Authors

Discussion

Trypanosoma cruzi is the etiologic agent of Chagas disease, which is transmitted by triatomine insects. As the life cycle of T. cruzi needs an invertebrate vector and a vertebrate host to completion, this parasite is highly adapted to different environments. For example, when glucose is limited in the insect vector’s gut, T. cruzi can degrade amino acids producing ammonia. Histidine (His) is present in high concentrations in the insect’s gut and the parasite can use it to generate ATP through its conversion in glutamate. The His degradation pathway consists of four enzymatic steps. The coding sequences for the first two enzymes and putative coding sequences for the last two enzymes are present in the T. cruzi’s genome. However, they are absent in Trypanosoma brucei what makes this parasite a useful model to investigate the biological role of this pathway. Aiming to generate T. brucei cell lines expressing the His degradation pathway from T. cruzi, we transfected T. brucei procyclic form with two plasmids that allow constitutive expression of histidine ammonia-lyase (TcHAL) and urocanate hydratase (TcUH). We confirmed the presence of the enzymes’ coding sequences by polymerase chain reaction (PCR), the expression of the enzymes by Western blotting and that the enzymes were expressed in their active forms by enzymatic activity assays. Then, we evaluated parasite proliferation and calculated the doubling time for wild type and transfected cell lines. We noticed that the simultaneous expression of TcHAL and TcUH in procyclic T. brucei affects the parasite proliferation rate while their individual expression does not affect parasite proliferation. Also, severe nutritional stress assay showed that parasites expressing TcHAL and TcUH simultaneously could not use His to maintain cell viability and that the presence of urocanate, the product of TcHAL, drastically reduces cell viability when compared to the negative control. Analysis of the complete His degradation pathway in T. brucei can contribute to elucidate which evolutionary advantages it brings to T. cruzi´s life cycle and provide valuable insights to understanding the parasite metabolism.