The procyclic form of Trypanosoma brucei is a parasitic protozoan that normally dwells in the midgut of its insect vector. In vitro, this parasite prefers D-glucose to L-proline as a carbon source, although this amino acid is the main carbon source available in its natural habitat. Here, we investigated how L-proline is metabolized in glucose-rich and glucose-depleted conditions. Analysis of the excreted end products of 13 C-enriched L-proline metabolism showed that the amino acid is converted into succinate or L-alanine depending on the presence or absence of D-glucose, respectively. The fact that the pathway of L-proline metabolism was truncated in glucose-rich conditions was confirmed by the analysis of 13 separate RNA interference-harboring or knock-out cell lines affecting different steps of this pathway. For instance, RNA interference studies revealed the loss of succinate dehydrogenase activity to be conditionally lethal only in the absence of D-glucose, confirming that in glucose-depleted conditions, L-proline needs to be converted beyond succinate. In addition, depletion of the F 0 /F 1 -ATP synthase activity by RNA interference led to cell death in glucose-depleted medium, but not in glucose-rich medium. This implies that, in the presence of D-glucose, the importance of the F 0 /F 1 -ATP synthase is diminished and ATP is produced by substrate level phosphorylation. We conclude that trypanosomes develop an elaborate adaptation of their energy production pathways in response to carbon source availability.Trypanosomatids are parasitic protozoa, among which several species cause serious diseases in humans such as sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and leishmaniasis (Leishmania spp.). These pathogenic trypanosomatids have developed a digenetic lifestyle with one or several vertebrate hosts (including humans) and a hematophagous insect vector that allows their transmission between vertebrate hosts. Recently, the genome sequencing projects of T. brucei (TREU927 strain) (1), T. cruzi (CL Brener strain) (2), and Leishmania major (Friedlin strain) (3) have been completed, providing wonderful tools to determine their metabolic complexities (1).Trypanosomatids depend on the carbon sources present in their hosts for their energy metabolism (4). For example, the trypomastigote forms of T. brucei and T. cruzi (bloodstream forms) use D-glucose, which is abundant in the fluids of their vertebrate host(s) (5, 6). In contrast, the insect vectors obtain their energy from L-proline and/or L-glutamine, the prominent constituent of their hemolymph and tissue fluids (7). Consequently, the insect stages of T. brucei and T. cruzi rely on amino acid catabolism, with a preference for L-proline. However, these parasites prefer D-glucose when grown in medium rich in this sugar. Because glucose-rich media are routinely used to grow these parasites, D-glucose metabolism has received the most attention, and relatively little is known about their amino acid metabolism. Recent advances in underst...
BackgroundAnimal African trypanosomosis, a disease mainly caused by the protozoan parasite Trypanosoma congolense, is a major constraint to livestock productivity and has a significant impact in the developing countries of Africa. RNA interference (RNAi) has been used to study gene function and identify drug and vaccine targets in a variety of organisms including trypanosomes. However, trypanosome RNAi studies have mainly been conducted in T. brucei, as a model for human infection, largely ignoring livestock parasites of economical importance such as T. congolense, which displays different pathogenesis profiles. The whole T. congolense life cycle can be completed in vitro, but this attractive model displayed important limitations: (i) genetic tools were currently limited to insect forms and production of modified infectious BSF through differentiation was never achieved, (ii) in vitro differentiation techniques lasted several months, (iii) absence of long-term bloodstream forms (BSF) in vitro culture prevented genomic analyses.Methodology/Principal FindingsWe optimized culture conditions for each developmental stage and secured the differentiation steps. Specifically, we devised a medium adapted for the strenuous development of stable long-term BSF culture. Using Amaxa nucleofection technology, we greatly improved the transfection rate of the insect form and designed an inducible transgene expression system using the IL3000 reference strain. We tested it by expression of reporter genes and through RNAi. Subsequently, we achieved the complete in vitro life cycle with dramatically shortened time requirements for various wild type and transgenic strains. Finally, we established the use of modified strains for experimental infections and underlined a host adaptation phase requirement.Conclusions/SignificanceWe devised an improved T. congolense model, which offers the opportunity to perform functional genomics analyses throughout the whole life cycle. It represents a very useful tool to understand pathogenesis mechanisms and to study potential therapeutic targets either in vitro or in vivo using a mouse model.
Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD ؉ /NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the ⌬pepck knock-out mutant cell line. In the absence of these two pathways (⌬pepck/ RNAi FAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the ⌬pepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the ⌬pepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.
Acetyl-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from 14 C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyl-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyl-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of 14 C-incorporation from radiolabeled glucose or acetate into de novo synthesized fatty acids. Thus, we demonstrate that the essential cytosolic enzyme AceCS of T. brucei is responsible for activation of acetate into acetyl-CoA to feed de novo biosynthesis of lipids. To date, Trypanosoma is the only known eukaryotic organism that uses acetate instead of citrate to transfer acetyl groups over the mitochondrial membrane for cytosolic lipid synthesis.acetyl-CoA synthetase ͉ citrate/malate shuttle ͉ de novo lipid biosynthesis ͉ mitochondrial acetate Trypanosoma brucei is an unicellular eukaryote, belonging to the protozoan order Kinetoplastida, that causes sleeping sickness in humans and economically important livestock diseases. This parasite undergoes a complex life cycle during transmission from the bloodstream of a mammalian host (bloodstream stages of the parasite) to the alimentary tract (procyclic stage) and salivary glands (epimastigote and metacyclic stages) of a bloodfeeding insect vector, the tse-tse fly. In addition to their relevance for health and development in subsaharan Africa, trypanosomes are famous for a variety of very unusual genetic and biochemical features that stimulate broad scientific and evolutionary interest. These include exotic mechanisms of gene expression like polycistronic transcription of genes (1), maturation of premessenger RNA by trans-splicing and extensive editing of mitochondrial RNAs (2, 3), and sophisticated mechanisms of immune evasion like antigenic variation and antibody endocytosis (4, 5). In the context of this report, metabolic peculiarities like the compartmentalization of glycolysis in glycosomes, which are peroxisomelike organelles (6), the presence of a single, developmentally regulated mitochondrion per cell with unusual enzyme activities (7, 8), energy metabolism (9) and unusual pathways for de novo synthesis of fatty acids (10) are noteworthy. Indeed, whereas ...
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