Análise e identificação de produtos do catabolismo de heme nas formas epimastigotas de Trypanosoma cruzi

Abstract

Trypanosoma cruzi, the causal agent of Chagas disease, has a complex life cycle and they must cope with diverse environmental conditions and depends on hosts for its nutritional needs. One of the nutritional characteristic is that they need a heme compound (Fe-protoporphyrin IX) as a growth factor. The mechanisms involved in these processes are crucial for their survival mainly because of trypanosomatids lack of the complete heme biosynthetic pathway and the cytotoxic activity of free heme. Following the fate of this porphyrin in the parasite we studied this missing pathway in T. cruzi. Here, we show that epimastigotes cultivated with heme yielded the compounds, α-meso hydroxyheme, verdoheme and biliverdin (as determined by HPLC with diode array detector). Furthermore, we observed ion species of m/z 583.4 and m/z 619.3 from epimastigotes extracts detected by direct infusion on LQT Orbitrap platform. A tipical biliverdin and verdoheme doughter-ion species were generated by m/z 583.4 and m/z 619.3 fragmentations, respectively. We also observed an ion species at m/z 1397.4 and m/z 1135. The subsequent fragmentation of this species produced a daughter-ions whose one with the same molecular mass as heme (m/z 616.4). This species, in turn, generated daughter species identical to an authentic heme, confirming that these intermediates were modified heme products. Based on these findings, we propose that heme catabolism in T. cruzi involves a additions of Bis(glutathionyl)spermine, a low molecular mass thiols occurring in trypanosomatids, to heme (m/z 1397.4), followed by removal of the glutamic residues (m/z 1135). Although the biochemical and physiological significance of the addition of thiol residues to heme molecule is underexplored, some works, already demonstrated their ability to bind heme and also this modified heme may resulting in the effective prevention of membrane damage and cytotoxicity by the heme accumulation. Taken together, these results offer new insights into heme metabolism in T. cruzi, revealing potential future therapeutic targets. We propose an active heme catabolism pathway that includes a trypanotione derivate additions and cleavage of the heme porphyring ring to biliverdin.