The persistence of the human malaria parasite Plasmodium falciparum during blood stage proliferation in its host depends on the successive expression of variant molecules at the surface of infected erythrocytes. This variation is mediated by the differential control of a family of surface molecules termed PfEMP1 encoded by approximately 60 var genes. Each individual parasite expresses a single var gene at a time, maintaining all other members of the family in a transcriptionally silent state. PfEMP1/var enables parasitized erythrocytes to adhere within the microvasculature, resulting in severe disease. This review highlights key regulatory mechanisms thought to be critical for monoallelic expression of var genes. Antigenic variation is orchestrated by epigenetic factors including monoallelic var transcription at separate spatial domains at the nuclear periphery, differential histone marks on otherwise identical var genes, and var silencing mediated by telomeric heterochromatin. In addition, controversies surrounding var genetic elements in antigenic variation are discussed.

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Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.

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...

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13 C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.Fumarate reductases (FRDs) 1 catalyze the reduction of fumarate to succinate and can be divided into two classes of enzymes: those belonging to a multimeric complex associated with the respiratory chain and transferring electrons from a quinol to fumarate and the soluble enzymes, which transfer electrons from a noncovalently bound cofactor (NADH or FADH 2 /FMNH 2 ) to fumarate. Most of the FRDs characterized so far belong to the first class (1). These FRDs are structurally similar to succinate dehydrogenases (SDH), complex II of the respiratory chain. The Shewanella putrefaciens FRD is functionally equivalent to the membrane-bound enzymes by accepting/transferring electrons from/to the respiratory chain but is a soluble enzyme lacking a membrane anchor (2). To date, only two examples of soluble FRDs not linked to the respiratory chain (second class) have been described. The yeast Saccharomyces cerevisiae expresses two soluble FRDs (cytosolic and promitochondrial) that use FADH 2 /FMNH 2 as an electron donor (3, 4), and the African trypanosome Trypanosoma brucei expresses a soluble NADH-dependent FRD located in the peroxisome-like organelle, called glycosome (5, 6). A phylogenetic analysis of FRDs and SDHs showed that the membrane-bound enzymes form a monophyletic group distantly related to the soluble enzymes, including the S. putrefaciens FRD (5). In 1980, Gest (7) proposed that the membrane-bou...

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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 ...

The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonine's contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end-products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7-fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8-fold reduction in threonine-derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.