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

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Presentation on theme: "“ “Tuberculosis Hypoxia” Max Planck Institute for Infection Biology MPIB-0202-10VSBL."— Presentation transcript:

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

2 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.

3 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?

4 Study Design 4 0Days1234567+1+2 HypoxiaReaeration Transcriptomics Lipidomics Pellet Proteomics/ Glycomics Metabolomics Pellet & Supernatant 2 Pellets & Supernatant +6 hours 2 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

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

6 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 Library Search for Biochemical ID

7 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

8 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

9 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

10  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

11 Statistical Analyses: Summary

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

13 13 Biochemical Data and Interpretation

14 M. tuberculosis and dormancy M. tuberculosis strains express a two component regulatory system (dosT/dosS) that is regulated by O 2 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 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

16 The TCA cycle and CO 2 loss pyruvate acetyl-CoA citrate cis-aconitate isocitrate succinyl-CoA succinate fumarate malate oxaloacetate glucose lactate α-ketoglutarate CO 2 The generation of CO 2 by the bacteria would acidify the environment The loss of carbon atoms would decrease the energy yield

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

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

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

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

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

22 Snow Globe: significant proteome changes Relative Counts Protein Descriptiongi NumberLocus TagDay 0Day 1Day 7Day 8 heat shock protein hspX [Mycobacterium tuberculosis H37Rv]15609168Rv2031c0.04550.461410.6198 serine protease PepA [Mycobacterium tuberculosis H37Rv]15607267Rv01250.0950.850.9751 isoniazid inductible gene protein INIB [Mycobacterium tuberculosis H37Rv]15607482Rv03410.15910.08330.60231 hypothetical protein Rv2744c [Mycobacterium tuberculosis H37Rv]57117019Rv2744c0.13420.208110.9195 succinyl-CoA synthetase subunit beta [Mycobacterium tuberculosis H37Rv]15608091Rv09510.10340.043110.931 transcription antitermination protein NusG [Mycobacterium tuberculosis H37Rv]15607779Rv06390.16670.26040.96881 dihydrolipoamide acetyltransferase [Mycobacterium tuberculosis H37Rv]15609352Rv22150.15290.094110.7176 hypothetical protein Rv0569 [Mycobacterium tuberculosis H37Rv]15607709Rv05690.06020.638610.5422 D-3-phosphoglycerate dehydrogenase [Mycobacterium tuberculosis H37Rv]57117042Rv2996c0.0290.0580.9711 hypothetical protein Rv1738 [Mycobacterium tuberculosis H37Rv]15608876Rv173800.730210.6667 hypothetical protein Rv2626c [Mycobacterium tuberculosis H37Rv]15609763Rv2626c00.220310.4576 transcriptional regulatory protein [Mycobacterium tuberculosis H37Rv]15608542Rv140400.358510.9623 malate dehydrogenase [Mycobacterium tuberculosis H37Rv]15608380Rv12400.14290.02380.92861 heat shock protein hsp (heat-stress-induced ribosome-binding protein A) [Mycobacterium tuberculosis H37Rv]15607392Rv0251c00.028610.6857 dehydrogenase [Mycobacterium tuberculosis H37Rv]15610525Rv3389c0.0769010.6923 glyceraldehyde-3-phosphate dehydrogenase [Mycobacterium tuberculosis H37Rv]15608574Rv14360.0455 0.51 hypothetical protein Rv2623 [Mycobacterium tuberculosis H37Rv]15609760Rv2623000.84621 short chain dehydrogenase [Mycobacterium tuberculosis H37Rv]15610360Rv32240.10.410.5 isocitrate dehydrogenase [Mycobacterium tuberculosis H37Rv]15607208Rv0066c0.11110.444410.8889 aspartate-semialdehyde dehydrogenase [Mycobacterium tuberculosis H37Rv]15610844Rv3708c000.71 50S ribosomal protein L18 [Mycobacterium tuberculosis H37Rv]15607860Rv072010.97060.08820.2059 50S ribosomal protein L19 [Mycobacterium tuberculosis H37Rv]15610041Rv2904c0.555610.05560.1389 Esat-6 like protein esxJ (Esat-6 like protein 2) [Mycobacterium tuberculosis H37Rv]15608178Rv1038c0.927410.33470.3992 low molecular weight antigen CFP2 (low molecular weight protein antigen 2) (CFP-2) [Mycobacterium tuberculosis H37Rv]15609513Rv2376c10.65770.26130.5315

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 pyruvate acetyl-CoA citrate cis-aconitate isocitrate succinate fumarate malate oxaloacetate glucose lactate glyoxylate

25 Aeration induces an increase in pentose phosphate pathway intermediates 6-Phosphogluconate Ribulose-5-P Xylulose-5-P Gluconate Glucose Glyceraldehyde-3-P + Sedoheptulose-7-P NADPH Fructose-6-P + Erythrose-4-P Xylulose-5-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 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 Acid Mononucleotide Nicotinic Acid Dinucleotide Nicotinamide Mononucleotide NAD Nicotinic AcidNicotinamide NAD(P) breakdown NADP Salvage Pathway Nicotinamide Riboside

28 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 Acid Mononucleotide Nicotinic Acid Dinucleotide Nicotinamide Mononucleotide NAD Nicotinic AcidNicotinamide NAD(P) breakdown NADP Salvage Pathway Nicotinamide Riboside

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 propionyl CoA levels pyruvate acetyl-CoA methylcitrate cis-aconitate methyl-isocitrate succinate fumarate malate oxaloacetate Propionyl-CoA 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

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 citrulline argininosuccinate arginine ornithine carbamoyl phosphate CO 2 + NH 4 + + ATP aspartate fumarate to Krebs Cycle Urea Cycle H2OH2O urea

33 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 OAA Aspartate

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

39

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 Summary of Biochemical Findings

45 Conclusion & Path Forward

46 46

47 The glyoxylate cycle reduces CO 2 loss pyruvate acetyl-CoA citrate cis-aconitate isocitrate succinate fumarate malate oxaloacetate glucose lactate glyoxylate


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