Metabolomic analysis of Trypanosoma cruzi parasite by 1H-NMR subjected to variations in heme availability: experimental setup

CECILIA B. DI CAPUA; LUCAS PAGURA; EVELYN TEVERE; JULIA A. CRICCO

Mar del Plata

Congreso; XXXI Reunión Anual de la Sociedad Argentina de Protozoología; 2019

Sociedad Argentina de Protozoología

Trypanosoma cruzi, the causative agent of Chagas disease, displays auxotrophy for heme cofactor being essential to acquire it from the hosts. According to our results, the parasite takes heme in the replicative stages, where TcHTE protein plays an essential role. It is well accepted that heme is imported to supply the cofactor for several hemeproteins, like mitochondrial cytochromes, however there is no evidence about which metabolic routes are modulated by heme. We designed and performed a non-targeted metabolomic assay as the key approach to answer that fundamental question. We described the experimental setup and preliminary results of the first metabolomics study by 1H-NMR spectroscopy reported in T. cruzi. Specimen preparation is a crucial step for metabolomics since rapid changes in metabolite levels may occur in response to external perturbations. Several protocols evaluating metabolic quenching, cell disruption and number of parasites were assayed to establish the most accurate method for sample preparation. Briefly, T. cruzi DM28c parasites growing in LIT-10% FBS with 5 uM hemin were collected and resuspended in fresh medium containing 0uM, 5uM or 20uM hemin for 48h. Then, 50x106 cells were pelleted and washed twice with ice cold NaCl 0.9% solution in order to quench cellular metabolism. Intracellular metabolites were extracted with chilled 50% acetonitrile aqueous solution. Samples were then subjected to freezing-unfreezing cycles and sonication in a water bath. Acetonitrile was dried under N2 flow and samples were lyophilized. Nine replicates of each condition were submitted for non-targeted metabolomics analysis by 1H-NMR. A multivariate study was performed using principal component analysis revealing metabolic differences among samples subjected to diverse hemin supplementation in the culture medium. Supervised methods will allowed us to establish the identity of the metabolites whose content was affected by variation in heme concentration.