Heme is an essential cofactor for many biological processes in aerobic organisms, which can synthesize it de novo through a conserved pathway. Trypanosoma cruzi, the etiological agent of Chagas disease, as well as other trypanosomatids relevant to human health, are heme auxotrophs, meaning they must import it from their mammalian hosts or insect vectors. However, how these species import and regulate heme levels is not fully defined yet. It is known that the membrane protein TcHTE is involved in T. cruzi heme transport, although its specific role remains unclear. In the present work, we studied endogenous TcHTE in the different life-cycle stages of the parasite to gain insight in its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote), in which heme transport activity was previously validated. We also showed that in epimastigotes, TcHTE protein and mRNA levels decrease in response to increments in heme concentration, confirming it as a member of the Heme Response Gene family. Finally, we demonstrated that T. cruzi epimastigotes can sense intracellular heme by an unknown mechanism and regulate heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity by adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of a critical pathway for T. cruzi biology allowing the identification of novel and essential proteins.

Heme is an essential cofactor for many biological processes in aerobic organisms. Unlike most organisms, which can synthesize it de novo through a conserved pathway, the etiological agent of Chagas disease, Trypanosoma cruzi, as well as other trypanosomatids relevant for human health, are heme auxotrophs; thereby they must import it from the hosts. TcHTE protein is involved in T. cruzi heme transport, although its specific role remains elusive. In the present work we studied endogenous TcHTE in the different life cycle stages of the parasite in order to gain insight in its function in heme transport and homeostasis. We have confirmed that TcHTE is predominantly detected in replicative stages (epimastigote and amastigote). We have also demonstrated that T. cruzi epimastigotes can sense intracellular heme content by an unknown mechanism and regulates heme transport to adapt to changing conditions. Based on these results, we propose a model in which T. cruzi senses intracellular heme and regulates heme transport activity adjusting the expression of TcHTE. The elucidation and characterization of heme transport and homeostasis will contribute to a better understanding of T. cruzi biology as well as other trypanosomatids, pointing out this pathway as a novel drug target for therapeutics.

Trypanosoma cruzi, a heme auxotrophic parasite, can control intracellular heme content by modulating TcHRG expression when a free heme source is added to axenic culture. Herein, we explore the role of TcHRG protein in regulating the uptake of heme derived from hemoglobin in epimastigotes. It was found that the parasite endogenous TcHRG (protein and mRNA) responds similarly to bound (hemoglobin) and free (hemin) heme. Additionally, the overexpression of TcHRG leads to an increase in intracellular heme content. The localization of TcHRG is also not affected in parasites supplemented with hemoglobin as the sole heme source. Endocytic null epimastigotes do not show a significant difference in growth profile, intracellular heme content and TcHRG protein accumulation compared to WT when feeding with hemoglobin or hemin as a source of heme. These results suggest that the uptake of hemoglobin-derived heme likely occurs through extracellular proteolysis of hemoglobin via the flagellar pocket, and this process is governed by TcHRG. In sum,T. cruziepimastigotes controls heme homeostasis by modulating TcHRG expression independently of the source of available heme.