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

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

Mar del Plata

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

Trypanosoma cruzi, the causative agent of Chagas disease, displays auxotrophy for hemecofactor being essential to acquire it from the hosts. According to our results, the parasitetakes heme in the replicative stages, where TcHTE protein plays an essential role. It is wellaccepted that heme is imported to supply the cofactor for several hemeproteins, likemitochondrial cytochromes, however there is no evidence about which metabolic routes aremodulated by heme. We designed and performed a non-targeted metabolomic assay as thekey approach to answer that fundamental question. We described the experimental setup andpreliminary results of the first metabolomics study by 1 H-NMR spectroscopy reported in T.cruzi. Specimen preparation is a crucial step for metabolomics since rapid changes inmetabolite levels may occur in response to external perturbations. Several protocolsevaluating metabolic quenching, cell disruption and number of parasites were assayed toestablish the most accurate method for sample preparation. Briefly, T. cruzi DM28c parasitesgrowing in LIT-10% FBS with 5 uM hemin were collected and resuspended in fresh mediumcontaining 0uM, 5uM or 20uM hemin for 48h. Then, 50x10 6 cells were pelleted and washedtwice with ice cold NaCl 0.9% solution in order to quench cellular metabolism. Intracellularmetabolites were extracted with chilled 50% acetonitrile aqueous solution. Samples were thensubjected to freezing-unfreezing cycles and sonication in a water bath. Acetonitrile was driedunder N 2 flow and samples were lyophilized. Nine replicates of each condition were submittedfor non-targeted metabolomics analysis by 1 H-NMR. A multivariate study was performed usingprincipal component analysis revealing metabolic differences among samples subjected todiverse hemin supplementation in the culture medium. Supervised methods will allowed us toestablish the identity of the metabolites whose content was affected by variation in hemeconcentration.