Authors

Discussion

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and is an important problem in public health in the Americas. This parasite uses amino acids for a variety of biological functions beyond the obvious one of being components of proteins. Our group is interested in describing how this parasite can metabolize branched chain amino acids (BCAA – Leu, Ile or Val) and their metabolic intermediates during the development of the parasite life cycle. In T. cruzi genome there are putative sequences annotated as encoding enzymes associated with BCAAs catabolism, including the enoyl-CoA hydratase. The enoyl-CoA hydratase (ECH) or crotonase is in the crossroads of BCAA oxidation and the β-oxidation pathways, since it participates of both by producing acetyl-CoA. In the present work, the coding sequence of ECH of T. cruzi (TcECH) was cloned, expressed and purified from Escherichia coli. We determined for the recombinant enzyme a K_M of 179.8 ± 31.2 μM and a V_max of 0.034 μM/min/µg using crotonoyl-CoA as the substrate. We produced specific polyclonal anti-TcECH antibodies and used it to determine its mitochondrial localization in epimastigote forms of the parasite. To assess the participation of this enzyme in the T. cruzi biology, we generated knock out mutants for both copies of tcech by using CRISPR/Cas9 technology in the CL Brener strain. After confirming TcECH gene ablation and the consequent deficiency in crotonase activity in the knock out lineages (Dtcech) we analyzed the proliferation rates of Dtcech epimastigotes which were not different from the controls (parasites constitutively expressing Cas9). We assayed the ability of knocked out parasites to survive to nutritional stress by incubating them in PBS supplemented or not (control) with Val, Ile or Leu for 96 and 120 hours. Parasites maintained in PBS supplemented with Leu recovered from starvation as well as controls. However, those supplemented with Val or Ile did not recover their proliferation rates indicating a deleterious effect of these metabolites in the absence of the enzyme. These results suggest the accumulation of a toxic metabolic intermediate under our experimental conditions. Finally, we show that knock out parasites differentiate into metacyclic tripomastigotes and infect mammalian cells with the same efficiency than controls. However, the complete deletion of TcECH produced a decrease in the trypomastigotes bursting. Altogether, these data indicate that, TcECH could be a potential new target for anti-T. cruzi drugs. In addition, our work will contribute to better understand the role of the BCAA oxidation for ATP production, and thus, for the biology of T. cruzi.