Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic


Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.


Vyšlo v časopise: Fumarate Reductase Activity Maintains an Energized Membrane in Anaerobic. PLoS Pathog 7(10): e32767. doi:10.1371/journal.ppat.1002287
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002287

Souhrn

Oxygen depletion of Mycobacterium tuberculosis engages the DosR regulon that coordinates an overall down-regulation of metabolism while up-regulating specific genes involved in respiration and central metabolism. We have developed a chemostat model of M. tuberculosis where growth rate was a function of dissolved oxygen concentration to analyze metabolic adaptation to hypoxia. A drop in dissolved oxygen concentration from 50 mmHg to 0.42 mmHg led to a 2.3 fold decrease in intracellular ATP levels with an almost 70-fold increase in the ratio of NADH/NAD+. This suggests that re-oxidation of this co-factor becomes limiting in the absence of a terminal electron acceptor. Upon oxygen limitation genes involved in the reverse TCA cycle were upregulated and this upregulation was associated with a significant accumulation of succinate in the extracellular milieu. We confirmed that this succinate was produced by a reversal of the TCA cycle towards the non-oxidative direction with net CO2 incorporation by analysis of the isotopomers of secreted succinate after feeding stable isotope (13C) labeled precursors. This showed that the resulting succinate retained both carbons lost during oxidative operation of the TCA cycle. Metabolomic analyses of all glycolytic and TCA cycle intermediates from 13C-glucose fed cells under aerobic and anaerobic conditions showed a clear reversal of isotope labeling patterns accompanying the switch from normoxic to anoxic conditions. M. tuberculosis encodes three potential succinate-producing enzymes including a canonical fumarate reductase which was highly upregulated under hypoxia. Knockout of frd, however, failed to reduce succinate accumulation and gene expression studies revealed a compensatory upregulation of two homologous enzymes. These major realignments of central metabolism are consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. This fermentative process may offer unique targets for the treatment of latent tuberculosis.


Zdroje

1. CorbettELWattCJWalkerNMaherDWilliamsBG 2003 The growing burden of tuberculosis: global trends and interactions with the HIV epidemic. Arch Intern Med 163 1009 1021

2. BoshoffHIBarryCEIii 2006 Is the mycobacterial cell wall a hopeless drug target for latent tuberculosis? Drug Discovery Today: Disease Mechanisms 3 237 245

3. 2000 Targeted tuberculin testing and treatment of latent tuberculosis infection. American Thoracic Society. MMWR Recomm Rep 49 1 51

4. BerryMPGrahamCMMcNabFWXuZBlochSA 2010 An interferon-inducible neutrophil-driven blood transcriptional signature in human tuberculosis. Nature 466 973 977

5. LeeSWJangYSParkCMKangHYKohWJ 2010 The role of chest CT scanning in TB outbreak investigation. Chest 137 1057 1064

6. BarryCE3rdBoshoffHIDartoisVDickTEhrtS 2009 The spectrum of latent tuberculosis: rethinking the biology and intervention strategies. Nat Rev Microbiol 7 845 855

7. GooJMImJGDoKHYeoJSSeoJB 2000 Pulmonary tuberculoma evaluated by means of FDG PET: findings in 10 cases. Radiology 216 117 121

8. HaraTKosakaNSuzukiTKudoKNiinoH 2003 Uptake rates of 18F-fluorodeoxyglucose and 11C-choline in lung cancer and pulmonary tuberculosis: a positron emission tomography study. Chest 124 893 901

9. YangCMHsuCHLeeCMWangFC 2003 Intense uptake of [F-18]-fluoro-2 deoxy-D-glucose in active pulmonary tuberculosis. Ann Nucl Med 17 407 410

10. ParkINRyuJSShimTS 2008 Evaluation of therapeutic response of tuberculoma using F-18 FDG positron emission tomography. Clin Nucl Med 33 1 3

11. RussellDG 2007 Who puts the tubercle in tuberculosis? Nat Rev Microbiol 5 39 47

12. NathanCShilohMU 2000 Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens. Proc Natl Acad Sci U S A 97 8841 8848

13. VoskuilMISchnappingerDViscontiKCHarrellMIDolganovGM 2003 Inhibition of respiration by nitric oxide induces a Mycobacterium tuberculosis dormancy program. J Exp Med 198 705 713

14. SchnappingerDEhrtSVoskuilMILiuYManganJA 2003 Transcriptional Adaptation of Mycobacterium tuberculosis within Macrophages: Insights into the Phagosomal Environment. J Exp Med 198 693 704

15. PaigeCBishaiWR 2010 Penitentiary or penthouse condo: the tuberculous granuloma from the microbe's point of view. Cell Microbiol 12 301 309

16. ViaLELinPLRaySMCarrilloJAllenSS 2008 Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates. Infect Immun 76 2333 2340

17. ParkHDGuinnKMHarrellMILiaoRVoskuilMI 2003 Rv3133c/dosR is a transcription factor that mediates the hypoxic response of Mycobacterium tuberculosis. Mol Microbiol 48 833 843

18. ShermanDRVoskuilMSchnappingerDLiaoRHarrellMI 2001 Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin. Proc Natl Acad Sci U S A 98 7534 7539

19. ShiLSohaskeyCDKanaBDDawesSNorthRJ 2005 Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration. Proc Natl Acad Sci U S A 102 15629 15634

20. RaoSPAlonsoSRandLDickTPetheK 2008 The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 105 11945 11950

21. BoshoffHIBarryCE3rd 2005 Tuberculosis - metabolism and respiration in the absence of growth. Nat Rev Microbiol 3 70 80

22. WayneLGHayesLG 1996 An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect Immun 64 2062 2069

23. DanielJDebCDubeyVSSirakovaTDAbomoelakB 2004 Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. J Bacteriol 186 5017 5030

24. MalmSTiffertYMicklinghoffJSchultzeSJoostI 2009 The roles of the nitrate reductase NarGHJI, the nitrite reductase NirBD and the response regulator GlnR in nitrate assimilation of Mycobacterium tuberculosis. Microbiology 155 1332 1339

25. BoshoffHIMyersTGCoppBRMcNeilMRWilsonMA 2004 The transcriptional responses of Mycobacterium tuberculosis to inhibitors of metabolism: novel insights into drug mechanisms of action. J Biol Chem 279 40174 40184

26. HonakerRWLeistikowRLBartekILVoskuilMI 2009 Unique roles of DosT and DosS in DosR regulon induction and Mycobacterium tuberculosis dormancy. Infect Immun 77 3258 3263

27. RustadTRHarrellMILiaoRShermanDR 2008 The enduring hypoxic response of Mycobacterium tuberculosis. PLoS One 3 e1502

28. YuanYCraneDDSimpsonRMZhuYQHickeyMJ 1998 The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci U S A 95 9578 9583

29. VoskuilMIViscontiKCSchoolnikGK 2004 Mycobacterium tuberculosis gene expression during adaptation to stationary phase and low-oxygen dormancy. Tuberculosis (Edinb) 84 218 227

30. TianJBrykRItohMSuematsuMNathanC 2005 Variant tricarboxylic acid cycle in Mycobacterium tuberculosis: identification of alpha-ketoglutarate decarboxylase. Proc Natl Acad Sci U S A 102 10670 10675

31. de CarvalhoLPZhaoHDickinsonCEArangoNMLimaCD 2010 Activity-based metabolomic profiling of enzymatic function: identification of Rv1248c as a mycobacterial 2-hydroxy-3-oxoadipate synthase. Chem Biol 17 323 332

32. GoodwinMBBoshoffHIBarryCE3rdDowdCS 2006 Quantification of small molecule organic acids from Mycobacterium tuberculosis culture supernatant using ion exclusion liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 20 3345 3350

33. MarreroJRheeKYSchnappingerDPetheKEhrtS 2010 Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc Natl Acad Sci U S A 107 9819 9824

34. GengenbacherMRaoSPPetheKDickT 2010 Nutrient-starved, non-replicating Mycobacterium tuberculosis requires respiration, ATP synthase and isocitrate lyase for maintenance of ATP homeostasis and viability. Microbiology 156 81 87

35. BesteDJHooperTStewartGBondeBAvignone-RossaC 2007 GSMN-TB: a web-based genome-scale network model of Mycobacterium tuberculosis metabolism. Genome Biol 8 R89

36. HirschCARasminskyMDavisBDLinEC 1963 A Fumarate Reductase in Escherichia Coli Distinct from Succinate Dehydrogenase. J Biol Chem 238 3770 3774

37. CecchiniGSchroderIGunsalusRPMaklashinaE 2002 Succinate dehydrogenase and fumarate reductase from Escherichia coli. Biochim Biophys Acta 1553 140 157

38. RachmanHStrongMUlrichsTGrodeLSchuchhardtJ 2006 Unique transcriptome signature of Mycobacterium tuberculosis in pulmonary tuberculosis. Infect Immun 74 1233 1242

39. GartonNJWaddellSJSherrattALLeeSMSmithRJ 2008 Cytological and transcript analyses reveal fat and lazy persister-like bacilli in tuberculous sputum. PLoS Med 5 e75

40. GallmetzerMMeranerJBurgstallerW 2002 Succinate synthesis and excretion by Penicillium simplicissimum under aerobic and anaerobic conditions. FEMS Microbiol Lett 210 221 225

41. EngelPKramerRUndenG 1994 Transport of C4-dicarboxylates by anaerobically grown Escherichia coli. Energetics and mechanism of exchange, uptake and efflux. Eur J Biochem 222 605 614

42. ChangGGTongL 2003 Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry 42 12721 12733

43. BolognaFPAndreoCSDrincovichMF 2007 Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure. J Bacteriol 189 5937 5946

44. EnchevaVShahHNGharbiaSE 2009 Proteomic analysis of the adaptive response of Salmonella enterica serovar Typhimurium to growth under anaerobic conditions. Microbiology 155 2429 2441

45. KwonYDKwonOHLeeHSKimP 2007 The effect of NADP-dependent malic enzyme expression and anaerobic C4 metabolism in Escherichia coli compared with other anaplerotic enzymes. J Appl Microbiol 103 2340 2345

46. WuHLiZMZhouLYeQ 2007 Improved succinic acid production in the anaerobic culture of an Escherichia coli pflB ldhA double mutant as a result of enhanced anaplerotic activities in the preceding aerobic culture. Appl Environ Microbiol 73 7837 7843

47. DubiniAMusFSeibertMGrossmanARPosewitzMC 2009 Flexibility in anaerobic metabolism as revealed in a mutant of Chlamydomonas reinhardtii lacking hydrogenase activity. J Biol Chem 284 7201 7213

48. ZhangXShanmugamKTIngramLO 2010 Fermentation of glycerol to succinate by metabolically engineered strains of Escherichia coli. Appl Environ Microbiol 76 2397 2401

49. JonesHMGunsalusRP 1987 Regulation of Escherichia coli fumarate reductase (frdABCD) operon expression by respiratory electron acceptors and the fnr gene product. J Bacteriol 169 3340 3349

50. WeinbergJMVenkatachalamMARoeserNFNissimI 2000 Mitochondrial dysfunction during hypoxia/reoxygenation and its correction by anaerobic metabolism of citric acid cycle intermediates. Proc Natl Acad Sci U S A 97 2826 2831

51. Amador-NoguezDFengXJFanJRoquetNRabitzH 2010 Systems-level metabolic flux profiling elucidates a complete, bifurcated tricarboxylic acid cycle in Clostridium acetobutylicum. J Bacteriol 192 4452 4461

52. SauerULaskoDRFiauxJHochuliMGlaserR 1999 Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181 6679 6688

53. GuytonA 1991 Textbook of medical physiology Philadelphia WB Saunders

54. SassettiCMBoydDHRubinEJ 2003 Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48 77 84

55. SliningerPJPetroskiRJBothastRJLadischMROkosMR 1989 Measurement of oxygen solubility in fermentation media: a colorimetric method. Biotechnol Bioeng 33 578 583

56. ParishTStokerNG 2000 Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146 Pt 8 1969 1975

57. BuescherJMMocoSSauerUZamboniN 2010 Ultrahigh performance liquid chromatography-tandem mass spectrometry method for fast and robust quantification of anionic and aromatic metabolites. Anal Chem 82 4403 4412

58. BoshoffHIReedMBBarryCE3rdMizrahiV 2003 DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113 183 193

59. BonnetGTyagiSLibchaberAKramerFR 1999 Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc Natl Acad Sci U S A 96 6171 6176

60. MunujosPColl-CantiJGonzalez-SastreFGellaFJ 1993 Assay of succinate dehydrogenase activity by a colorimetric-continuous method using iodonitrotetrazolium chloride as electron acceptor. Anal Biochem 212 506 509

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Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

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