Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase

The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase (PEPCK) gene knockout (Δpepck) leads to the selection of a deletion event between two tandemly arranged fumarate reductase (FRDg and FRDm2) genes to produce a chimeric FRDg-m2 gene in the Δpepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δpepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δpepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δpepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this proces

PLoS Biol

Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as "catabolite repression," allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named "metabolic contest" for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This "metabolic contest" depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met

Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1–2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly

UDP-glucose pyrophosphorylase (UGP) : import into glycosomes and implication in glycosomal and cytosolic nucleotide sugar biosynthetic pathways in Trypanosoma brucei

Trypanosoma brucei, un protiste responsable de la Trypanosomose Humaine Africaine, également connue sous le nom de la maladie du sommeil, est transmis par la mouche tsé-tsé (Glossina sp.). La découverte d'organites de type peroxysome spécialisés dans la glycolyse, appelés glycosomes, a soulevé un certain nombre de questions sur le rôle de cet organite dans la biologie des trypanosomes. Plusieurs voies métaboliques présentes dans le cytosol d'autres eucaryotes, comme la glycolyse et la biosynthèse des sucres nucléotidiques, sont compartimentées dans les glycosomes. Les raisons et les avantages de la présence des enzymes glycolytiques dans l'organite ont été largement discutés, mais la fonctionnalité et le rôle des voies de biosynthèse des sucres nucléotidiques glycosomales ne sont pas connus. Notre étude s'est focalisée sur l'UDP-glucose pyrophosphorylase (UGP), une enzyme impliquée dans la synthèse de l'UDP-glucose (UDP-Glc). Sur la base de la double localisation glycosomale et cytosolique de l'UGP mise en évidence ici à l'aide de plusieurs techniques de localisation subcellulaire, nous avons abordé deux questions en utilisant comme modèle les formes procycliques de T. brucei présentes dans l'insecte vecteur. La première est liée au mécanisme d'import de l'UGP dans les glycosomes, car cette protéine ne possède aucun signal d'adressage aux peroxysomes de type PTS1 ou PTS2. Nous avons montré que l'UGP est importée dans les glycosomes par "piggybacking" en s'associant à la phosphoénolpyruvate décarboxylase (PEPCK) possédant un signal d’adressage PTS1. Les interactions entre l'UGP et la PEPCK ont été montrées in situ et l'identification les régions impliquées dans ces interactions ont été identifiées. Nos résultats suggèrent que le complexe UGP-PEPCK est formé de manière transitoire lors de son import dans les glycosomes nouvellement produits et compétents pour l'import des protéines. La seconde question concerne le rôle de l'UGP dans les glycosomes. Nous avons montré que l'UGP est essentielle à la croissance des trypanosomes et que les voies métaboliques glycosomales et cytosoliques dont l'UGP fait partie sont fonctionnelles. En effet, des mutants viables contenant l'UGP exclusivement dans les glycosomes ou dans le cytosol sont viables et produisent des quantités similaires d'UDP-Glc. La raison d'être de la production glycosomale d'UDP-Glc par l'UGP reste inconnue, mais n'est probablement pas liée aux réactions de glycosylation, étant donné qu'aucune glycosyltransférase n'a été détectée dans l'organite.Un autre aspect de ce travail concerne le rôle des intermédiaires du cycle de l'acide tricarboxylique (TCA) dans le métabolisme mitochondrial des formes procycliques. Dans le tractus digestif de son insecte vecteur, les trypanosomes dépendent de la proline pour alimenter leur métabolisme énergétique. Cependant, la disponibilité d'éventuelles autres sources de carbone pouvant être utilisées par le parasite est actuellement inconnue. Nous avons montré que les intermédiaires du cycle TCA, i.e. succinate, malate et a-cétoglutarate, stimulent la croissance des formes procycliques incubées dans un milieu contenant 2 mM de proline, concentration se situant dans la gamme des quantités mesurées dans l'intestin de la mouche. De plus, le développement de nouvelles approches ont permis d'étudier une branche peu explorée du cycle TCA convertissant le malate en a-cétoglutarate, précédemment décrite comme peu ou pas utilisée par le parasite, quellles que soient les quantités de glucose disponibles. L'activité de cette branche suggère qu'un cycle TCA complet peut être mis en œuvre dans les formes procycliques et probablement dans les autres formes parasitaires de l'insecte. Nos données élargissent le potentiel métabolique des trypanosomes et ouvrent la voie vers une meilleure compréhension du métabolisme de ce parasite dans divers organes de la mouche tsé-tsé, où il évolue.Trypanosoma brucei, a protist responsible for human African trypanosomiasis, also known as sleeping sickness, is transmitted by the tsetse fly (Glossina sp.). The discovery of peroxisome-like organelles specialized in glycolysis called glycosomes, has raised a number of questions about the role of this organelle in the biology of trypanosomes. Several metabolic pathways present in the cytosol of eukaryotes, like glycolysis and sugar nucleotides biosynthesis, are compartmentalized within glycosomes. While the reasons and advantages of having glycolytic enzymes compartmentalized in the organelle have been extensively discussed, little is proposed for sugar nucleotides biosynthetic pathways. This study is focused on the UDP-glucose pyrophosphorylase (UGP), an enzyme involved in the synthesis of UDP-glucose (UDP-Glc). Based on the UGP's dual glycosomal and cytosolic localization evidenced here using several subcellular localization techniques, we addressed two questions using as a model the procyclic forms of T. brucei present in the insect vector. The first one is related to the mechanism of UGP import into glycosomes, since this protein lacks any known peroxisomal targeting signal (PTS1 and PTS2). We demonstrated that UGP is imported into the organelle by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate decarboxylase (PEPCK). Interactions between UGP and PEPCK have been showed in situ and the interacting regions have been identified. Our data suggest that the complex UGP-PEPCK is formed transiently to facilitate the import of UGP and that it is detected in newly formed import-competent glycosomes. The second question concerns the role of UGP in glycosomes. We demonstrated that UGP is essential for the growth of trypanosomes and that mutants containing UGP exclusively in glycosomes or in the cytosol still produce UDP-Glc at similar levels and are viable, which implies that the glycosomal and cytosolic metabolic pathways involving UGP are functional. The glycosomal function of UDP-Glc is currently unknown and probably not related to glycosylation reactions, since no glycosyltransferases have been detected in the organelle.Another aspect of this work concerns the role of tricarboxylic acid (TCA) cycle intermediates in the mitochondrial metabolism of the procyclic trypanosomes. In the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. We showed that TCA cycle intermediates, i.e. succinate, malate and a-ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. In addition, we have implemented new approaches to study cell growth and metabolic pathways in order to investigate mitochondrial metabolism. These new tools have allowed us to study a poorly explored branch of the TCA cycle converting malate to a-ketoglutarate, which was previously described as non-functional or little used in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes and probably in the other trypanosome forms present in the fly. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasite's metabolism in various organ systems of the tsetse fly, where it evolves

UDP-glucose pyrophosphorylase (UGP) : import dans les glycosomes et implication dans la biosynthèse glycosomale et cytosolique des sucres nucléotidiques chez Trypanosoma brucei

Trypanosoma brucei, a protist responsible for human African trypanosomiasis, also known as sleeping sickness, is transmitted by the tsetse fly (Glossina sp.). The discovery of peroxisome-like organelles specialized in glycolysis called glycosomes, has raised a number of questions about the role of this organelle in the biology of trypanosomes. Several metabolic pathways present in the cytosol of eukaryotes, like glycolysis and sugar nucleotides biosynthesis, are compartmentalized within glycosomes. While the reasons and advantages of having glycolytic enzymes compartmentalized in the organelle have been extensively discussed, little is proposed for sugar nucleotides biosynthetic pathways. This study is focused on the UDP-glucose pyrophosphorylase (UGP), an enzyme involved in the synthesis of UDP-glucose (UDP-Glc). Based on the UGP's dual glycosomal and cytosolic localization evidenced here using several subcellular localization techniques, we addressed two questions using as a model the procyclic forms of T. brucei present in the insect vector. The first one is related to the mechanism of UGP import into glycosomes, since this protein lacks any known peroxisomal targeting signal (PTS1 and PTS2). We demonstrated that UGP is imported into the organelle by piggybacking on the glycosomal PTS1-containing phosphoenolpyruvate decarboxylase (PEPCK). Interactions between UGP and PEPCK have been showed in situ and the interacting regions have been identified. Our data suggest that the complex UGP-PEPCK is formed transiently to facilitate the import of UGP and that it is detected in newly formed import-competent glycosomes. The second question concerns the role of UGP in glycosomes. We demonstrated that UGP is essential for the growth of trypanosomes and that mutants containing UGP exclusively in glycosomes or in the cytosol still produce UDP-Glc at similar levels and are viable, which implies that the glycosomal and cytosolic metabolic pathways involving UGP are functional. The glycosomal function of UDP-Glc is currently unknown and probably not related to glycosylation reactions, since no glycosyltransferases have been detected in the organelle.Another aspect of this work concerns the role of tricarboxylic acid (TCA) cycle intermediates in the mitochondrial metabolism of the procyclic trypanosomes. In the midgut of its insect vector, trypanosomes rely on proline to feed their energy metabolism. However, the availability of other potential carbon sources that can be used by the parasite is currently unknown. We showed that TCA cycle intermediates, i.e. succinate, malate and a-ketoglutarate, stimulate growth of procyclic trypanosomes incubated in medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. In addition, we have implemented new approaches to study cell growth and metabolic pathways in order to investigate mitochondrial metabolism. These new tools have allowed us to study a poorly explored branch of the TCA cycle converting malate to a-ketoglutarate, which was previously described as non-functional or little used in the parasite, regardless of the glucose levels available. The discovery of this branch reveals that a full TCA cycle can operate in procyclic trypanosomes and probably in the other trypanosome forms present in the fly. Our data broaden the metabolic potential of trypanosomes and pave the way for a better understanding of the parasite's metabolism in various organ systems of the tsetse fly, where it evolves.Trypanosoma brucei, un protiste responsable de la Trypanosomose Humaine Africaine, également connue sous le nom de la maladie du sommeil, est transmis par la mouche tsé-tsé (Glossina sp.). La découverte d'organites de type peroxysome spécialisés dans la glycolyse, appelés glycosomes, a soulevé un certain nombre de questions sur le rôle de cet organite dans la biologie des trypanosomes. Plusieurs voies métaboliques présentes dans le cytosol d'autres eucaryotes, comme la glycolyse et la biosynthèse des sucres nucléotidiques, sont compartimentées dans les glycosomes. Les raisons et les avantages de la présence des enzymes glycolytiques dans l'organite ont été largement discutés, mais la fonctionnalité et le rôle des voies de biosynthèse des sucres nucléotidiques glycosomales ne sont pas connus. Notre étude s'est focalisée sur l'UDP-glucose pyrophosphorylase (UGP), une enzyme impliquée dans la synthèse de l'UDP-glucose (UDP-Glc). Sur la base de la double localisation glycosomale et cytosolique de l'UGP mise en évidence ici à l'aide de plusieurs techniques de localisation subcellulaire, nous avons abordé deux questions en utilisant comme modèle les formes procycliques de T. brucei présentes dans l'insecte vecteur. La première est liée au mécanisme d'import de l'UGP dans les glycosomes, car cette protéine ne possède aucun signal d'adressage aux peroxysomes de type PTS1 ou PTS2. Nous avons montré que l'UGP est importée dans les glycosomes par "piggybacking" en s'associant à la phosphoénolpyruvate décarboxylase (PEPCK) possédant un signal d’adressage PTS1. Les interactions entre l'UGP et la PEPCK ont été montrées in situ et l'identification les régions impliquées dans ces interactions ont été identifiées. Nos résultats suggèrent que le complexe UGP-PEPCK est formé de manière transitoire lors de son import dans les glycosomes nouvellement produits et compétents pour l'import des protéines. La seconde question concerne le rôle de l'UGP dans les glycosomes. Nous avons montré que l'UGP est essentielle à la croissance des trypanosomes et que les voies métaboliques glycosomales et cytosoliques dont l'UGP fait partie sont fonctionnelles. En effet, des mutants viables contenant l'UGP exclusivement dans les glycosomes ou dans le cytosol sont viables et produisent des quantités similaires d'UDP-Glc. La raison d'être de la production glycosomale d'UDP-Glc par l'UGP reste inconnue, mais n'est probablement pas liée aux réactions de glycosylation, étant donné qu'aucune glycosyltransférase n'a été détectée dans l'organite.Un autre aspect de ce travail concerne le rôle des intermédiaires du cycle de l'acide tricarboxylique (TCA) dans le métabolisme mitochondrial des formes procycliques. Dans le tractus digestif de son insecte vecteur, les trypanosomes dépendent de la proline pour alimenter leur métabolisme énergétique. Cependant, la disponibilité d'éventuelles autres sources de carbone pouvant être utilisées par le parasite est actuellement inconnue. Nous avons montré que les intermédiaires du cycle TCA, i.e. succinate, malate et a-cétoglutarate, stimulent la croissance des formes procycliques incubées dans un milieu contenant 2 mM de proline, concentration se situant dans la gamme des quantités mesurées dans l'intestin de la mouche. De plus, le développement de nouvelles approches ont permis d'étudier une branche peu explorée du cycle TCA convertissant le malate en a-cétoglutarate, précédemment décrite comme peu ou pas utilisée par le parasite, quellles que soient les quantités de glucose disponibles. L'activité de cette branche suggère qu'un cycle TCA complet peut être mis en œuvre dans les formes procycliques et probablement dans les autres formes parasitaires de l'insecte. Nos données élargissent le potentiel métabolique des trypanosomes et ouvrent la voie vers une meilleure compréhension du métabolisme de ce parasite dans divers organes de la mouche tsé-tsé, où il évolue

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. Proximity ligation assays revealed that this interaction occurs in 3 to 10% of glycosomes, suggesting that these correspond to organelles competent for protein import. We also showed that UGP is essential for the growth of trypanosomes and that both the glycosomal and cytosolic metabolic pathways involving UGP are functional, since the lethality of the knockdown UGP mutant cell line (RNAi UGP, where RNAi indicates RNA interference) was rescued by expressing a recoded UGP (rUGP) in the organelle (RNAi UGP/ EXP rUGP-GPDH, where GPDH is glycerol-3-phosphate dehydrogenase). Our conclusion was supported by targeted metabolomic analyses (ion chromatography-high-resolution mass spectrometry [IC-HRMS]) showing that UDPglucose is no longer detectable in the RNAi UGP mutant, while it is still produced in cells expressing UGP exclusively in the cytosol (PEPCK null mutant) or glycosomes (RNAi UGP/ EXP rUGP-GPDH). Trypanosomatids are the only known organisms to have selected functional peroxisomal (glycosomal) sugar nucleotide biosynthetic pathways in addition to the canonical cytosolic ones. IMPORTANCE Unusual compartmentalization of metabolic pathways within organelles is one of the most enigmatic features of trypanosomatids. These unicellular eukaryotes are the only organisms that sequestered glycolysis inside peroxisomes (glycosomes), although the selective advantage of this compartmentalization is still no

Gluconeogenesis is essential for trypanosome development in the tsetse fly vector

In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomesMetabolisme de l'acetyl-CoA et de l'acetate chez les trypanosomes: identification de nouvelles voies métaboliques spécifiques aux parasitesAlliance française contre les maladies parasitaire