Caracterização e avaliação do papel da degradação de hisitdina na bioenergética de Trypanosoma cruzi
BARISON, M. J. Characterization and evaluation of the role of histidine degradation in Trypanosoma cruzi bioenergetics. 2015. 154 p. Ph. D. Thesis (Parasitology) -Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo 2015. Trypanosoma cruzi is able to use amino acids as an eficient energy source. Histidine, an abundant amino acid in the insect vector, is an essential metabolite for T. cruzi, so it must be incorporated from the extracelular medium. In the present work we show
... ent work we show that T. cruzi epimastigotes incorporate His through a single and saturable transport system. His uptake system is highly specific and is dependent on intracellular ATP content. Once in the cytoplasm, His can be degraded to glutamate through a metabolic pathway not described in other trypanosomatids. The first enzyme, histidine ammonia-lyase (TcHAL) catalizes the nonoxidative deamination of His to urocanate, which in turn is converted into the intermediate 4imidazolone-5-propionate by an urocanate hydratase. Afterwards, through the action of two additional enzymes (imidazolone propionase and formimino glutamase) glutamate is produced. The produced glutamate can be deaminated to α-ketoglutarate, a Tricarboxylic Acids Cycle (TCA) intermediate. In the present work, the first two enzymes of the pathway, TcHAL and TcUH were expressed and purified in heterologous system and their kinetic and biochemical parameters were determined. We also observed that parasites incubated with [ 14 C(U)]-His produce radiolabeled CO 2 , demonstrating that the complete oxidation pathway of His is operative in T. cruzi. In addition, the characterization of His as an energy source for epimastigotes was performed by measuring parameters such as ATP production, mitochondrial inner membrane potential (ΔΨ m ) and oxygen consumption. We also observed that parasites submitted to nutritional stress and then recovered with His, had their ΔΨ m restored, promoted oxygen consumption and ATP synthesis by oxidative phosphorylation. When His metabolism of epimastigotes in exponential and stationary phase of growth were compared we observed that His is degraded more actively in exponential phase, according to the proposed role of His in T. cruzi bioenergetic. In contrast, His uptake was higher in stationary phase, and conhsequently intracellular levels of His and its first degradation intermediate urocanate, were also increased, suggesting a probable accumulation of these metabolites in that phase of growth. Furthermore, we observed that His can stimulate metacyclogenesis, in comparable levels to that obtained in standard differentiation media, TAU-3AAG. A metabolomic approach showed that His can be used by the parasite as energy source, particularly, in the early stages of differentiation. Our results show the relevance of this amino acid in T. cruzi bioenergetics, indicating the important role of His for the parasite, more speciffically in the insect vector stages.