Biochemical studies on enzymes involved in the intermediary metabolism of Leishmania parasites and trypanosomes. From sequence to function.

NOWICKI CRISTINA

Mar del Plata

Congreso; IX Congreso Argentino de Protozoología y Enfermedades Parasitarias; 2011

Conferencia dictada en el IX Congreso Argentino de Protozoología y Enfermedades Parasitarias: Biochemical studies on enzymes involved in the intermediary metabolism of Leishmania parasites and trypanosomes. From sequence to function. Nowicki Cristina. Fac. de Farmacia y Bioquímica, UBA-CONICET, Junín 956, Bs As, Argentina Trypanosomes and Leishmania have adapted to parasitism by scavenging the nutrients available in the different niches they colonize during their life cycles. Amino acids represent the main energy and carbon source for the insect stages of trypanosomatids since usually little glucose is available in the midgut of their specific hematophagous vectors, the milieu where the insect stages of these pathogens thrive. Particularly, the insect stages of Leishmania sp appear to have adapted to utilize disaccharides and various sugars present in the nectar upon which the sandflies feed. When each of these parasites infects the mammals, they colonize very different microenvironments. For instance, the bloodstream forms of Trypanosoma brucei actively grow in the mammalian host´s blood, which is a naturally rich glucose medium. This nutrient is very actively consumed through the glycolytic pathway that is nearly the sole catabolic route functional in T. brucei bloodstream trypomastigotes, at this stage the mitochondrion becomes metabolically almost inactive. Hence, dramatic variations in the energy metabolism occur along the life cycle of T. brucei [Bringaud et al., 2006]. However, unlike african trypanosomes, the mammalian stages of T. cruzi and Leishmania spp colonize intracellular environments where glucose is normally scarce. Under these circumstances, the mammalian stages Leishmania and T. cruzi are expected to exhibit more extensive metabolic machinery and seem to depend on non-glucose carbon sources, preferring in vivo amino acids for energy production [Naderer et al., 2006, Silber et al., 2009]. Although the fully sequenced genomes are a powerful tool for the predictions in silico of metabolic maps, the substrates specificities assigned to the putative enzymes as well as their subcellular localization need to be experimentally validated. On the other hand, among the major limitations to extend the knowledge of the energy metabolism is the lower availability of the mammalian stages of T. cruzi and Leishmania parasites to examine the essentiality for parasite surviving. We have embarked in the comparative biochemical characterization of putative enzymes related with amino acid metabolism and oxidoreductases linked to NAD and NADP. The latter could be play important roles in maintaining the intracellular redox balance and providing the reducing equivalents required for biosynthetic processes, defence against oxidative stress, etc. Our results showed that TriTryps express a varied battery of aminotransferases, when functionally characterized most of these aminotransferases exhibit notably broader substrate specificities than their mammalian counterparts, and are developmentally regulated at the protein level during the life cycles of these parasites. Presumably due to the glucose scarcity in the intracellular environments where the mammalian forms of T. cruzi and Leishmania parasites thrive, the intermediates to be derived into the oxidative branch of the pentose phosphate pathway are also limited, and therefore, alternative sources of NADPH are required. Glutamate dehydrogenases, malic enzymes and isocitrate dehydrogenases could play this pivotal role. Our results showed that T. cruzi exhibits functional cytosolic and mitochondrial isoforms of malic enzymes and isocitrate dehydrogenase, being the expression of these enzymes developmentally regulated along the life cycle of this pathogen. Moreover, the presence of mitochondrial isoform of isocitrate dehydrogenase specifically linked to NADP suggest that as it has been reported for T. brucei, also in T. cruzi the Krebs cycle could correspond to a non-cyclic pathway, being the conversion of pyruvate into citrate via acetyl-CoA in addition to the conversion of 2-oxoglutarate into succinate the two main branches of this route.