Advertisement

Use of 13C Stable Isotope Labelling for Pathway and Metabolic Flux Analysis in Leishmania Parasites

  • Eleanor C. Saunders
  • David P. de Souza
  • Jennifer M. Chambers
  • Milica Ng
  • James Pyke
  • Malcolm J. McConvilleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)

Abstract

This protocol describes the combined use of metabolite profiling and stable isotope labelling to define pathways of central carbon metabolism in the protozoa parasite, Leishmania mexicana. Parasite stages are cultivated in standard or completely defined media and then rapidly transferred to chemically equivalent media containing a single 13C-labelled nutrient. The incorporation of label can be followed over time or after establishment of isotopic equilibrium by harvesting parasites with rapid metabolic quenching. 13C enrichment of multiple intracellular polar and apolar (lipidic) metabolites can be quantified using gas chromatography-mass spectrometry (GC-MS), while the uptake and secretion of 13C-labelled metabolites can be measured by 13C-NMR. Analysis of the mass isotopomer distribution of key metabolites provides information on pathway structure, while analysis of labelling kinetics can be used to infer metabolic fluxes. This protocol is exemplified using L. mexicana labelled with 13C-U-glucose. The method can be used to measure perturbations in parasite metabolism induced by drug inhibition or genetic manipulation of enzyme levels and is broadly applicable to any cultured parasite stages.

Key words

Leishmania spp. Metabolomics Stable isotope Central carbon metabolism Gas chromatography Mass spectrometry 

References

  1. 1.
    Stuart K, Brun R, Croft S, Fairlamb A, Gürtler RE, McKerrow J, Reed S, Tarleton R (2008) Kinetoplastids: related protozoan pathogens, different diseases. J Clin Invest 118:1301–1310CrossRefPubMedCentralPubMedGoogle Scholar
  2. 2.
    Creek DJ, Anderson J, McConville MJ, Barrett MP (2012) Metabolomic analysis of trypanosomatid protozoa. Mol Biochem Parasitol 181:73–84CrossRefPubMedGoogle Scholar
  3. 3.
    Saunders EC, DE Souza DP, Naderer T, Sernee MF, Ralton JE, Doyle MA, Macrae JI, Chambers JL, Heng J et al (2010) Central carbon metabolism of Leishmania parasites. Parasitology 137:1303–1313CrossRefPubMedGoogle Scholar
  4. 4.
    T'kindt R, Scheltema RA, Jankevics A, Brunker K, Rijal S, Dujardin J-C, Breitling R, Watson DG, Coombs GH, Decuypere S (2010) Metabolomics to unveil and understand phenotypic diversity between pathogen populations. PLoS Negl Trop Dis 4:e904CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Murray HW, Berman JD, Davies CR, Saravia NG (2005) Advances in leishmaniasis. Lancet 366:1561–1577CrossRefPubMedGoogle Scholar
  6. 6.
    Bern C, Maguire JH, Alvar J (2008) Complexities of assessing the disease burden attributable to leishmaniasis. PLoS Negl Trop Dis 2:e313CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Croft SL, Sundar S, Fairlamb AH (2006) Drug resistance in leishmaniasis. Clin Microbiol Rev 19:111–126CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, Sisk E, Rajandream MA, Adlem E et al (2005) The genome of the kinetoplastid parasite, Leishmania major. Science 309:436–442CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Doyle MA, MacRae JI, De Souza DP, Saunders EC, McConville MJ, Likić VA (2009) LeishCyc: a biochemical pathways database for Leishmania major. BMC Syst Biol 3:57CrossRefPubMedCentralPubMedGoogle Scholar
  10. 10.
    Opperdoes FR, Szikora J-P (2006) In silico prediction of the glycosomal enzymes of Leishmania major and trypanosomes. Mol Biochem Parasitol 147:193–206CrossRefPubMedGoogle Scholar
  11. 11.
    Chavali AK, Whittemore JD, Eddy JA, Williams KT, Papin JA (2008) Systems analysis of metabolism in the pathogenic trypanosomatid Leishmania major. Mol Syst Biol 4:177CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, Harris D, Her Y, Herzyk P et al (2011) Chromosome and gene copy number variation allow major structural change between species and strains of Leishmania. Genome Res 21:2129–2142CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Cohen-Freue G, Holzer TR, Forney JD, McMaster WR (2007) Global gene expression in Leishmania. Int J Parasitol 37:1077–1086CrossRefPubMedGoogle Scholar
  14. 14.
    Kramer S (2012) Developmental regulation of gene expression in the absence of transcriptional control: the case of kinetoplastids. Mol Biochem Parasitol 181:61–72CrossRefPubMedGoogle Scholar
  15. 15.
    McConville MJ, Naderer T (2011) Metabolic pathways required for the intracellular survival of Leishmania. Annu Rev Microbiol 65:543–561CrossRefPubMedGoogle Scholar
  16. 16.
    Eisenreich W, Slaghuis J, Laupitz R, Bussemer J, Stritzker J, Schwarz C, Schwarz R, Dandekar T, Goebel W, Bacher A (2006) 13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA. Proc Natl Acad Sci U S A 103:2040–2045CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Eisenreich W, Dandekar T, Heesemann J, Goebel W (2010) Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat Rev Microbiol 8:401–412CrossRefPubMedGoogle Scholar
  18. 18.
    Eylert E, Schär J, Mertins S, Stoll R, Bacher A, Goebel W, Eisenreich W (2008) Carbon metabolism of Listeria monocytogenes growing inside macrophages. Mol Microbiol 69:1008–1017CrossRefPubMedGoogle Scholar
  19. 19.
    Saunders EC, Ng WW, Chamber JM, Ng M, Naderer T, Kroemer JO, Likic VA, McConville MJ (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 286:27706–27717CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    De Souza DP, Saunders EC, McConville MJ, Likić VA (2006) Progressive peak clustering in GC-MS metabolomic experiments applied to Leishmania parasites. Bioinformatics 22:1391–1396CrossRefPubMedGoogle Scholar
  21. 21.
    Zamboni N, Fendt SM, Ruhl M, Sauer U (2009) 13C-based metabolic flux analysis. Nat Protoc 4:878–892CrossRefPubMedGoogle Scholar
  22. 22.
    Bartek T, Blombach B, Lang S, Eikmanns BJ, Wiechert W, Oldiges M, Nöh K, Noack S (2011) Comparative 13C metabolic flux analysis of pyruvate dehydrogenase complex-deficient, L-valine-producing Corynebacterium glutamicum. Appl Environ Microbiol 77:6644–6652CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Tang YJ, Martin HG, Myers S, Rodriguez S, Baidoo EEK, Keasling JD (2009) Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling. Mass Spectrom Rev 28:362–375CrossRefPubMedGoogle Scholar
  24. 24.
    Quek L-E, Wittmann C, Nielsen LK, Krömer JO (2009) OpenFLUX: efficient modelling software for 13C-based metabolic flux analysis. Microb Cell Fact 8:25CrossRefPubMedCentralPubMedGoogle Scholar
  25. 25.
    Rantanen A, Rousu J, Jouhten P, Zamboni N, Maaheimo H, Ukkonen E (2008) An analytic and systematic framework for estimating metabolic flux ratios from 13C tracer experiments. BMC Bioinformatics 9:266CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Yuan J, Bennett BD, Rabinowitz JD (2008) Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nat Protoc 3:1328–1340CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Clasquin MF, Melamud E, Singer A, Gooding JR, Xu X, Dong A, Cui H, Campagna SR, Savchenko A et al (2011) Riboneogenesis in yeast. Cell 145:969–980CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Lemons JMS, Feng X-J, Bennett BD, Legesse-Miller A, Johnson EL, Raitman I, Pollina EA, Rabitz HA, Rabinowitz JD, Coller HA (2010) Quiescent fibroblasts exhibit high metabolic activity. PLoS Biol 8:e1000514CrossRefPubMedCentralPubMedGoogle Scholar
  29. 29.
    Merlen T, Sereno D, Brajon N, Rostand F, Lemesre JL (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 60:41–50PubMedGoogle Scholar
  30. 30.
    Jennings ME, Matthews DE (2005) Determination of complex isotopomer patterns in isotopically labeled compounds by mass spectrometry. Anal Chem 77:6435–6444CrossRefPubMedCentralPubMedGoogle Scholar
  31. 31.
    van Winden WA, Wittmann C, Heinzle E, Heijnen JJ (2002) Correcting mass isotopomer distributions for naturally occurring isotopes. Biotechnol Bioeng 80:477–479CrossRefPubMedGoogle Scholar
  32. 32.
    Lee WN, Byerley LO, Bergner EA, Edmond J (1991) Mass isotopomer analysis: theoretical and practical considerations. Biol Mass Spectrom 20:451–458CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Eleanor C. Saunders
    • 1
  • David P. de Souza
    • 2
  • Jennifer M. Chambers
    • 1
  • Milica Ng
    • 1
  • James Pyke
    • 2
  • Malcolm J. McConville
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and BiotechnologyUniversity of MelbourneParkvilleAustralia
  2. 2.Metabolomics Australia, Bio21 Institute of Molecular Science and BiotechnologyThe University of MelbourneParkvilleAustralia

Personalised recommendations