Fatty acid oxidation participates in resistance to nutrient-depleted environments in the insect stages of Trypanosoma cruzi.

Trypanosoma cruzi, the parasite causing Chagas disease, is a digenetic flagellated protist that infects mammals (including humans) and reduviid insect vectors. Therefore, T. cruzi must colonize different niches in order to complete its life cycle in both hosts. This fact determines the need of adaptations to face challenging environmental cues. The primary environmental challenge, particularly in the insect stages, is poor nutrient availability. In this regard, it is well known that T. cruzi has a flexible metabolism able to rapidly switch from carbohydrates (mainly glucose) to amino acids (mostly proline) consumption. Also established has been the capability of T. cruzi to use glucose and amino acids to support the differentiation process occurring in the insect, from replicative non-infective epimastigotes to non-replicative infective metacyclic trypomastigotes. However, little is known about the possibilities of using externally available and internally stored fatty acids as resources to survive in nutrient-poor environments, and to sustain metacyclogenesis. In this study, we revisit the metabolic fate of fatty acid breakdown in T. cruzi. Herein, we show that during parasite proliferation, the glucose concentration in the medium can regulate the fatty acid metabolism. At the stationary phase, the parasites fully oxidize fatty acids. [U-14C]-palmitate can be taken up from the medium, leading to CO2 production. Additionally, we show that electrons are fed directly to oxidative phosphorylation, and acetyl-CoA is supplied to the tricarboxylic acid (TCA) cycle, which can be used to feed anabolic pathways such as the de novo biosynthesis of fatty acids. Finally, we show as well that the inhibition of fatty acids mobilization into the mitochondrion diminishes the survival to severe starvation, and impairs metacyclogenesis.

The Uptake and Metabolism of Amino Acids, and Their Unique Role in the Biology of Pathogenic Trypanosomatids.

Trypanosoma brucei, as well as Trypanosoma cruzi and more than 20 species of the genus Leishmania, form a group of flagellated protists that threaten human health. These organisms are transmitted by insects that, together with mammals, are their natural hosts. This implies that during their life cycles each of them faces environments with different physical, chemical, biochemical, and biological characteristics. In this work we review how amino acids are obtained from such environments, how they are metabolized, and how they and some of their intermediate metabolites are used as a survival toolbox to cope with the different conditions in which these parasites should establish the infections in the insects and mammalian hosts.

Deletion of transketolase triggers a stringent metabolic response in promastigotes and loss of virulence in amastigotes of Leishmania mexicana.

Transketolase (TKT) is part of the non-oxidative branch of the pentose phosphate pathway (PPP). Here we describe the impact of removing this enzyme from the pathogenic protozoan Leishmania mexicana. Whereas the deletion had no obvious effect on cultured promastigote forms of the parasite, the Δtkt cells were not virulent in mice. Δtkt promastigotes were more susceptible to oxidative stress and various leishmanicidal drugs than wild-type, and metabolomics analysis revealed profound changes to metabolism in these cells. In addition to changes consistent with those directly related to the role of TKT in the PPP, central carbon metabolism was substantially decreased, the cells consumed significantly less glucose, flux through glycolysis diminished, and production of the main end products of metabolism was decreased. Only minor changes in RNA abundance from genes encoding enzymes in central carbon metabolism, however, were detected although fructose-1,6-bisphosphate aldolase activity was decreased two-fold in the knock-out cell line. We also showed that the dual localisation of TKT between cytosol and glycosomes is determined by the C-terminus of the enzyme and by engineering different variants of the enzyme we could alter its sub-cellular localisation. However, no effect on the overall flux of glucose was noted irrespective of whether the enzyme was found uniquely in either compartment, or in both.

Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector.

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly's midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

Crystallographic substrate binding studies of Leishmania mexicana SCP2-thiolase (type-2): unique features of oxyanion hole-1.

C: Structures of the C123A variant of the dimeric Leishmania mexicana SCP2-thiolase (type-2) (Lm-thiolase), complexed with acetyl-CoA and acetoacetyl-CoA, respectively, are reported. The catalytic site of thiolase contains two oxyanion holes, OAH1 and OAH2, which are important for catalysis. The two structures reveal for the first time the hydrogen bond interactions of the CoA-thioester oxygen atom of the substrate with the hydrogen bond donors of OAH1 of a CHH-thiolase. The amino acid sequence fingerprints ( xS, EAF, G P) of three catalytic loops identify the active site geometry of the well-studied CNH-thiolases, whereas SCP2-thiolases (type-1, type-2) are classified as CHH-thiolases, having as corresponding fingerprints xS, DCF and G P. In all thiolases, OAH2 is formed by the main chain NH groups of two catalytic loops. In the well-studied CNH-thiolases, OAH1 is formed by a water (of the Wat-Asn(NEAF) dyad) and NE2 (of the GHP-histidine). In the two described liganded Lm-thiolase structures, it is seen that in this CHH-thiolase, OAH1 is formed by NE2 of His338 (HDCF) and His388 (GHP). Analysis of the OAH1 hydrogen bond networks suggests that the GHP-histidine is doubly protonated and positively charged in these complexes, whereas the HDCF histidine is neutral and singly protonated.

Pyruvate phosphate dikinase and pyrophosphate metabolism in the glycosome of Trypanosoma cruzi epimastigotes.

Pyruvate phosphate dikinase (PPDK) was recently reported in trypanosomatids, but its metabolic function is not yet known. The present work deals with the cellular localization and the function of the Trypanosoma cruzi enzyme. First, we show by digitonin titration and cell fractionation that the enzyme was essentially present in the glycosome matrix of the epimastigote form. Second, we address the issue of the direction of the reaction inside the glycosome for one part, our bibliographic survey evidenced a quite exergonic DeltaGo' (at least -5.2 kcal/mol at neutral pH and physiologic ionic strength); for another part, no pyrophosphatase (PPase) could be detected in fractions corresponding to the glycosomes; therefore, glycosomal PPDK likely works in the direction of pyruvate production. Third, we address the issue of the origin of the glycosomal pyrophosphate (PPi): several synthetic pathways known to produce PPi are already considered to be glycosomal. This work also indicates the presence of an NADP(+)-dependent beta-oxidation of palmitoyl-CoA in the glycosome. Several pyruvate-consuming activities, in particular alanine dehydrogenase (ADH) and pyruvate carboxylase (PC), were detected in the glycosomal fraction. PPDK appears therefore as a central enzyme in the metabolism of the glycosome of T. cruzi by providing a link between glycolysis, fatty acid oxidation and biosynthetic PPi-producing pathways. Indeed, PPDK seems to replace pyrophosphatase in its classical thermodynamic role of displacing the equilibrium of PPi-producing reactions, as well as in its role of eliminating the toxic PPi.