Study Overview

1. “Biochemical Profiles of Mycobacterium tuberculosis Grown Under Hypoxic Conditions (Snow Globe Model)” Max Planck Institute for Infection Biology MPIB-0206-09VSBL

2. Study Overview 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-D n=1; Sauton's media no Tween (SnoT) Objective 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. Hypoxia Reaeration Pellet & Supernatant Metabolomics Study Design 1 2 3 4 5 6 7 +1 +2 0 Days +3 +4 +5 +6 hours 2 hours

3. Metabolyzer™ Statistical Analysis UHPLC-MS/MS (+ESI) Peak Detection UHPLC-MS/MS (-ESI) Peak Integration Biochemical Extraction Library Search RT, Mass, MS/MS QA/QC GC-MS (+EI) Metabolon Platform Technology • Biochemical • Interpretation • Pathway analysis • Literature Global Biochemical Pathway Changes Disease Biomarkers Mechanistic Toxicology Drug MOA Cellular Characteristics Heat Maps by Pathway

4. cholesterol 143,789 Database Of Standards cholesterol 3.17 min Mass spectrum Biochemical ID Automated Biochemical Identification Biochemical Amount Metabolyzer Software 14.43 4.01 5.84 4.38 10.66 8.46 10.18 4.55 11.76 6.52 6.73 7.74 9.34 8.01 11.03 11.79 13.05 9.47 7.50 5.34 11.21 3.17 12.89 13.30 4 5 6 7 8 9 10 11 12 13 14 Time (min)

5. Quality Control Processes 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 CMTRX These processes allow for monitoring platform and process variability

6. Platform QC and Metabolite Summary Data Quality and Precision 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’sQC specifications. Number of Biochemicals

7. 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, q≤0.10 was taken as significant Sample Statistics Table The full t-test table is supplied as a separate excel file

8. Statistical Comparisons Cells Media Welch's Two Total number of Biochemicals Total number of Biochemicals Total number of Biochemicals Total number of Biochemicals Sample t-Tests biochemicals ( ? ? ) biochemicals with ( ? ? ) biochemicals ( ? ? ) biochemicals with ( ? ? ) with p ?0.05 p?0.05 0.05< p <0.10 0.05<p<0.10 with p ?0.05 p?0.05 0.05< p <0.10 0.05<p<0.10 SG7-0 40 31 | 9 15 10 | 5 10 1| 9 7 1| 6 SG7-1 SG7-0 69 52 | 17 11 8 | 3 24 1| 23 2 0| 2 SG7-7 SG7-0 54 40 | 14 14 9 | 5 24 1| 23 6 0| 6 SG7-8 SG7-0 33 14 | 19 18 7 | 11 28 1| 27 8 0| 8 SG7-12 SG7-7 42 17 | 25 19 5 | 14 2 1| 1 2 0| 2 SG7-8 SG7-7 65 15 | 50 14 6|8 11 1| 10 7 4 | 3 SG7-12 Statistical Analyses: Summary

9. Visualization with Box Plots Media Cells Metabolite Name , Snow Globe Metabolite Name , Snow Globe Box and Whiskers Legend Scaled Intensity Scaled Intensity + Mean Value ___ Median Value Extreme Data Points Upper Quartile Lower Quartile Timepoint Timepoint “Max” of distribution “Min” of distribution

11. Summary of Biochemical Findings

12. Hypoxia and the Glyoxylate Cycle Citrate – SG7 Cis-aconitate – SG7 glucose pyruvate lactate acetyl-CoA citrate Isocitrate – SG7 Succinate – SG7 Fumarate – SG7 oxaloacetate cis-aconitate glyoxylate malate isocitrate acetyl-CoA fumarate Malate – SG7 succinate • Several glyoxylate intermediates accumulate during hypoxia • Succinate and acetyl-CoA are major entry points for anaplerotic reactions.

13. Anaplerosis and the Glyoxylate Cycle glucose Cis-aconitate – SG7 pyruvate lactate acetyl-CoA citrate Succinate – SG7 Isocitrate – SG7 oxaloacetate cis-aconitate glyoxylate malate isocitrate acetyl-CoA fumarate succinate • 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.

14. Hypoxia and Anaplerotic Reactions: A Summary Adenosine – SG7 Tyrosine – SG7 • Cellular energetics plays a critical role in M. tbduring hypoxia. • The bacterium has at least three possible stores of metabolites for utilization in energy production if carbon sources are depleted.

15. Glutamine – SG7 Amino Acid Levels Change with Oxygen Status Serine – SG7 Glutamate – SG7 Tryptophan – SG7 • 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.

16. Amino Acid Levels Change with Oxygen Status Lysine – SG6 2-aminoadipate – SG6 Tryptophan – SG6 5-methoxytryptamine – SG6 • 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.

17. Glyoxylate Intermediates Accumulate in Spent Media glucose Citrate– SG7 pyruvate lactate acetyl-CoA citrate Cis-aconitate – SG7 oxaloacetate cis-aconitate Malate – SG7 malate glyoxylate isocitrate acetyl-CoA fumarate succinate Isocitrate – SG7 Succinate – SG7 • Glyoxylate pathway intermediates accumulate in spent media. • These intermediates reach highest levels during hypoxia and may result from active excretion of metabolites

18. Malate and Aspartate Metabolism During the Snow Globe Culture Period Glucose Hexose-P Glycerate-P Serine Aspartate – SG7 PEP Pyruvate Alanine Acetyl-CoA Aspartate OAA Malate TCA/Glyoxylate Cycle Malate – SG7 • Malate levels decrease during hypoxia and may suggest shuttling of malate from the glyoxylate pathway.

19. Glycolysis and Hypoxia in M. tb Glucose – SG7 glucose glucose 6-P fructose 6-P Glucose-6-P – SG7 fructose 1,6-bisP 3-phosphoglycerate – SG7 Dihydroacetone phosphate glyceraldehyde-3-P 1,3-bisphosphoglycerate Fructose-6-P – SG7 3-phosphoglycerate 2-phosphoglycerate Pyruvate – SG7 phosphoenolpyruvate pyruvate Acetyl CoA • 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

20. Pentose Phosphate Pathway (PPP) Intermediates Accumulate During Reaeration Gluconate – SG7 glucose 6-phosphate 6-phosphogluconolactone 6-phosphogluconate ribulose 5-phosphate ribulose ribose Sedoheptulose-7-P– SG7 ribose 5-phosphate xylulose 5-phosphate glyceraldehyde 3-phosphate sedoheptulose 7-phosphate xylitol fructose 6-phosphate erythrose 4-phosphate xylulose 5-phosphate xylulose • 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. fructose 6-phosphate glyceraldehyde 3-phosphate

21. Nucleotide Levels During Hypoxia Nucleotides/ Nucleosides Adenosine – SG7 Hypoxic Conditions Oxygen-rich Conditions DNA Synthesis RNA Synthesis (Cell growth/division and increased transcriptional activity) Nitrogen source Carbon source High energy phosphate bonds Less cellular growth and DNA replication 2’deoxyguanosine – SG7 • 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.

22. Purines and Pyrimidines Show Equivalent Profiles During Hypoxia Adenine – SG7 Purines Adenosine – SG7 Guanine – SG7 Guanosine – SG7 2’deoxyguanosine – SG7 Pyrimidines Thymine – SG7 Uracil – SG7 • 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.

23. NAD Metabolism Nicotinamide - SG7 NAD+- SG7 Salvage Pathway NAD(P) breakdown Nicotinic Acid Nicotinamide NADP+ - SG7 Nicotinateribonucleoside*- SG7 Nicotinamide Riboside Nicotinic Acid Mononucleotide Nicotinamide Mononucleotide Nicotinic Acid Dinucleotide NAD NADP • NAD+ starvation is a cidal event in tubercle bacilli • NAD+ production is tightly regulated • The balance of NAD levels in M. tbis critical for survival in granulomas • Depletion of adenine may drive lower amounds of NAD+ and NADP+

24. Hypoxia and Fatty Acid Metabolism Palmitate (16:0) – SG7 Lipid Metabolism Synthesis of high mol. wt. species (triglycerides) to increase rigidity of granuloma cell wall Catabolism or rearrangement of cell wall/plasma membrane and degradation of components for energy Tuberculostearate – SG7 • 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.

25. Hypoxia and Fatty Acid Metabolism Palmitate (16:0) – SG7 Margarate (17:0) – SG7 Hexacosanoate (26:0) – SG7 Stearate (18:0) – SG7 Tuberculostearate – SG7 • 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.

26. The Methylcitrate cycle 2-methylcitrate – SG7 Margarate (17:0) – SG7 Pelargonate(9:0) – SG7 methylcitrate propionylCoA oxaloacetate cis-aconitate malate glyoxylate methyl-isocitrate acetyl-CoA fumarate pyruvate succinate • 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.

27. Trehalose – SG7 A possible source of glucose: Trehalose Cell Wall/Lipid Reorganization Glucose – SG7 Free Trehalose (α,α linked glucose) Glucose Glycolysis • 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.

28. Mycothione Levels During Hypoxia Mycothione (MSSM) – SG7 • 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.

29. Major Components for Sauton’s Media Citrate – SG7 Glycerol – SG7 Asparagine – SG7 Phosphate – SG7 • 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.

30. Media Analysis Trehalose – SG7 Tyrosine – SG7 Valine – SG7 • 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.

31. Conclusion & Path Forward