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1. Confining Trypanosoma brucei in emulsion droplets reveals population variabilities in division rates and improves in vitro cultivation

Author

Allmann, Stefan, Wargnies, Marion, Plazolles, Nicolas, Cahoreau, Edern, Biran, Marc, Morand, Pauline, Pineda, Erika, Kulyk, Hanna, Asencio, Corinne, Villafraz, Oriana, Rivière, Loïc, Tetaud, Emmanuel, Rotureau, Brice, Mourier, Arnaud, Portais, Jean-Charles, Dé Ric Bringaud, Fré, Oldenburg, Simone, Buisson, Lionel, Beneyton, Thomas, Pekin, Deniz, Thonnus, Magali, Bringaud, Frédéric, Baret, Jean-Christophe, Microbiologie Fondamentale et Pathogénicité [Bordeaux] (MFP), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche Paul Pascal (CRPP), Université de Bordeaux (UB)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Universitaire de France (IUF), and Ministère de l'Education nationale, de l’Enseignement supérieur et de la Recherche (M.E.N.E.S.R.)

Subjects
Science, Microfluidics, Trypanosoma brucei brucei, Cell, Population, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Trypanosoma brucei, 01 natural sciences, Article, 03 medical and health sciences, [CHIM.ANAL]Chemical Sciences/Analytical chemistry, medicine, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, education, Droplet size, 030304 developmental biology, 0303 health sciences, education.field_of_study, Multidisciplinary, Lab-on-a-chip, Host (biology), 010401 analytical chemistry, Tsetse fly, biology.organism_classification, In vitro, 0104 chemical sciences, Cell biology, Parasite biology, medicine.anatomical_structure, Biological Variation, Population, Medicine, Emulsions, Single-Cell Analysis, Emulsion droplet, Cell Division
Abstract

Trypanosome parasites are infecting mammals in Sub-Saharan Africa and are transmitted between hosts through bites of the tsetse fly. The transmission from the insect vector to the mammal host causes a number of metabolic and physiological changes. A fraction of the population continuously adapt to the immune system of the host, indicating heterogeneity at the population level. Yet, the cell to cell variability in populations is mostly unknown. We develop here an analytical method for quantitative measurements at the single cell level based on encapsulation and cultivation of single-cell Trypanosoma brucei in emulsion droplets. We first show that mammalian stage trypanosomes survive for several hours to days in droplets, with an influence of droplet size on both survival and growth. We unravel various growth patterns within a population and find that droplet cultivation of trypanosomes results in 10-fold higher cell densities of the highest dividing cell variants compared to standard cultivation techniques. Some variants reach final cell titers in droplets closer to what is observed in nature than standard culture, of practical interest for cell production. Droplet microfluidics is therefore a promising tool for trypanosome cultivation and analysis with further potential for high-throughput single cell trypanosome analysis.

Published
2021

2. Glycerol, a possible new player in the biology of trypanosomes

Author

Bringaud, Frédéric, Plazolles, Nicolas, Pineda, Erika, Asencio, Corinne, Villafraz, Oriana, Millerioux, Yoann, Rivière, Loïc, and Tetaud, Emmanuel

Subjects
Glycerol, Pyruvate, Trypanosoma, QH301-705.5, Enzyme Metabolism, Trypanosoma brucei brucei, Carbohydrates, Disease Vectors, Biochemistry, Pearls, Medical Conditions, Glucose Metabolism, Trypanosoma Brucei, Medicine and Health Sciences, Humans, Biology (General), Enzyme Chemistry, Biology, Protozoans, Organic Compounds, Monomers, Monosaccharides, Organic Chemistry, Organisms, Chemical Compounds, Biology and Life Sciences, Eukaryota, Ketones, RC581-607, Polymer Chemistry, Parasitic Protozoans, Insect Vectors, Chemistry, Species Interactions, Metabolism, Glucose, Infectious Diseases, Trypanosomiasis, African, Physical Sciences, Enzymology, Carbohydrate Metabolism, Immunologic diseases. Allergy, Acids, Trypanosoma Brucei Gambiense
Published
2021

3. Glycerol suppresses glucose consumption in trypanosomes through metabolic contest

Author

Allmann, Stefan, primary, Wargnies, Marion, additional, Plazolles, Nicolas, additional, Cahoreau, Edern, additional, Biran, Marc, additional, Morand, Pauline, additional, Pineda, Erika, additional, Kulyk, Hanna, additional, Asencio, Corinne, additional, Villafraz, Oriana, additional, Rivière, Loïc, additional, Tetaud, Emmanuel, additional, Rotureau, Brice, additional, Mourier, Arnaud, additional, Portais, Jean-Charles, additional, and Bringaud, Frédéric, additional

Published
2021
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4. Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline

Author

Villafraz, Oriana, Biran, Marc, Pineda, Erika, Plazolles, Nicolas, Cahoreau, Edern, Ornitz Oliveira Souza, Rodolpho, Thonnus, Magali, Allmann, Stefan, Tetaud, Emmanuel, Rivière, Loïc, Silber, Ariel M., Barrett, Michael P., Zíková, Alena, Boshart, Michael, Portais, Jean-Charles, Bringaud, Frédéric, Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Centre de résonance magnétique des systèmes biologiques (CRMSB), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), MetaToul FluxoMet (TBI-MetaToul), MetaboHUB-MetaToul, MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidade de São Paulo = University of São Paulo (USP), Ludwig-Maximilians-Universität München (LMU), University of Glasgow, Institute of Parasitology [České Budějovice] (BIOLOGY CENTRE CAS), Biology Centre of the Czech Academy of Sciences (BIOLOGY CENTRE CAS), Czech Academy of Sciences [Prague] (CAS)-Czech Academy of Sciences [Prague] (CAS), Geroscience and rejuvenation research center (RESTORE), Université de Toulouse (UT)-Université de Toulouse (UT)-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Bordeaux, CNRS, ANR-11-INBS-0010,METABOHUB,Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovation(2011), ANR-15-CE15-0025,GLYCONOV,Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomes(2015), ANR-19-CE15-0004,AdipoTryp,Interactions métaboliques entre les adipocytes et les trypanosomes, un nouveau paradigme pour les trypanosomoses(2019), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), Université de Bordeaux (UB), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Universidade de São Paulo (USP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), ANR-15-CE15-002501, ANR19-CE15-0004-01, Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), VIAUD, Karine, Développement d'une infrastructure française distribuée pour la métabolomique dédiée à l'innovation - - METABOHUB2011 - ANR-11-INBS-0010 - INBS - VALID, Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomes - - GLYCONOV2015 - ANR-15-CE15-0025 - AAPG2015 - VALID, Interactions métaboliques entre les adipocytes et les trypanosomes, un nouveau paradigme pour les trypanosomoses - - AdipoTryp2019 - ANR-19-CE15-0004 - AAPG2019 - VALID, and Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID

Subjects
Metabolic Processes, Pyruvate, Trypanosoma, Proline, Tsetse Flies, QH301-705.5, Physiology, Citric Acid Cycle, Trypanosoma brucei brucei, Excretion, Carbohydrates, [SDV.MP.PRO] Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, Biochemistry, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, Glucose Metabolism, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Biology (General), Amino Acids, Protozoans, Alanine, Organic Compounds, Organic Chemistry, Monosaccharides, fungi, Organisms, Chemical Compounds, Biology and Life Sciences, Eukaryota, Proteins, Cyclic Amino Acids, RC581-607, Ketones, Parasitic Protozoans, Insect Vectors, Chemistry, Glucose, Trypanosomiasis, African, Metabolism, Aliphatic Amino Acids, Physical Sciences, Carbohydrate Metabolism, RNA Interference, Immunologic diseases. Allergy, Physiological Processes, Oxidation-Reduction, Acids, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Research Article
Abstract

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., Author summary 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. Here we show that tricarboxylic acid (TCA) cycle intermediates, i.e. succinate, malate and α-ketoglutarate, stimulate growth of procyclic trypanosomes incubated in a medium containing 2 mM proline, which is in the range of the amounts measured in the midgut of the fly. Some of these additional carbon sources are needed for the development of epimastigotes, which differentiate from procyclics in the midgut of the fly, since their growth defect observed in the presence of 2 mM proline is rescued by addition of α-ketoglutarate. In addition, we have implemented new approaches to study a poorly explored branch of the TCA cycle converting malate to α-ketoglutarate, which was previously described as non-functional 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. 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 develops.

Published
2021

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

Author

Wargnies, Marion, Plazolles, Nicolas, Schenk, Robin, Villafraz, Oriana, Dupuy, Jean-William, Biran, Marc, Bachmaier, Sabine, Baudouin, Hélène, Clayton, Christine, Boshart, Michael, Bringaud, Frédéric, Université de Bordeaux (UB), Centre de résonance magnétique des systèmes biologiques (CRMSB), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Ludwig-Maximilians-Universität München (LMU), Plateforme Protéome [Bordeaux], Centre Génomique Fonctionnelle Bordeaux [Bordeaux] (CGFB), Institut Polytechnique de Bordeaux-Université de Bordeaux Ségalen [Bordeaux 2]-Institut Polytechnique de Bordeaux-Université de Bordeaux Ségalen [Bordeaux 2], and Universität Heidelberg [Heidelberg]

Subjects
phosphoenolpyruvate carboxykinase (PEPCK), Trypanosoma brucei brucei, homologous recombination, Microbodies, ROS, reactive oxygen species, PPDK, pyruvate phosphate dikinase, positive selection, Flavins, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, trypanosoma, Cytb5R domain, reactive oxygen species, parasite differentiation, PEP, phosphoenolpyruvate, PCF, procyclic form, ACN, acetonitrile, covalent flavinylation, FRD, fumarate reductase, Succinate Dehydrogenase, Glucose, genomic rearrangement, PEPCK, phosphoenolpyruvate carboxykinase, NADH-dependent fumarate reductase (FRD), Research Article
Abstract

International audience; 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 process

Published
2021

6. Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes

Author

Riviere, Loic, Moreau, Patrick, Allmann, Stefan, Hahn, Matthias, Biran, Marc, Plazolles, Nicolas, Franconi, Jean-Michel, Boshart, Michael, and Bringaud, Frederic

Subjects
Mitochondria -- Research, Trypanosoma -- Research, Lipids -- Synthesis, Lipids -- Research, Science and technology
Abstract

AcetyI-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 [sup.14]C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyI-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyI-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 [sup.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

Published
2009

7. Fly stage trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in near physiological conditions

Author

Villafraz, Oriana, primary, Biran, Marc, additional, Pineda, Erika, additional, Plazolles, Nicolas, additional, Cahoreau, Edern, additional, Souza, Rodolpho Ornitz Oliveira, additional, Thonnus, Magali, additional, Allmann, Stefan, additional, Tetaud, Emmanuel, additional, Rivière, Loïc, additional, Silber, Ariel M., additional, Barrett, Michael P., additional, Zíková, Alena, additional, Boshart, Michael, additional, Portais, Jean-Charles, additional, and Bringaud, Frédéric, additional

Published
2020
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8. Metabolic selection of a homologous recombination mediated loss of glycosomal fumarate reductase inTrypanosoma brucei

Author

Wargnies, Marion, primary, Plazolles, Nicolas, additional, Schenk, Robin, additional, Villafraz, Oriana, additional, Dupuy, Jean-William, additional, Biran, Marc, additional, Bachmaier, Sabine, additional, Baudouin, Hélène, additional, Clayton, Christine, additional, Boshart, Michael, additional, and Bringaud, Frédéric, additional

Published
2020
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9. Gluconeogenesis is essential for trypanosome development in the tsetse fly vector

Author

Wargnies, Marion, Bertiaux, Eloise, Cahoreau, Edern, Ziebart, Nicole, Crouzols, Aline, Morand, Pauline, Biran, Marc, Allmann, Stefan, Hubert, Jane, Villafraz, Oriana, Millerioux, Yoann, Plazolles, Nicolas, Asencio, Corinne, Rivière, Loïc, Rotureau, Brice, Boshart, Michael, Portais, Jean-Charles, Bringaud, Frédéric, Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Centre de résonance magnétique des systèmes biologiques (CRMSB), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Biologie cellulaire des Trypanosomes - Trypanosome Cell Biology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), MetaToul FluxoMet (TBI-MetaToul), MetaboHUB-MetaToul, MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Ecole Nationale Vétérinaire de Toulouse (ENVT), Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-MetaboHUB-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Ludwig-Maximilians-Universität München (LMU), The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. FB's and BR's group were funded by the Agence Nationale de la Recherche (ANR) through GLYCONOV (grant number ANR-15-CE15-0025-01) of the ANR-BLANC-2015 call. FB's group was supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the ANR through the grants ACETOTRYP (grant number ANR-2010-BLAN-1319-02) of the ANR-BLANC-2010 call, the Laboratoire d’Excellence (LabEx) ParaFrap ANR-11-LABX-0024 and the ParaMet PhD programme of Marie Curie Initial Training Network. BR’s group was supported by the Institut Pasteur, the Institut National de la Santé et de la Recherche Médicale (INSERM). EB is funded by a doctoral fellowship from French National Ministry for Research and Technology (Doctoral School CDV515). MB was funded by the University of Munich and MB and FB were supported by a research cooperation grant of the Franco-Bavarian University Cooperation Center (BFHZ/CCUFB)., ANR-15-CE15-0025,GLYCONOV,Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomes(2015), ANR-10-BLAN-1319,ACETOTRYP,Metabolisme de l'acetyl-CoA et de l'acetate chez les trypanosomes: identification de nouvelles voies métaboliques spécifiques aux parasites(2010), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Résonance magnétique des systèmes biologiques (RMSB), Biologie cellulaire des Trypanosomes, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Ecole Nationale Vétérinaire de Toulouse (ENVT), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Génopole Toulouse Midi-Pyrénées [Auzeville] (GENOTOUL), Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Rotureau, Brice, Voies métaboliques glycosomales non glycolytiques: nouvelles fonctions pour le développement et la virulence des trypanosomes - - GLYCONOV2015 - ANR-15-CE15-0025 - AAPG2015 - VALID, BLANC - Metabolisme de l'acetyl-CoA et de l'acetate chez les trypanosomes: identification de nouvelles voies métaboliques spécifiques aux parasites - - ACETOTRYP2010 - ANR-10-BLAN-1319 - BLANC - VALID, and Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID

Subjects
Glycerol, gène codant, Disease Vectors, Biochemistry, Salivary Glands, Glucose Metabolism, Medicine and Health Sciences, Biology (General), Amino Acids, Protozoans, surexpression, Organic Compounds, Microbiology and Parasitology, Monosaccharides, Monomers, Eukaryota, Microbiologie et Parasitologie, Chemistry, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, protéine, Physical Sciences, Carbohydrate Metabolism, Anatomy, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Research Article, Trypanosoma, Tsetse Flies, Proline, QH301-705.5, Trypanosoma brucei brucei, Carbohydrates, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, Phosphates, Exocrine Glands, Parasitic Diseases, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, gluconeogénèse, Organic Chemistry, Gluconeogenesis, Organisms, Chemical Compounds, Biology and Life Sciences, Proteins, Cyclic Amino Acids, RC581-607, mouche tsé tsé, Polymer Chemistry, Parasitic Protozoans, enzyme, Trypanosomiasis, African, Metabolism, Glucose, Immunologic diseases. Allergy, Digestive System, trypanosoma brucei
Abstract

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., Author summary Trypanosoma brucei, the parasite responsible for sleeping sickness in humans, is transmitted by the tsetse fly that primarily uses amino acids for its energy production. In the glucose-free environment encountered between the insect blood meals, T. brucei needs to produce through gluconeogenesis glucose 6-phosphate, a key precursor for several essential metabolic pathways. We have shown here that two key gluconeogenic steps, which produce phosphoenolpyruvate and fructose 6-phosphate, respectively, are performed by redundant enzymes (PPDK and PEPCK for phosphoenolpyruvate production; FBPase and a yet unknown enzyme for fructose 6-phosphate production), which highlights the importance of this metabolic pathway for the insect stages of the parasite. Interestingly, deletion of the parasite FBPase gene abolished both the colonisation of the insect salivary glands and the in vitro differentiation of the epimastigote forms into the mammalian infective form of the parasite. Altogether, these data demonstrate for the first time that gluconeogenesis is essential for development of T. brucei in its insect vector and that early development stages of the parasite present in the tsetse midgut are not affected by the absence of FBPase, probably by developing an alternative yet unknown approach to produce fructose 6-phosphate.

Published
2018

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

Author

Millerioux, Yoann, Mazet, Muriel, Bouyssou, Guillaume, Allmann, Stefan, Kiema, Tiila-Riikka, Bertiaux, Eloise, Fouillen, Laetitia, Thapa, Chandan, Biran, Marc, Plazolles, Nicolas, Dittrich-Domergue, Franziska, Crouzols, Aline, Wierenga, Rik, Rotureau, Brice, MOREAU, Patrick, Bringaud, Frédéric, Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Centre de résonance magnétique des systèmes biologiques (CRMSB), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biogenèse membranaire (LBM), University of Oulu, Biologie cellulaire des Trypanosomes - Trypanosome Cell Biology, Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), This research was supported by the Centre National de la Recherche Scientifique (CNRS), the Université de Bordeaux, the Agence Nationale de la Recherche (ANR) through grants ACETOTRYP (grant number ANR-2010-BLAN-1319-02) of the ANR-BLANC-2010 call and GLYCONOV (grant number ANR-15-CE15-0025-01) of the 'Générique' 2015 call, the Laboratoire d’Excellence (LabEx) ParaFrap (grant number ANR-11-LABX-0024), the Institut Pasteur and by the European Commission FP7 Marie Curie Initial Training Network 'ParaMet' (grant number 290080). EB was funded by a doctoral fellowship from French National Ministry for Research and Technology (doctoral school CDV515)., ANR-10-BLAN-1319,ACETOTRYP,Metabolisme de l'acetyl-CoA et de l'acetate chez les trypanosomes: identification de nouvelles voies métaboliques spécifiques aux parasites(2010), ANR-11-LABX-0024,ParaFrap,Alliance française contre les maladies parasitaires(2011), European Project: 290080,EC:FP7:PEOPLE,FP7-PEOPLE-2011-ITN,PARAMET(2012), Résonance magnétique des systèmes biologiques (RMSB), Laboratoire Microorganismes : Génome et Environnement (LMGE), Université Blaise Pascal - Clermont-Ferrand 2 (UBP)-Université d'Auvergne - Clermont-Ferrand I (UdA)-Centre National de la Recherche Scientifique (CNRS), Microbiologie cellulaire et moléculaire et pathogénicité (MCMP), Biologie cellulaire des Trypanosomes, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université de Bordeaux (UB), Rotureau, Brice, BLANC - Metabolisme de l'acetyl-CoA et de l'acetate chez les trypanosomes: identification de nouvelles voies métaboliques spécifiques aux parasites - - ACETOTRYP2010 - ANR-10-BLAN-1319 - BLANC - VALID, Laboratoires d'excellence - Alliance française contre les maladies parasitaires - - ParaFrap2011 - ANR-11-LABX-0024 - LABX - VALID, and A systematic analysis of parasite metabolism - from metabolism to intervention - PARAMET - - EC:FP7:PEOPLE2012-06-01 - 2016-05-31 - 290080 - VALID

Subjects
Threonine, [SDV]Life Sciences [q-bio], blood-sream forms, Acetates, Biochemistry, molecular characterization, Gene Knockout Techniques, dependent enzyme, Glucose Metabolism, proline metabolism, MESH: Animals, Amino Acids, MESH: Threonine, lcsh:QH301-705.5, MESH: Gene Knockout Techniques, Protozoans, MESH: Tsetse Flies, Organic Compounds, Fatty Acids, Monosaccharides, Eukaryota, cell-cycle, acetyl-coa, energy-metabolism, lipbiosyntesis, leishmania-mexicana, succinate coa-transferase, MESH: Mevalonic Acid, Lipids, MESH: Gene Expression Regulation, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Fatty Acids, [SDV] Life Sciences [q-bio], MESH: Glucose, Sterols, Chemistry, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Physical Sciences, Carbohydrate Metabolism, MESH: Acetates, [SDV.MP.PAR] Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, Research Article, lcsh:Immunologic diseases. Allergy, Trypanosoma, Proline, Tsetse Flies, Trypanosoma brucei brucei, Carbohydrates, Mevalonic Acid, MESH: Carbon, MESH: Insect Vectors, Biosynthesis, [SDV.MP.PRO]Life Sciences [q-bio]/Microbiology and Parasitology/Protistology, MESH: Alcohol Oxidoreductases, Acetyl Coenzyme A, Acetyltransferases, Leucine, Hydroxyl Amino Acids, MESH: Acyl Coenzyme A, Animals, [SDV.MP.PAR]Life Sciences [q-bio]/Microbiology and Parasitology/Parasitology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, MESH: Proline, Organic Chemistry, Organisms, Chemical Compounds, Biology and Life Sciences, Proteins, MESH: Trypanosoma brucei brucei, MESH: Acetyltransferases, Carbon, Parasitic Protozoans, Insect Vectors, Alcohol Oxidoreductases, Glucose, Metabolism, MESH: Leucine, Gene Expression Regulation, Aliphatic Amino Acids, lcsh:Biology (General), MESH: Sterols, Acyl Coenzyme A, lcsh:RC581-607, MESH: Acetyl Coenzyme A
Abstract

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. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway., Author summary In this study, we have (i) determined the carbon sources used by the Trypanosoma brucei procyclic insect form to feed the essential lipid biosynthetic pathways, (ii) further characterized the metabolic pathways leading to their degradation into acetyl-CoA (fatty acid precursor) and 3-hydroxy-3-methylglutaryl-CoA (sterol precursor) and (iii) showed that reduction of the ketogenic carbon sources degradation, favors their incorporation into fatty acids, instead of sterols. This fatty acid preference is compensated by an increase of leucine incorporation into sterols, which highlights the parasite adaptation capacity regarding carbon source availability by modulating the metabolic flux between branches within the network. This metabolic flexibility is particularly relevant for the insect stages of trypanosomes that evolve in the midgut and the salivary glands of their blood-feeding insect vector. One may also consider that, the metabolic flow redistribution towards the mevalonate pathway (sterol production) described in vitro also occurs in vivo, depending on the carbon source composition of the tsetse fly micro-environment, which may considerably vary along the digestive tract and depending on the fly feeding status, as well as in the other infected fly organs.

Published
2018

11. “Metabolic contest”, a new way to control carbon source preference

Author

Allmann, Stefan, primary, Wargnies, Marion, additional, Cahoreau, Edern, additional, Biran, Marc, additional, Plazolles, Nicolas, additional, Morand, Pauline, additional, Pineda, Erika, additional, Kulyk, Hanna, additional, Asencio, Corinne, additional, Villafraz, Oriana, additional, Rivière, Loïc, additional, Tétaud, Emmanuel, additional, Rotureau, Brice, additional, Mourier, Arnaud, additional, Portais, Jean-Charles, additional, and Bringaud, Frédéric, additional

Published
2019
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15. Ablation of Succinate Production from Glucose Metabolism in the Procyclic Trypanosomes Induces Metabolic Switches to the Glycerol 3-Phosphate/Dihydroxyacetone Phosphate Shuttle and to Proline Metabolism

Author

Ebikeme, Charles, primary, Hubert, Jane, additional, Biran, Marc, additional, Gouspillou, Gilles, additional, Morand, Pauline, additional, Plazolles, Nicolas, additional, Guegan, Fabien, additional, Diolez, Philippe, additional, Franconi, Jean-Michel, additional, Portais, Jean-Charles, additional, and Bringaud, Frédéric, additional

Published
2010
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18. Erythrophagocytosis of desialylated red blood cells is responsible for anaemia during Trypanosoma vivax infection.

Author

Guegan, Fabien, Plazolles, Nicolas, Baltz, Théo, and Coustou, Virginie

Subjects
*TRYPANOSOMA, *PHAGOCYTOSIS, *ERYTHROCYTES, *ANEMIA, *BLOOD serum analysis, *GLYCOPHORIN
Abstract

Trypanosomal infection-induced anaemia is a devastating scourge for cattle in widespread regions. Although Trypanosoma vivax is considered as one of the most important parasites regarding economic impact in Africa and South America, very few in-depth studies have been conducted due to the difficulty of manipulating this parasite. Several hypotheses were proposed to explain trypanosome induced-anaemia but mechanisms have not yet been elucidated. Here, we characterized a multigenic family of trans-sialidases in T. vivax, some of which are released into the host serum during infection. These enzymes are able to trigger erythrophagocytosis by desialylating the major surface erythrocytes sialoglycoproteins, the glycophorins. Using an ex vivo assay to quantify erythrophagocytosis throughout infection, we showed that erythrocyte desialylation alone results in significant levels of anaemia during the acute phase of the disease. Characterization of virulence factors such as the trans-sialidases is vital to develop a control strategy against the disease or parasite. [ABSTRACT FROM AUTHOR]

Published
2013
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19. Procyclic trypanosomes recycle glucose catabolites and TCA cycle intermediates to stimulate growth in the presence of physiological amounts of proline.

Author

Villafraz O, Biran M, Pineda E, Plazolles N, Cahoreau E, Ornitz Oliveira Souza R, Thonnus M, Allmann S, Tetaud E, Rivière L, Silber AM, Barrett MP, Zíková A, Boshart M, Portais JC, and Bringaud F

Subjects
Animals, Citric Acid Cycle drug effects, Insect Vectors parasitology, Oxidation-Reduction drug effects, Proline metabolism, RNA Interference physiology, Trypanosoma metabolism, Trypanosoma brucei brucei metabolism, Trypanosomiasis, African drug therapy, Tsetse Flies parasitology, Glucose metabolism, Proline pharmacology, Trypanosoma drug effects, Trypanosoma brucei brucei drug effects, Tsetse Flies drug effects
Abstract

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., Competing Interests: The authors have declared that no competing interests exist.

Published
2021
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20. Metabolic selection of a homologous recombination-mediated gene loss protects Trypanosoma brucei from ROS production by glycosomal fumarate reductase.

Author

Wargnies M, Plazolles N, Schenk R, Villafraz O, Dupuy JW, Biran M, Bachmaier S, Baudouin H, Clayton C, Boshart M, and Bringaud F

Subjects
Cells, Cultured, Flavins metabolism, Succinate Dehydrogenase genetics, Trypanosoma brucei brucei genetics, Trypanosoma brucei brucei growth & development, Glucose metabolism, Homologous Recombination, Microbodies enzymology, Reactive Oxygen Species metabolism, Succinate Dehydrogenase metabolism, Trypanosoma brucei brucei metabolism
Abstract

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 process., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Published
2021
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21. Gluconeogenesis is essential for trypanosome development in the tsetse fly vector.

Author

Wargnies M, Bertiaux E, Cahoreau E, Ziebart N, Crouzols A, Morand P, Biran M, Allmann S, Hubert J, Villafraz O, Millerioux Y, Plazolles N, Asencio C, Rivière L, Rotureau B, Boshart M, Portais JC, and Bringaud F

Subjects
Animals, Disease Vectors, Trypanosomiasis, African, Gluconeogenesis physiology, Trypanosoma brucei brucei metabolism, Tsetse Flies parasitology
Abstract

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., Competing Interests: The authors have declared that no competing interests exist.

Published
2018
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22. De novo biosynthesis of sterols and fatty acids in the Trypanosoma brucei procyclic form: Carbon source preferences and metabolic flux redistributions.

Author

Millerioux Y, Mazet M, Bouyssou G, Allmann S, Kiema TR, Bertiaux E, Fouillen L, Thapa C, Biran M, Plazolles N, Dittrich-Domergue F, Crouzols A, Wierenga RK, Rotureau B, Moreau P, and Bringaud F

Subjects
Acetates metabolism, Acetyl Coenzyme A metabolism, Acetyltransferases metabolism, Acyl Coenzyme A metabolism, Alcohol Oxidoreductases metabolism, Animals, Gene Expression Regulation, Gene Knockout Techniques, Glucose metabolism, Insect Vectors parasitology, Leucine metabolism, Mevalonic Acid metabolism, Proline metabolism, Threonine metabolism, Trypanosoma brucei brucei genetics, Tsetse Flies parasitology, Carbon metabolism, Fatty Acids biosynthesis, Sterols biosynthesis, Trypanosoma brucei brucei metabolism
Abstract

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. For instance, (i) deletion of the gene encoding isovaleryl-CoA dehydrogenase, involved in the leucine degradation pathway, abolished leucine incorporation into sterols, and (ii) RNAi-mediated down-regulation of the SCP2-thiolase gene expression abolished incorporation of the three ketogenic carbon sources into sterols. The SCP2-thiolase is part of a unidirectional two-step bridge between the fatty acid precursor, acetyl-CoA, and the precursor of the mevalonate pathway leading to sterol biosynthesis, 3-hydroxy-3-methylglutaryl-CoA. Metabolic flux through this bridge is increased either in the isovaleryl-CoA dehydrogenase null mutant or when the degradation of the ketogenic carbon sources is affected. We also observed a preference for fatty acids synthesis from ketogenic carbon sources, since blocking acetyl-CoA production from both glucose and threonine abolished acetate incorporation into sterols, while incorporation of acetate into fatty acids was increased. Interestingly, the growth of the isovaleryl-CoA dehydrogenase null mutant, but not that of the parental cells, is interrupted in the absence of ketogenic carbon sources, including lipids, which demonstrates the essential role of the mevalonate pathway. We concluded that procyclic trypanosomes have a strong preference for fatty acid versus sterol biosynthesis from ketogenic carbon sources, and as a consequence, that leucine is likely to be the main source, if not the only one, used by trypanosomes in the infected insect vector digestive tract to feed the mevalonate pathway., Competing Interests: The authors have declared that no competing interests exist.

Published
2018
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