Metabolomic Analysis of Toxoplasma gondii Tachyzoites

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.

Keywords: 13C-flux analysis; Intracellular pathogen; Mass spectrometry; Metabolomics; Toxoplasmosis.