The Trypanosome UDP-Glucose Pyrophosphorylase Is Imported by Piggybacking into Glycosomes, Where Unconventional Sugar Nucleotide Synthesis Takes Place.

Glycosomes are peroxisome-related organelles of trypanosomatid parasites containing metabolic pathways, such as glycolysis and biosynthesis of sugar nucleotides, usually present in the cytosol of other eukaryotes. UDP-glucose pyrophosphorylase (UGP), the enzyme responsible for the synthesis of the sugar nucleotide UDP-glucose, is localized in the cytosol and glycosomes of the bloodstream and procyclic trypanosomes, despite the absence of any known peroxisome-targeting signal (PTS1 and PTS2). The questions that we address here are (i) is the unusual glycosomal biosynthetic pathway of sugar nucleotides functional and (ii) how is the PTS-free UGP imported into glycosomes? We showed that UGP is imported into glycosomes by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate carboxykinase (PEPCK) and identified the domains involved in the UGP/PEPCK interaction.

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei.

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient.

Glycerol supports growth of the Trypanosoma brucei bloodstream forms in the absence of glucose: Analysis of metabolic adaptations on glycerol-rich conditions.

The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet.

De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions.

De novo biosynthesis of lipids is essential for Trypanosoma brucei, a protist responsible for the sleeping sickness. Here, we demonstrate that the ketogenic carbon sources, threonine, acetate and glucose, are precursors for both fatty acid and sterol synthesis, while leucine only contributes to sterol production in the tsetse fly midgut stage of the parasite. Degradation of these carbon sources into lipids was investigated using a combination of reverse genetics and analysis of radio-labelled precursors incorporation into lipids.

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

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.

NADH dehydrogenase of Trypanosoma brucei is important for efficient acetate production in bloodstream forms.

In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite's second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion.

The SCP2-thiolase-like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism.

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2-thiolase-like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2-Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose.

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities.

Combining reverse genetics and nuclear magnetic resonance-based metabolomics unravels trypanosome-specific metabolic pathways.

Numerous eukaryotes have developed specific metabolic traits that are not present in extensively studied model organisms. For instance, the procyclic insect form of Trypanosoma brucei, a parasite responsible for sleeping sickness in its mammalian-specific bloodstream form, metabolizes glucose into excreted succinate and acetate through pathways with unique features. Succinate is primarily produced from glucose-derived phosphoenolpyruvate in peroxisome-like organelles, also known as glycosomes, by a soluble NADH-dependent fumarate reductase only described in trypanosomes so far.

Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei.

Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T.

Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form.

Trypanosoma brucei belongs to a group of protists that sequester the first six or seven glycolytic steps inside specialized peroxisomes, named glycosomes. Because of the glycosomal membrane impermeability to nucleotides, ATP molecules consumed by the first glycolytic steps need to be regenerated in the glycosomes by kinases, such as phosphoenolpyruvate carboxykinase (PEPCK). The glycosomal pyruvate phosphate dikinase (PPDK), which reversibly converts phosphoenolpyruvate into pyruvate, could also be involved in this process.

Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability.

Background: The bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.

The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control.

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.

Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux.

All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media.

Naphthoquinone derivatives exert their antitrypanosomal activity via a multi-target mechanism.

Background And Methodology: Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED(50) of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action.

ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma.

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source.

The characterization and evolutionary relationships of a trypanosomal thiolase.

Thiolases are enzymes that remove an acetyl-coenzyme A group from acyl-CoA in the catabolic β-oxidation of fatty acids, or catalyse the reverse condensation reaction for anabolic processes such as the biosynthesis of sterols and ketone bodies. In humans, six homologous isoforms of thiolase have been described, differing from each other in sequence, oligomeric state, substrate specificity and subcellular localization. A bioinformatics analysis of parasite genomes, being (i) different species of African trypanosomes, (ii) Trypanosoma cruzi and (iii) Leishmania spp.

Glycosomal ABC transporters of Trypanosoma brucei: characterisation of their expression, topology and substrate specificity.

Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1-3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei.

The human microsporidian Encephalitozoon hellem synthesizes two spore wall polymorphic proteins useful for epidemiological studies.

Microsporidia are obligate intracellular fungus-related parasites considered as emerging opportunistic human pathogens. Their extracellular infective and resistance stage is a spore surrounded by a unique plasma membrane protected by a thick cell wall consisting of two layers: the electron-lucent inner endospore which contains chitin and protein components and the outer-electron-dense and mainly proteinaceous exospore. We identified the whole sequences of two spore wall proteins in the microsporidian species Encephalitozoon hellem, designated EhSWP1a and EhSWP1b.

First molecular characterisation of hydrogenosomes in the protozoan parasite Histomonas meleagridis.

Histomonas meleagridis is a trichomonad species that undergoes a flagellate-to-amoeba transformation during tissue invasion and causes a serious disease in gallinaceous birds (blackhead disease or histomoniasis). Living in the avian cecum, the flagellated form can be grown in vitro in the presence of an ill-defined bacterial flora. Its cytoplasm harbours numerous spherical bodies which structurally resemble hydrogenosomes.

Major O-glycans in the spores of two microsporidian parasites are represented by unbranched manno-oligosaccharides containing alpha-1,2 linkages.

Protein glycosylation in microsporidia, a fungi-related group comprising exclusively obligate intracellular parasitic species, is still poorly documented. Here, we have studied glycoconjugate localization and glycan structures in spores of Encephalitozoon cuniculi and Antonospora locustae, two distantly related microsporidians invading mammalian and insect hosts, respectively. The polar sac-anchoring disc complex or polar cap, an apical element of the sporal invasion apparatus, was strongly periodic acid-thiocarbohydrazide-Ag proteinate-positive.