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Metabolomic Analysis of Toxoplasma gondii Tachyzoites

  • Elizabeth F. B. King
  • Simon A. Cobbold
  • Alessandro D. Uboldi
  • Christopher J. Tonkin
  • Malcolm J. McConvilleEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2071)

Abstract

This protocol describes the use of 13C-stable isotope labeling, combined with metabolite profiling, to investigate the metabolism of the tachyzoite stage of the protozoan parasite Toxoplasma gondii. T. gondii tachyzoites can infect any nucleated cell in their vertebrate (including human) hosts, and utilize a range of carbon sources that freely permeate across the limiting membrane of the specialized vacuole within which they proliferate. Methods for cultivating tachyzoites in human foreskin fibroblasts and metabolically labeling intracellular and naturally egressed tachyzoites with a range of 13C-labeled carbon sources are described. Parasites are harvested and purified from host metabolites, with rapid metabolic quenching and 13C-enrichment in intracellular polar metabolites quantified by gas chromatography–mass spectrometry (GC-MS) and liquid chromatography–mass spectrometry (LC-MS). The mass isotopomer distribution of key metabolites is determined using DExSI software. This 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 other cultured or intracellular parasite stages.

Key words

Metabolomics 13C-flux analysis Intracellular pathogen Toxoplasmosis Mass spectrometry 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Elizabeth F. B. King
    • 1
  • Simon A. Cobbold
    • 1
  • Alessandro D. Uboldi
    • 2
    • 3
  • Christopher J. Tonkin
    • 4
  • Malcolm J. McConville
    • 1
    Email author
  1. 1.Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and BiotechnologyThe University of MelbourneParkvilleAustralia
  2. 2.Division of Infection and ImmunityThe Walter and Eliza Hall Institute of Medical ResearchMelbourneAustralia
  3. 3.Department of Medical BiologyThe University of MelbourneParkvilleAustralia
  4. 4.Division of Infectious Disease and Immune DefenseThe Walter and Eliza Hall Institute of Medical ResearchMelbourneAustralia

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