A novel heme-binding protein identified in Trypanosoma cruzi

MERLI ML; TEVERE E; DI CAPUA C; ACOSTA OVIEDO H; MEDIAVILLA MG; CRICCO JA

Rosario

Congreso; L Reunión Anual de la Sociedad Argentina de Biofísica; 2022

Sociedad Argentina de Biofísica

Trypanosoma cruzi, the etiological agent of Chagas disease, presents nutritional requirements for heme, but contains several heme-proteins playing relevant functions for its proliferation, for instance the mitochondrial respiratory complexes. This parasite regulates heme homeostasis controlling heme import activity where the function of TcHRG protein is relevant. We have demonstrated that the presence and accumulation of TcHRG, as mRNA and protein, depends on the intracellular heme concentration. TcHRG detection increases under heme starving conditions (where intracellular heme is low) and decreases with sufficient intracellular heme. However, a certain identification of a heme transporter, heme-carriers and chaperons remains elusive. To identify genes that respond to heme in a similar way as TcHRG, we conducted a global transcriptional analysis in epimastigotes of T. cruzi using hemin (as free heme) or hemoglobin as heme sources. We analyzed the differential expressed genes (DEGs) at 4 and 24 hours after refeeding heme-starved epimastigotes with 5 μM heme. We observed that changes in intracellular heme caused a wide but very mild perturbation in hundreds of genes (up and downregulating them). Instead, few genes responded rapidly and strongly to heme. One of them (C4B63_18g39) encoded for a hypothetical protein with a predicted CRAL-TRIO domain. An extensive bioinformatic search indicated that this protein presents sequence homology to S. cerevisiae Sfh5 protein, a heme-protein presenting a Sec14-fold which would play a role in organic oxidant-induced stress responses. The T. cruzi gene was cloned and expressed in E. coli as a recombinant his-tagged fusion protein. The recombinant T. cruzi CRAL-TRIO protein presented a brownish color when purified, and the UV-Vis spectrum revealed that it binds heme, suggesting a similar role to Sfh5 and/or controling T. cruzi heme homeostasis. This is the first described T. cruzi heme-protein, so far, whose expression is regulated by heme.