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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Alday PH, Doggett JS (2017) Drugs in development for toxoplasmosis: advances, challenges, and current status. Drug Des Devel Ther 11:273–293
Gamo F-J (2014) Antimalarial drug resistance: new treatments options for plasmodium. Drug Discov Today Technol 11:81–88
Miyamoto Y, Eckmann L (2015) Drug development against the major diarrhea-causing parasites of the small intestine, cryptosporidium and giardia. Front Microbiol 6:1208
Jang C, Chen L, Rabinowitz JD (2018) Metabolomics and isotope tracing. Cell 173:822–837
Dumas M-E (2012) Metabolome 2.0: quantitative genetics and network biology of metabolic phenotypes. Mol Biosyst 8:2494
Kloehn J, Blume M, Cobbold S et al (2016) Using metabolomics to dissect host–parasite interactions. Curr Opin Microbiol 32:59–65
MacRae JI, Sheiner L, Nahid A et al (2012) Mitochondrial metabolism of glucose and glutamine is required for intracellular growth of Toxoplasma gondii. Cell Host Microbe 12:682–692
Oppenheim RD, Creek DJ, Macrae JI et al (2014) BCKDH: the missing link in apicomplexan mitochondrial metabolism is required for full virulence of Toxoplasma gondii and plasmodium berghei. PLoS Pathog 10:e1004263
Blume M, Nitzsche R, Sternberg U et al (2015) A Toxoplasma gondii gluconeogenic enzyme contributes to robust central carbon metabolism and is essential for replication and virulence. Cell Host Microbe 18:210–220
Dagley MJ, McConville MJ, Wren J (2018) DExSI: a new tool for the rapid quantitation of 13C-labelled metabolites detected by GC-MS. 34:1957–1958
Junker BH, Klukas C, Schreiber F VANTED: a system for advanced data analysis and visualization in the context of biological networks. BMC Bioinformatics 7:109
Clasquin MF, Melamud E, Rabinowitz JD (2012) LC-MS data processing with MAVEN: a metabolomic analysis and visualization engine. Curr Protoc Bioinforma:1–23
Melamud E, Vastag L, Rabinowitz JD (2010) Metabolomic analysis and visualization engine for LC-MS data. Anal Chem 82:9818–9826
Smith CA, O’Maille G, Want EJ et al (2005) METLIN: a metabolite mass spectral database. Ther Drug Monit 27:747–751
Zhu Z-J, Schultz AW, Wang J et al (2013) Liquid chromatography quadrupole time-of-flight mass spectrometry characterization of metabolites guided by the METLIN database. Nat Protoc 8:451–460
Yuan J, Bennett BD, Rabinowitz JD (2008) Kinetic flux profiling for quantitation of cellular metabolic fluxes. Nat Protoc 3:1328–1340
Kell DB, Brown M, Davey HM et al (2005) Metabolic footprinting and systems biology: the medium is the message. Nat Rev Microbiol 3:557–565
Wu L, Chen S x, Jiang X g et al (2012) Separation and purification of Toxoplasma gondii tachyzoites from in vitro and in vivo culture systems. Exp Parasitol 130:91–94
Carey MA, Covelli V, Brown A et al (2018) Influential parameters for the analysis of intracellular parasite metabolomics. 3(2):e00097-18
Fang M, Ivanisevic J, Benton HP et al (2015) Thermal degradation of small molecules: a global metabolomic investigation. Anal Chem 87:10935–10941
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
King, E.F.B., Cobbold, S.A., Uboldi, A.D., Tonkin, C.J., McConville, M.J. (2020). Metabolomic Analysis of Toxoplasma gondii Tachyzoites. In: Tonkin, C. (eds) Toxoplasma gondii. Methods in Molecular Biology, vol 2071. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9857-9_22
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9857-9_22
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9856-2
Online ISBN: 978-1-4939-9857-9
eBook Packages: Springer Protocols