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 Objective 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. 0Days 1234567+1+2 Metabolomics Pellet & Supernatant +6 hours 2 hours +3+4+5 HypoxiaReaeration Time Point / ConditionCell Pellet (n; Rep)Supernatant (n; Rep) T=0 (log phase; hypox start)n=4; Reps A-D T=1d (1d hypoxia)n=4; Reps A-D T=7d (7d hypoxia)n=4; Reps A-D T=8d (1d re-aeration)n=4; Reps A-D T=12d (5d re-aeration)n=4; Reps A-D n=1; Sauton's media no Tween (SnoT)

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

4. 4 Metabolyzer Software 4567891011121314 Time (min) 4.01 14.43 5.84 4.38 10.66 8.46 10.18 11.764.55 6.52 6.737.74 9.34 11.79 11.03 13.05 9.47 7.50 11.21 5.34 12.893.1713.30 8.01 Mass spectrum 3.17 min Biochemical Amount cholesterol143,789 Database Of Standards cholesterol Biochemical ID Automated Biochemical Identification

5. Quality Control Processes 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

6. Platform QC and Metabolite Summary 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 Data Quality and Precision These data are within Metabolon’s QC specifications. Number of Biochemicals

7. Study Design Samples from five time points were collected during the hypoxia and reaeration phases. The day +5 time point is an additional time point compared to previous experiments. These samples were added to give increased resolution to the response of M. tuberculosis after reaeration of the culture. 0Days 1234567+1+2 HypoxiaReaeration Metabolomics Pellet & Supernatant +6 hours 2 hours +3+4+5

8.  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 Fold of Change Welch's Two Sample t-Tests SUB PATHWAYBIOCHEMICAL NAMEPLATFORMCOMP ID SG7-0 SG7-1 SG7-0 SG7-7 SG7-0 SG7-8 SG7-0 SG7-12 SG7-7 SG7-8 SG7-7 SG7-12 Glutamate metabolism glutamateLC/MS pos572.424.151.530.980.370.24 glutamineLC/MS pos533.7815.232.631.120.170.07 gamma-aminobutyrate (GABA) LC/MS pos14167.3711.636.340.670.550.06 N-acetylglutamateLC/MS pos1572014.3313.383.581.640.270.12

9. Statistical Analyses: Summary Total number of biochemicals withp?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 withp?0.05 Biochemicals (??) p?0.05 Total number of biochemicals with 0.05<p<0.10 Biochemicals (??) 0.05<p<0.10 SG7-0 SG7-1 4031|91510|5 1|97 6 SG7-0 SG7-7 6952|17118|3241|2320|2 SG7-0 SG7-8 5440|14 9|5241|2360|6 SG7-0 SG7-12 3314|19187|11281|2780|8 SG7-7 SG7-8 4217|25195|1421|120|2 SG7-7 SG7-12 6515|50146|8111|1074|3 Statistical Comparisons CellsMedia Welch's Two Sample t-Tests

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

12. 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.

13. 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 Succinate – SG7 Fumarate – SG7 Citrate – SG7 Malate – SG7 Isocitrate – SG7

14. 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 Succinate – SG7 Isocitrate – SG7

15. 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 Adenosine – SG7

16. 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 Glutamate – SG7 Tryptophan – SG7 Glutamine – SG7

17. 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 – SG62-aminoadipate – SG6 Tryptophan – SG65-methoxytryptamine – SG6

18. 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 Succinate – SG7 Citrate– SG7 Cis-aconitate – SG7 Malate – SG7

19. 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 OAA Aspartate Aspartate – SG7 Malate – SG7  Malate levels decrease during hypoxia and may suggest shuttling of malate from the glyoxylate pathway.

20. 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 Fructose-6-P – SG7 Glucose-6-P – SG7 Pyruvate – SG7 3-phosphoglycerate – SG7

21. 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 O 2 environment, the bacteria will divide and have an increased need for nucleotides. Sedoheptulose-7-P– SG7 Gluconate – SG7 glucose 6-phosphate 6-phosphogluconate ribulose 5-phosphate ribose 5-phosphate xylulose 5-phosphate glyceraldehyde 3-phosphate sedoheptulose 7-phosphate fructose 6-phosphate erythrose 4-phosphate fructose 6-phosphate glyceraldehyde 3-phosphate xylulose 5-phosphate 6-phosphogluconolactone ribose xylulose ribulose xylitol

22. 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 source Carbon source High energy phosphate bonds Less cellular growth and DNA replication DNA Synthesis RNA Synthesis (Cell growth/division and increased transcriptional activity) Hypoxic Conditions Oxygen-rich Conditions Adenosine – SG7 2’deoxyguanosine – SG7

23. 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 – SG7Adenosine – SG7 Guanine – SG7Guanosine – SG7 2’deoxyguanosine – SG7 Thymine – SG7 Uracil – SG7 Purines Pyrimidines

24. 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+ NADP+ - SG7 Nicotinate ribonucleoside*- SG7 Nicotinamide - SG7 NAD+- SG7 Nicotinic Acid Mononucleotide Nicotinic Acid Dinucleotide Nicotinamide Mononucleotide NAD Nicotinic AcidNicotinamide NAD(P) breakdown NADP Salvage Pathway Nicotinamide Riboside

25. 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. Lipid Metabolism Catabolism or rearrangement of 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 Tuberculostearate – SG7

26. 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. Palmitate (16:0) – SG7 Margarate (17:0) – SG7 Stearate (18:0) – SG7Tuberculostearate – SG7 Hexacosanoate (26:0) – SG7

27. 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 O 2. methylcitrate cis-aconitate methyl-isocitrate succinate fumarate malate oxaloacetate glyoxylate acetyl-CoA pyruvate propionyl CoA 2-methylcitrate – SG7 Pelargonate (9:0) – SG7Margarate (17:0) – SG7

28. 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 Glucose – SG7 Cell Wall/Lipid Reorganization Free Trehalose (α,α linked glucose) Glucose Glycolysis

29. 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 O 2. Mycothione (MSSM) – SG7

30. 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 Glycerol – SG7 Asparagine – SG7 Phosphate – SG7

31. 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 Tyrosine – SG7Valine – SG7

32. 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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