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

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Parasite Genomics Protocols

Part of the book series: Methods in Molecular Biology ((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.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, University of Melbourne, 30 Flemington Rd, Parkville, VIC, 3010, Australia

    Eleanor C. Saunders, Jennifer M. Chambers, Milica Ng & Malcolm J. McConville

  2. Metabolomics Australia, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, VIC, Australia

    David P. de Souza & James Pyke

Authors
  1. Eleanor C. Saunders
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  2. David P. de Souza
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  3. Jennifer M. Chambers
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  4. Milica Ng
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  5. James Pyke
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  6. Malcolm J. McConville
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Corresponding author

Correspondence to Malcolm J. McConville .

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Editors and Affiliations

  1. University of Western Australia, Nedlands, West Australia, Australia

    Christopher Peacock

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Saunders, E.C., de Souza, D.P., Chambers, J.M., Ng, M., Pyke, J., McConville, M.J. (2015). Use of 13C Stable Isotope Labelling for Pathway and Metabolic Flux Analysis in Leishmania Parasites. In: Peacock, C. (eds) Parasite Genomics Protocols. Methods in Molecular Biology, vol 1201. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1438-8_18

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  • DOI: https://doi.org/10.1007/978-1-4939-1438-8_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1437-1

  • Online ISBN: 978-1-4939-1438-8

  • eBook Packages: Springer Protocols

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