“ Tuberculosis Hypoxia” Max Planck Institute for Infection Biology MPIB-0202-10VSBL

1. “Tuberculosis Hypoxia” Max Planck Institute for Infection Biology MPIB-0202-10VSBL

2. 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.

3. 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?

4. Study Design Pellet Lipidomics Pellet & Supernatant Metabolomics Proteomics/ Glycomics 2 Pellets & Supernatant Pellet Transcriptomics Hypoxia Reaeration 0 1 2 3 4 5 6 7 +1 +2 Days +6 hours 2 hours 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

5. Metabolon Platform Technology Metabolyzer™ UHPLC-MS/MS (+ESI) Instrumentation Peak Detection Peak Integration Biochemical Extraction UHPLC-MS/MS (-ESI) Library Search RT, Mass, MS/MS Biochemical Analysis QA/QC GC-MS (+EI)

6. Library Search for Biochemical ID cholesterol 143,789 Database Of Standards cholesterol 3.17 min Mass spectrum Biochemical ID 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)

7. 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

8. 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

9. 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

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

11. Statistical Analyses: Summary

12. Visualization with Box/Line Plots Box and Whiskers Legend Metabolite Name, Matrix “C” = cells; “M” = media Scaled Intensity + Mean Value ___ Median Value Extreme Data Points Day Experiment Upper Quartile Snow Globe (Box Plots) Lower Quartile “Max” of distribution Metabolite Name, Matrix “Min” of distribution Scaled Intensity Day Experiment Fermentor(Line Plots)

13. Biochemical Data and Interpretation 13

14. 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

15. Oxygen status affects the glycolytic pathway glucose glucose 6-P fructose 6-P fructose 1,6-bisP Dihydroacetone phosphate glyceraldehyde-3-P 1,3-bisphosphoglycerate 3-phosphoglycerate 2-phosphoglycerate phosphoenolpyruvate pyruvate Acetyl CoA

16. The TCA cycle and CO2 loss glucose pyruvate lactate acetyl-CoA The generation of CO2 by the bacteria would acidify the environment The loss of carbon atoms would decrease the energy yield citrate oxaloacetate cis-aconitate malate isocitrate CO2 fumarate α-ketoglutarate CO2 succinyl-CoA succinate

17. Glyoxylate Cycle glucose pyruvate lactate acetyl-CoA citrate oxaloacetate cis-aconitate malate glyoxylate isocitrate acetyl-CoA fumarate succinate

18. Glyoxylate Cycle glucose pyruvate lactate acetyl-CoA citrate oxaloacetate cis-aconitate malate glyoxylate isocitrate acetyl-CoA fumarate succinate

19. Glyoxylate Cycle glucose pyruvate lactate acetyl-CoA citrate oxaloacetate cis-aconitate malate glyoxylate isocitrate acetyl-CoA fumarate succinate

20. Glyoxylate Cycle glucose pyruvate lactate acetyl-CoA citrate oxaloacetate cis-aconitate malate glyoxylate isocitrate acetyl-CoA fumarate succinate

21. Methylcitrate cycle pyruvate acetyl-CoA methylcitrate propionylCoA oxaloacetate cis-aconitate malate glyoxylate methyl-isocitrate acetyl-CoA fumarate pyruvate succinate

22. Snow Globe: significant proteome changes

23. 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.

24. TCA cycle intermediates accumulate in media glucose pyruvate lactate acetyl-CoA citrate cis-aconitate oxaloacetate glyoxylate malate isocitrate fumarate succinate

25. Aeration induces an increase in pentose phosphate pathway intermediates Glucose NADPH Gluconate 6-Phosphogluconate NADPH Ribulose-5-P Xylulose-5-P Ribulose-5-P Glyceraldehyde-3-P + Sedoheptulose-7-P Xylulose-5-P Fructose-6-P + Erythrose-4-P Glyceraldehyde-3-P + Fructose-6-P

26. Glucose utilization in bacteria Fuhrer et al., J Bacteriol. 187(5): 1581-1590

27. NAD+ synthesis tightly regulated in M. tb Salvage Pathway NAD(P) breakdown Nicotinic Acid Nicotinamide 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 • Enzymes common to the de novo and salvage pathways are hypothesized to • be good drug targets

28. NAD+ synthesis tightly regulated in M. tb Salvage Pathway NAD(P) breakdown Nicotinic Acid Nicotinamide 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 • Enzymes common to the de novo and salvage pathways are hypothesized to • be good drug targets

29. β-oxidation and hypoxia • 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

30. The methylcitrate cycle regulates propionylCoA levels pyruvate acetyl-CoA methylcitrate Propionyl-CoA cis-aconitate oxaloacetate malate methyl-isocitrate fumarate pyruvate succinate • PropionylCoA is generated during the β-oxidation of odd-chain length fatty acids • PropionylCoA is toxic at high concentrations. • The methylcitrate cycle consumes propionylCoA in order to maintain homeostasis

31. Long chain fatty acids accumulate during hypoxia • 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

32. Urea cycle: production of arginine CO2 + NH4+ + ATP carbamoyl phosphate aspartate citrulline Urea Cycle ornithine argininosuccinate urea to Krebs Cycle fumarate arginine H2O

33. Metabolite biosynthesis during hypoxia Glucose Hexose-P Glycerate-P Serine PEP Pyruvate Alanine Acetyl-CoA Aspartate OAA Malate TCA/Glyoxylate Cycle • Alanine and aspartate levels increased during hypoxia possibly from the increased • levels of their precursor metabolites

34. 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

35. 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

36. Unknown Compounds that Decrease with Aeration

37. Unknown Compounds that Increase with Aeration

38. Unknown Compounds that Increase with Aeration

39. Unknown Compounds that Increase with Aeration

40. Media Components Throughout the Time course • 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.

41. Amino Acids Accumulate in the Media: Possible Cell Death

42. Unknown Compounds Found in Media and Cells

43. Unknown Compounds Found in Media and Cells (Fermentor)

44. Conclusion & Path Forward

46. The glyoxylate cycle reduces CO2 loss glucose pyruvate lactate acetyl-CoA citrate cis-aconitate oxaloacetate glyoxylate malate isocitrate fumarate succinate