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1. Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei.

Author

Tounkara M, Boulangé A, Thonnus M, Bringaud F, Bélem AMG, Bengaly Z, Thévenon S, Berthier D, and Rivière L

Subjects
Animals, Cattle, Glycerol Kinase genetics, Glycerol Kinase immunology, Lysophospholipase genetics, Lysophospholipase immunology, Protozoan Proteins genetics, Protozoan Proteins immunology, Trypanosoma classification, Trypanosoma enzymology, Trypanosoma genetics, Trypanosomiasis, Bovine blood, Trypanosomiasis, Bovine parasitology, Enzyme-Linked Immunosorbent Assay methods, Glycerol Kinase blood, Lysophospholipase blood, Protozoan Proteins blood, Serologic Tests methods, Trypanosoma immunology, Trypanosomiasis, Bovine diagnosis
Abstract

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45-55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87-96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT., Competing Interests: The authors have declared that no competing interests exist.

Published
2021
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2. 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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3. Streptococcus pyogenes Cas9 ribonucleoprotein delivery for efficient, rapid and marker‐free gene editing in Trypanosoma and Leishmania.

Author

Asencio, Corinne, Hervé, Perrine, Morand, Pauline, Oliveres, Quentin, Morel, Chloé Alexandra, Prouzet‐Mauleon, Valérie, Biran, Marc, Monic, Sarah, Bonhivers, Mélanie, Robinson, Derrick Roy, Ouellette, Marc, Rivière, Loïc, Bringaud, Frédéric, and Tetaud, Emmanuel

Subjects
TRYPANOSOMA, GENOME editing, AFRICAN trypanosomiasis, STREPTOCOCCUS pyogenes, ECONOMIC impact of disease, LIFE cycles (Biology), LEISHMANIA
Abstract

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro‐synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker‐free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild‐type parasite. [ABSTRACT FROM AUTHOR]

Published
2024
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4. 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

5. 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

6. Les méthodes de diagnostic des trypanosomiases animales africaines : avantages, limites et nouvelles pistes d’amélioration

Author

TOUNKARA, Magamba, BOULANGÉ, Alain, BRINGAUD, Frédéric, Thonnus, Magali, Belem, Adrien Marie Gaston, Bengaly, Zakaria, THÉVENON, Sophie, Berthier, David, RIVIÈRE, Loïc, TOUNKARA, Magamba, Microbiologie Fondamentale et Pathogénicité (MFP), Université Bordeaux Segalen - Bordeaux 2-Centre National de la Recherche Scientifique (CNRS), Interactions hôtes-vecteurs-parasites-environnement dans les maladies tropicales négligées dues aux trypanosomatides (UMR INTERTRYP), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)-Institut de Recherche pour le Développement (IRD)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université de Bordeaux (UB), Département Systèmes Biologiques (Cirad-BIOS), Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad), Université Polytechnique Nazi Boni Bobo-Dioulasso (UNB), Centre international de recherche-développement sur l'élevage en zone sub-humide (CIRDES), Microbiologie Fondamentale et Pathogénicité [Bordeaux] (MFP), Université de Bordeaux (UB)-Centre National de la Recherche Scientifique (CNRS), Université de Bordeaux (UB)-Institut de Recherche pour le Développement (IRD)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre de Coopération Internationale en Recherche Agronomique pour le Développement (Cirad)

Subjects
Trypanosoma, [SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, [SDV.IMM] Life Sciences [q-bio]/Immunology, Diagnosis, [SDV.BA.MVSA] Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health, [SDV.IMM]Life Sciences [q-bio]/Immunology, African anmal trypanosomiasis, Diagnostic, trypanosomiase animale africaine, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology
Abstract

The animal trypanosomiases of African origin are a rather typical example of a neglected tropical disease. They are caused by blood parasites of the Trypanosoma genus transmitted by haematophagous insects, cyclically by tsetse flies for Trypanosoma congolense, T. vivax and T. brucei brucei, which cause nagana, mechanically by T. evansi, the agent of surra, and finally by T. equiperdum, adapted to sexual transmission, which causes dourine. Nagana affects 38 African countries in the sub-Saharan region with 55 million cattle at risk, not counting small ruminants. These diseases deteriorate the animal’s physical condition, leading to a sharp drop in productivity; in the most severe cases, and in the absence of treatment, the animal dies within a few weeks. The socio-economic impact is estimated at US$ 4.75 billion in annual losses, making these diseases one of the major obstacles to livestock development in Africa. The control of these diseases is hampered by the lack of vaccines, a handful of molecules for treatment against which parasites are increasingly developing resistance, and finally, molecular or serological diagnostics whose robustness needs to be greatly improved. Clinical signs are not specific, parasitological diagnosis is often not very sensitive, and molecular methods are not applicable in the field. Immunodiagnostic methods in the form of rapid diagnostic tests are the only ones suitable for the field, but the few existing tests are poorly available and lack specificity and/or sensitivity. Here we address the advantages and limitations of current diagnostic methods and propose new ways of improving them for better disease control., Les trypanosomiases animales d’origine africaine représentent typiquement une maladie tropicale négligée. Elles sont causées par des parasites sanguicoles du genre Trypanosoma transmis par des insectes hématophages, de manière cyclique par les mouches tsé-tsé pour Trypanosoma congolense, T. vivax et T. brucei brucei, qui causent la nagana, de manière mécanique pour T. evansi, agent de la surra, et enfin par transmission sexuelle pour T. equiperdum, qui cause la dourine. La nagana est présente dans 38 pays d’Afrique subsaharienne où elle peut affecter de nombreuses espèces animales, sauvages et domestiques. Près de 55 millions de bovins vivent sous risque trypanosomien, sans compter les petits ruminants. Ces maladies détériorent la condition physique de l’animal, induisant une forte baisse de productivité. Dans les cas les plus sévères, et en l’absence de traitement, la mort survient en quelques semaines. L’impact socio-économique est estimé à 4,75 milliards de dollars de pertes annuelles, ce qui fait de ces maladies l’un des obstacles majeurs au développement de l’élevage en Afrique. Le contrôle de ces maladies se heurte à l’absence de vaccin, à des traitements dont les molécules sont peu nombreuses et contre lesquelles se développent de plus en plus de résistances de la part des parasites, et enfin à un diagnostic, moléculaire ou sérologique, dont la robustesse demande à être grandement améliorée. Les signes cliniques ne sont pas spécifiques, le diagnostic parasitologique est souvent peu sensible, et les méthodes moléculaires ne sont pas applicables sur le terrain. Les méthodes d’immunodiagnostic sous forme de tests de diagnostic rapide sont les seules adaptées au terrain, mais les rares tests existants sont très peu accessibles (disponibilité, prix), et manquent de spécificité et/ou de sensibilité. Ici, nous traiterons des limites et des avantages comparatifs des méthodes actuelles de diagnostic et proposerons de nouvelles pistes d’amélioration de ces dernières pour un meilleur contrôle de la maladie.

Published
2021

7. 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

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

Author

Pineda, Erika, Thonnus, Magali, Mazet, Muriel, Mourier, Arnaud, Cahoreau, Edern, Kulyk, Hanna, Dupuy, Jean-William, Biran, Marc, Masante, Cyril, Allmann, Stefan, Rivière, Loïc, Rotureau, Brice, Portais, Jean-Charles, and Bringaud, Frédéric

Subjects
TRYPANOSOMA brucei, GLYCERIN, GLUCONEOGENESIS, WESTERN immunoblotting, FAT cells, MONOSACCHARIDES
Abstract

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. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues. [ABSTRACT FROM AUTHOR]

Published
2018
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9. Acetate produced in the mitochondrion is the essential precursor for lipid biosynthesis in procyclic trypanosomes.

Author

Rivière, Loïc, Moreau, Patrick, Allmann, Stefan, Hahn, Matthias, Biran, Marc, Plazolles, Nicolas, Franconi, Jean-Michel, Boshart, Michael, and Bringaud, Frédéric

Subjects
*BIOCHEMISTRY, *LIPIDS, *TRYPANOSOMA, *BIOSYNTHESIS, *ACETATES, *MITOCHONDRIA
Abstract

Acetyl-CoA produced in mitochondria from carbohydrate or amino acid catabolism needs to reach the cytosol to initiate de novo synthesis of fatty acids. All eukaryotes analyzed so far use a citrate/malate shuttle to transfer acetyl group equivalents from the mitochondrial matrix to the cytosol. Here we investigate how this acetyl group transfer occurs in the procyclic life cycle stage of Trypanosoma brucei, a protozoan parasite responsible of human sleeping sickness and economically important livestock diseases. Deletion of the potential citrate lyase gene, a critical cytosolic enzyme of the citrate/malate shuttle, has no effect on de novo biosynthesis of fatty acids from 14C-labeled glucose, indicating that another route is used for acetyl group transfer. Because acetate is produced from acetyl-CoA in the mitochondrion of this parasite, we considered genes encoding cytosolic enzymes producing acetyl-CoA from acetate. We identified an acetyl-CoA synthetase gene encoding a cytosolic enzyme (AceCS), which is essential for cell viability. Repression of AceCS by inducible RNAi results in a 20-fold reduction of 14C-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. [ABSTRACT FROM AUTHOR]

Published
2009
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10. Energy metabolism of trypanosomatids: Adaptation to available carbon sources

Author

Bringaud, Frédéric, Rivière, Loïc, and Coustou, Virginie

Subjects
*TRYPANOSOMATIDAE, *PROTOZOAN diseases, *TRYPANOSOMA, *FERMENTATION
Abstract

Abstract: Some development stages of the trypanosomatid protozoan parasites are well adapted to in vitro culture. They can be maintained in rich medium containing large excess of glucose and amino acids, which they use as carbon sources for ATP production. Under these growth conditions, carbon sources are converted into partially oxidized end products by so-called aerobic fermentation. Surprisingly, some species, such as the Trypanosoma brucei, Trypanosoma cruzi and Crithidia insect stages, prefer consuming glucose to amino acids, although their natural habitat is l-proline-rich. This review focuses on recent progress in understanding glucose and l-proline metabolism of insect stages, how these metabolic processes are regulated, and the rationale of the aerobic fermentation strategies developed by these parasites. [Copyright &y& Elsevier]

Published
2006
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11. Fumarate Is an Essential Intermediary Metabolite Produced by the Procyclic Trypanosoma brucei.

Author

Coustou, Virginie, Biran, Marc, Besteiro, Sébastien, Rivière, Loïc, Baltz, Théo, Franconi, Jean-Michel, and Bringaud, Frédéric

Subjects
*SUCCINATE dehydrogenase, *METABOLITES, *TRYPANOSOMA brucei, *ENZYMES, *TRYPANOSOMA
Abstract

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189 -200). [ABSTRACT FROM AUTHOR]

Published
2006
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