pyruvate - SG7

0 1 7 8 120

0.2

0.4

0.6

0.8

1Pyruvate – SG7

3-phosphoglycerate - SG7

0 1 7 8 120

0.4

0.8

1.2

1.6

3-phosphoglycerate – SG7

Pentose Phosphate Pathway (PPP) Intermediates Accumulate During Reaeration

The increase in PPP intermediates during reaeration may indicate higher glucose metabolism and shunting of G6P to the PPP.

In high O2 environment, the bacteria will divide and have an increased need for nucleotides.

sedoheptulose-7-phosphate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Sedoheptulose-7-P– SG7

gluconate - SG7

0 1 7 8 120

2

4

6

8

Gluconate – SG7

glucose 6-phosphate

6-phosphogluconate

ribulose 5-phosphate

ribose5-phosphate

xylulose5-phosphate

glyceraldehyde 3-phosphate

sedoheptulose7-phosphate

fructose6-phosphate

erythrose4-phosphate

fructose6-phosphate

glyceraldehyde 3-phosphate

xylulose5-phosphate

6-phosphogluconolactone

ribose

xylulose

ribulose

xylitol

Nucleotide Levels During Hypoxia

The increase in nucleotides correlates with increased pentose phosphate pathway activity.

The result may be the production of more 5-carbon species for nucleotide production.

Nucleotides/Nucleosides

Nitrogen sourceCarbon source

High energy phosphate bondsLess cellular growth and DNA replication

DNA SynthesisRNA Synthesis

(Cell growth/division and increased transcriptional activity)

HypoxicConditions

Oxygen-richConditions

adenosine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Adenosine – SG7 2'-deoxyguanosine - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

2’deoxyguanosine – SG7

Purines and Pyrimidines Show Equivalent Profiles During Hypoxia

Purine and pyrimidine synthesis is tightly regulated. The increase in these metabolites with reaeration of the

culture may signify an increased need during DNA replication or transcription.

adenine - SG7

0 1 7 8 120

0.5

1

1.5

2Adenine – SG7 adenosine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Adenosine – SG7

guanine - SG7

0 1 7 8 120

1

2

3

4

5Guanine – SG7 guanosine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5Guanosine – SG7 2'-deoxyguanosine - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

2’deoxyguanosine – SG7

thymine - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Thymine – SG7uracil - SG7

0 1 7 8 120

0.4

0.8

1.2

1.6

Uracil – SG7

Purines

Pyrimidines

NAD Metabolism

• NAD+ starvation is a cidal event in tubercle bacilli

• NAD+ production is tightly regulated

• The balance of NAD levels in M. tb is critical for survival in granulomas

• Depletion of adenine may drive lower amounds of NAD+ and NADP+

nicotinamide adenine dinucleotide phosphate (NADP+) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3NADP+ - SG7

nicotinate ribonucleoside* - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Nicotinate ribonucleoside*- SG7

nicotinamide - SG7

0 1 7 8 120

0.20.40.60.8

11.21.41.6

Nicotinamide - SG7

nicotinamide adenine dinucleotide (NAD+) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3NAD+- SG7

Nicotinic AcidMononucleotide

Nicotinic AcidDinucleotide

NicotinamideMononucleotide

NAD

Nicotinic Acid Nicotinamide

NAD(P) breakdown

NADP

Salvage Pathway

NicotinamideRiboside

Hypoxia and Fatty Acid Metabolism

Free fatty acids accumulate to highest levels during hypoxia M. Tuberculosis may rely on β-oxidation of fatty acids for

energy production during hypoxia Alternatively, free fatty acids may be utilized for synthesis

of higher molecular weight lipid species (e.g. triglycerides) in order to strenghten the cellular wall of granuloma-like structures.

LipidMetabolism

Catabolism or rearrangementof cell wall/plasma membrane and

degradation of components for energy

Synthesis of high mol. wt.species (triglycerides) to increase

rigidity of granuloma cell wall

palmitate (16:0) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Palmitate (16:0) – SG7 tuberculostearate - SG7

0 1 7 8 120

0.5

1

1.5

2Tuberculostearate – SG7

Hypoxia and Fatty Acid Metabolism

Free fatty acids accumulate to highest levels during hypoxia.

This profile was reproduced in Snow globe 6 as well. Many of the fatty acids in M. tuberculosis are >30 carbons

in length so the accumulation of these “shorter” chain fatty acids may be indicative of metabolism of these molecules.

margarate (17:0) - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

palmitate (16:0) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Palmitate (16:0) – SG7 Margarate (17:0) – SG7

stearate (18:0) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Stearate (18:0) – SG7 tuberculostearate - SG7

0 1 7 8 120

0.5

1

1.5

2Tuberculostearate – SG7hexacosanoate (26:0) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Hexacosanoate (26:0) – SG7

The Methylcitrate cycle

The methylcitrate cycle is utilized to metabolize odd-chain fatty acids.

2-methylcitrate increases during hypoxia and may suggest increased b-oxidation of fatty acids during low O2.

methylcitrate

cis-aconitate

methyl-isocitrate

succinate

fumarate

malate

oxaloacetate

glyoxylate

acetyl-CoA

pyruvate

propionyl CoA

2-methylcitrate - SG7

0 1 7 8 120

0.5

1

1.5

2

2.52-methylcitrate – SG7pelargonate (9:0) - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5Pelargonate (9:0) – SG7margarate (17:0) - SG7

0 1 7 8 120

0.51

1.52

2.53

3.54

Margarate (17:0) – SG7

A possible source of glucose: Trehalose

Trehalose is a major component of mycolic lipids. Mycolic lipids give the cell wall structural integrity.

During cell wall/membrane rearrangement, trehalose may be liberated and then metabolised to glucose.

This glucose could be a good source for glycolytic metabolism.

trehalose - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Trehalose – SG7

glucose - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3Glucose – SG7

Cell Wall/Lipid Reorganization

Free Trehalose (α,α linked glucose)

Glucose

Glycolysis

Mycothione Levels During Hypoxia

Mycothione levels increased during reaeration of the culture suggesting increased oxidative stress during this time frame.

This increased need for mycothione may be due to increased oxidative metabolism or other cellular processes in the presence of O2.

mycothione (MSSM)* - SG7

0 1 7 8 120

0.5

1

1.5

2

2.5

3

3.5Mycothione (MSSM) – SG7

Major Components for Sauton’s Media

The major constituents for the media are represented and do not appear limiting throughout the culture process.

The relatively high levels of citrate in the media are likely the reason for the static levels of citrate in the cellular fraction.

citrate - SG7

S Ctrl 0 1 7 8 120

0.2

0.4

0.6

0.8

1

1.2Citrate – SG7

glycerol - SG7

S Ctrl 0 1 7 8 120

0.20.40.60.8

11.21.41.6

Glycerol – SG7

asparagine - SG7

S Ctrl 0 1 7 8 120

0.2

0.4

0.6

0.8

1

1.2

1.4Asparagine – SG7

phosphate - SG7

S Ctrl 0 1 7 8 120

0.2

0.4

0.6

0.8

1

1.2

1.4Phosphate – SG7

Media Analysis

In addition to glyoxylate pathway intermediates, several other metabolites accumulate in media during hypoxia.

Trehalose and amino acids accumulate to significantly higher levels.

Given the context of lower metabolic activity during hypoxia, this could mean that metabolites are excreted during cell death or other processes during which the cell wall/membrane is porous.

trehalose - SG7

S Ctrl 0 1 7 8 120

0.5

1

1.5

2

2.5

3Trehalose – SG7

tyrosine - SG7

S Ctrl 0 1 7 8 120

0.5

1

1.5

2Tyrosine – SG7 valine - SG7

S Ctrl 0 1 7 8 120

0.4

0.8

1.2

1.6

Valine – SG7

Conclusion & Path Forward

•The results for Snow Globe 7, and Snow Globe 6, show very good reproducibility compared with earlier snow globe analyses.

•During hypoxia, metabolic profiles of carbon sources suggest that M. tb is relying on amino acids and lipids as sources of energy.

•Nucleotide profiles suggest that in an oxygen rich environment M. tb may synthesize nucleotides for cell division.

•NAD metabolites also increased during reaeration of the culture suggesting more oxidative metabolism during this time. This may also be linked to nucleotide metabolism.

Main biochemical findings:

•Compare snow globe/in vitro results to metabolome of granulomas from mice infected with M. tb.

•Test compounds used to treat M.tb infections to determine metabolic effects of treatment on the bacteria.

•Obtain metabolic profile of M.tb grown in macrophage cell lines.

Possible path forward:

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