#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Induction of a Stringent Metabolic Response in Intracellular Stages of Leads to Increased Dependence on Mitochondrial Metabolism


Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.


Vyšlo v časopise: Induction of a Stringent Metabolic Response in Intracellular Stages of Leads to Increased Dependence on Mitochondrial Metabolism. PLoS Pathog 10(1): e32767. doi:10.1371/journal.ppat.1003888
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1003888

Souhrn

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.


Zdroje

1. BernC, MaguireJH, AlvarJ (2008) Complexities of assessing the disease burden attributable to leishmaniasis. PLoS Negl Trop Dis 2: e313.

2. MurrayHW, BermanJD, DaviesCR, SaraviaNG (2005) Advances in leishmaniasis. Lancet 366: 1561–1577.

3. KayeP, ScottP (2011) Leishmaniasis: complexity at the host-pathogen interface. Nat Rev Microbiol 9: 604–615.

4. BurchmoreRJ, BarrettMP (2001) Life in vacuoles-nutrient acquisition by Leishmania amastigotes. Int. J Parasitol 31: 1311–1320.

5. McConvilleMJ, NadererT (2011) Metabolic pathways required for the intracellular survival of Leishmania. Annu Rev Microbiol 65: 543–561.

6. NadererT, EllisMA, SerneeMF, De SouzaDP, CurtisJ, et al. (2006) Virulence of Leishmania major in macrophages and mice requires the gluconeogenic enzyme fructose-1,6-bisphosphatase. Proc Natl Acad Sci U S A 103: 5502–5507.

7. NadererT, McConvilleMJ (2008) The Leishmania-macrophage interaction: a metabolic perspective. Cell Microbiol 10: 301–308.

8. DuncanRC, SalotraP, GoyalN, AkopyantsNS, BeverleySM, NakhasiHL (2004) The application of gene expression microarray technology to kinetoplastid research. Curr Mol Med 4: 611–621.

9. Cohen-FreueG, HolzerTR, ForneyJD, McMasterWR (2007) Global gene expression in Leishmania. Int J Parasitol 37: 1077–1086.

10. PaapeD, LippunerC, SchmidM, AckermannR, Barrios-LlerenaME, et al. (2008) Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes. Mol Cell Proteomics 7: 1688–1701.

11. RosenzweigD, SmithD, OpperdoesF, SternS, OlafsonRW, ZilbersteinD (2008) Retooling Leishmania metabolism: from sand fly gut to human macrophage. FASEB J 22: 590–602.

12. LahavT, SivamD, VolpinH, RonenM, TsigankovP, et al. (2010) Multiple levels of gene regulation mediate differentiation of the intracellular pathogen Leishmania. FASEB J 25: 515–525.

13. TsigankovP, GherardiniPF, Helmer-CitterichM, ZilbersteinD (2012) What has proteomics taught us about Leishmania development? Parasitology 139: 1146–1157.

14. BringaudF, RivièreL, CoustouV (2006) Energy metabolism of trypanosomatids: adaptation to available carbon sources. Mol Biochem Parasitol 149: 1–9.

15. TielensAGM, van HellemondJJ (2009) Surprising variety in energy metabolism within Trypanosomatidae. Trends Parasitol 25: 482–490.

16. CreekDJ, AndersonJ, McConvilleMJ, BarrettMP (2012) Metabolomic analysis of trypanosomatid protozoa. Mol Biochem Parasitol 181: 73–84.

17. OpperdoesFR, CoombsGH (2007) Metabolism of Leishmania: proven and predicted. Trends Parasitol 23: 149–158.

18. SaundersEC, NgWW, ChamberJM, NgM, NadererT, et al. (2011) Isoptopomer profiling of Leishmania mexicana promastigotes reveals important roles for succinate fermentation and aspartate uptake in TCA cycle anaplerosis, glutamate synthesis and growth. J Biol Chem 286: 27706–27717.

19. SaundersEC, DE SouzaDP, NadererT, SerneeMF, RaltonJE, et al. (2010) Central carbon metabolism of Leishmania parasites. Parasitology 137: 1303–1313.

20. HartDT, CoombsGH (1982) Leishmania mexicana: energy metabolism of amastigotes and promastigotes. Exp. Parasitol 54: 397–409.

21. BurchmoreRJS, Rodriguez-ContrerasD, McBrideK, MerkelP, BarrettMP, et al. (2003) Genetic characterization of glucose transporter function in Leishmania mexicana. Proc Natl Acad Sci U S A 100: 3901–3906.

22. FengX, Rodriguez-ContrerasD, BuffaloC, BouwerHGA, KruvandE, et al. (2009) Amplification of an alternate transporter gene suppresses the avirulent phenotype of glucose transporter null mutants in Leishmania mexicana. Mol Microbiol 71: 369–381.

23. NadererT, HengJ, McConvilleMJ (2010) Evidence that intracellular stages of Leishmania major utilize amino sugars as a major carbon source. PLoS Pathog 6: e1001245.

24. NeeseRA, MisellLM, TurnerS, ChuA, KimJ, et al. (2002) Measurement in vivo of proliferation rates of slow turnover cells by 2H2O labeling of the deoxyribose moiety of DNA. Proc Natl Acad Sci U S A 99: 15345–15350.

25. Castanys-MuñozE, BrownE, CoombsGH, MottramJC (2012) Leishmania mexicana metacaspase is a negative regulator of amastigote proliferation in mammalian cells. Cell Death Dis 3: e385.

26. NadererT, VinceJE, McConvilleMJ (2004) Surface determinants of Leishmania parasites and their role in infectivity in the mammalian host. Curr Mol Med 4: 649–665.

27. GaramiA, MehlertA, IlgT (2001) Glycosylation defects and virulence phenotypes of Leishmania mexicana phosphomannomutase and dolicholphosphate-mannose synthase gene deletion mutants. Mol Cell Biol 21: 8168–8183.

28. ProudfootAT, BradberrySM, ValeJA (2006) Sodium fluoroacetate poisoning. Toxicol Rev 25: 213–219.

29. CarterNS, YatesP, ArendtCS, BoitzJM, UllmanB (2008) Purine and pyrimidine metabolism in Leishmania. Adv Exp Med Biol 625: 141–154.

30. NadererT, WeeE, McConvilleMJ (2008) Role of hexosamine biosynthesis in Leishmania growth and virulence. Mol Microbiol 69: 858–869.

31. RaineyPM, MacKenzieNE (1991) A carbon-13 nuclear magnetic resonance analysis of the products of glucose metabolism in Leishmania pifanoi amastigotes and promastigotes. Mol Biochem Parasitol 45: 307–315.

32. CloutierS, LaverdièreM, ChouM-N, BoilardN, ChowC, PapadopoulouB (2012) Translational control through eIF2alpha phosphorylation during the Leishmania differentiation process. PLoS One 7: e35085.

33. RaltonJE, NadererT, PirainoHL, BashtannykTA, CallaghanJM, McConvilleMJ (2003) Evidence that intracellular β1-2 mannan is a virulence factor in Leishmania parasites. J Biol Chem 278: 40757–40763.

34. GannavaramS, ConnellyPS, DanielsMP, DuncanR, SalotraP, NakhasiHL (2012) Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β-oxidation of fatty acids and blocks cell division in the amastigote stage. Mol Microbiol 86: 187–198.

35. MukherjeeA, RoyG, GuimondC, OuelletteM (2009) The γ-glutamylcysteine synthetase gene of Leishmania is essential and involved in response to oxidants. Mol Microbiol 74: 914–927.

36. TeodoroJS, RoloAP, PalmeiraCM (2013) The NAD ratio redox paradox: why does too much reductive power cause oxidative stress? Toxicol Mech Methods 23: 297–302.

37. MurphyMP (2009) How mitochondria produce reactive oxygen species. Biochem J 417: 1–13.

38. DeyR, MenesesC, SalotraP, KamhawiS, NakhasiHL, DuncanR (2010) Characterization of a Leishmania stage-specific mitochondrial membrane protein that enhances the activity of cytochrome c oxidase and its role in virulence. Mol Microbiol 77: 399–414.

39. UboldiAD, LuederFB, WalshP, SpurckT, McFaddenGI, et al. (2006) A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania. Int J Parasitol 36: 1499–1514.

40. KramerS (2012) Developmental regulation of gene expression in the absence of transcriptional control: The case of kinetoplastids. Mol Biochem Parasitol 181: 61–72.

41. VinceJE, TullD, LandfearS, McConvilleMJ (2011) Lysosomal degradation of Leishmania hexose and inositol transporters is regulated in a stage-, nutrient- and ubiquitin-dependent manner. Int J Parasitol 41: 791–800.

42. OrtizD, SanchezMA, PierceS, HerrmannT, KimblinN, et al. (2007) Molecular genetic analysis of purine nucleobase transport in Leishmania major. Mol Microbiol 64: 1228–1243.

43. BlumJJ (1990) Effects of culture age and hexoses on fatty acid oxidation by Leishmania major. J Protozool 37: 505–510.

44. BrauerMJ, HuttenhowerC, AiroldiEM, RosensteinR, MateseJC, et al. (2008) Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. Mol Biol Cell 19: 352–367.

45. LuC, BrauerMJ, BotsteinD (2009) Slow growth induces heat-shock resistance in normal and respiratory-deficient yeast. Mol Biol Cell 20: 891–903.

46. RittershausESC, BaekS-H, SassettiCM (2013) The normalcy of dormancy: common themes in microbial quiescence. Cell Host Microbe 13: 643–651.

47. LewisK (2010) Persister cells. Annu Rev Microbiol 64: 357–372.

48. BesteDJV, BondeB, HawkinsN, WardJL, BealeMH, et al. (2011) 13C metabolic flux analysis identifies an unusual route for pyruvate dissimilation in mycobacteria which requires isocitrate lyase and carbon dioxide fixation. PLoS Pathog 7: e1002091.

49. MarreroJ, TrujilloC, RheeKY, EhrtS (2013) Glucose phosphorylation is required for Mycobacterium tuberculosis persistence in mice. PLoS Pathog 9: e1003116.

50. BatesPA, RobertsonCD, TetleyL, CoombsGH (1992) Axenic cultivation and characterization of Leishmania mexicana amastigote-like forms. Parasitology 105(Pt 2): 193–202.

51. GlaserTA, WellsSJ, SpithillTW, PettittJM, HumphrisDC, MukkadaAJ (1990) Leishmania major and L. donovani: a method for rapid purification of amastigotes. Exp Parasitol 71: 343–345.

52. MerlenT, SerenoD, BrajonN, RostandF, LemesreJL (1999) Leishmania spp: completely defined medium without serum and macromolecules (CDM/LP) for the continuous in vitro cultivation of infective promastigote forms. Am J Trop Med Hyg 60: 41–50.

53. RotureauB, GegoA, CarmeB (2005) Trypanosomatid protozoa: a simplified DNA isolation procedure. Exp Parasitol 111: 207–209.

54. VoogtJN, AwadaM, MurphyEJ, HayesGM, BuschR, HellersteinMK (2007) Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis. Nat Protoc 2: 3058–3062.

55. WuM-Y, ChenB-G, ChangCD, HuangM-H, WuT-G, et al. (2008) A novel derivatization approach for simultaneous determination of glyoxal, methylglyoxal, and 3-deoxyglucosone in plasma by gas chromatography-mass spectrometry. J Chromatogr A 1204: 81–86.

56. ZamboniN, FendtSM, RuhlM, SauerU (2009) (13)C-based metabolic flux analysis. Nat Protoc 4: 878–892.

57. LutzNW, YahiN, FantiniJ, CozzonePJ (1996) A new method for the determination of specific 13C enrichment in phosphorylated [1-13C]glucose metabolites. 13C-coupled, 1H-decoupled 31P-NMR spectroscopy of tissue perchloric acid extracts. Eur J Biochem 238: 470–475.

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2014 Číslo 1
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#