Metabolomic Analysis of Toxoplasma gondii Tachyzoites

This protocol describes the use of C-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 ut...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 2071; p. 435
Main Authors King, Elizabeth F B, Cobbold, Simon A, Uboldi, Alessandro D, Tonkin, Christopher J, McConville, Malcolm J
Format Journal Article
LanguageEnglish
Published United States 01.01.2020
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Abstract This protocol describes the use of C-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 C-labeled carbon sources are described. Parasites are harvested and purified from host metabolites, with rapid metabolic quenching and C-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.
AbstractList This protocol describes the use of C-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 C-labeled carbon sources are described. Parasites are harvested and purified from host metabolites, with rapid metabolic quenching and C-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.
Author Cobbold, Simon A
King, Elizabeth F B
Tonkin, Christopher J
Uboldi, Alessandro D
McConville, Malcolm J
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  givenname: Elizabeth F B
  surname: King
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  organization: Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria 3010, Australia
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  givenname: Simon A
  surname: Cobbold
  fullname: Cobbold, Simon A
  organization: Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria 3010, Australia
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  givenname: Alessandro D
  surname: Uboldi
  fullname: Uboldi, Alessandro D
  organization: Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia
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  givenname: Christopher J
  surname: Tonkin
  fullname: Tonkin, Christopher J
  organization: Division of Infectious Disease and Immune Defense, The Walter and Eliza Hall Institute of Medical Research, Melbourne, VIC, Australia
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  givenname: Malcolm J
  surname: McConville
  fullname: McConville, Malcolm J
  email: malcolmm@unimelb.edu.au
  organization: Department of Biochemistry and Molecular Biology, Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria 3010, Australia. malcolmm@unimelb.edu.au
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Keywords Metabolomics
13C-flux analysis
Toxoplasmosis
Intracellular pathogen
Mass spectrometry
Language English
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Snippet This protocol describes the use of C-stable isotope labeling, combined with metabolite profiling, to investigate the metabolism of the tachyzoite stage of the...
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StartPage 435
SubjectTerms Animals
Chromatography, Liquid
Fibroblasts - parasitology
Foreskin - cytology
Gas Chromatography-Mass Spectrometry
Humans
Male
Mass Spectrometry
Metabolomics
Software
Toxoplasma - metabolism
Toxoplasma - pathogenicity
Toxoplasmosis
Title Metabolomic Analysis of Toxoplasma gondii Tachyzoites
URI https://www.ncbi.nlm.nih.gov/pubmed/31758465
Volume 2071
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