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.

Fakultät für Biologie Genetik Ludwig Maximilians Universität München Grosshadernerstrasse 2 4 Martinsried Germany

Glasgow Polyomics Wolfson Wohl Cancer Research Centre Garscube Campus College of Medical Veterinary and Life Sciences University of Glasgow Glasgow United Kingdom

Institute of Parasitology Biology Center Czech Academy of Sciences České Budějovice Czech Republic

Laboratory of Biochemistry of Tryps LaBTryps Department of Parasitology Institute of Biomedical Sciences University of São Paulo São Paulo Brazil

MetaToul MetaboHub National Infrastructure of Metabolomics and Fluxomics Toulouse France

RESTORE Université de Toulouse Inserm U1031 CNRS 5070 UPS EFS ENVT Toulouse France

Toulouse Biotechnology Institute TBI INSA de Toulouse INSA CNRS 5504 UMR INSA INRA 792 Toulouse France

Univ Bordeaux CNRS Centre de Résonance Magnétique des Systèmes Biologiques UMR 5536 Bordeaux France

Univ Bordeaux CNRS Microbiologie Fondamentale et Pathogénicité UMR 5234 Bordeaux France

Wellcome Centre for Integrative Parasitology Institute of Infection Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow Glasgow United Kingdom

See more in PubMed

Buscher P, Cecchi G, Jamonneau V, Priotto G. Human African trypanosomiasis. Lancet. 2017;390: 2397–2409. 10.1016/S0140-6736(17)31510-6 PubMed DOI

Opperdoes FR, Borst P. Localization of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett. 1977;80: 360–4. 0014-5793(77)80476-6 [pii] 10.1016/0014-5793(77)80476-6 PubMed DOI

Visser N, Opperdoes FR. Glycolysis in Trypanosoma brucei. Eur J Biochem. 1980;103: 623–32. 10.1111/j.1432-1033.1980.tb05988.x PubMed DOI

Mazet M, Morand P, Biran M, Bouyssou G, Courtois P, Daulouede S, et al.. Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability. PLoS Negl Trop Dis. 2013;7: e2587. 10.1371/journal.pntd.0002587 PNTD-D-13-01131 [pii] PubMed DOI PMC

Bringaud F, Riviere L, Coustou V. Energy metabolism of trypanosomatids: adaptation to available carbon sources. Mol Biochem Parasitol. 2006;149: 1–9. S0166-6851(06)00115-0 [pii] 10.1016/j.molbiopara.2006.03.017 PubMed DOI

Lamour N, Riviere L, Coustou V, Coombs GH, Barrett MP, Bringaud F. Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose. J Biol Chem. 2005;280: 11902–11910. 10.1074/jbc.M414274200 PubMed DOI

Coustou V, Biran M, Breton M, Guegan F, Riviere L, Plazolles N, et al.. Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei. J Biol Chem. 2008;283: 16342–54. M709592200 [pii] 10.1074/jbc.M709592200 PubMed DOI

Mantilla BS, Marchese L, Casas-Sanchez A, Dyer NA, Ejeh N, Biran M, et al.. Proline metabolism is essential for Trypanosoma brucei brucei survival in the tsetse vector. PLoS Pathog. 2017;13: e1006158. 10.1371/journal.ppat.1006158 PubMed DOI PMC

Bursell E. The role of proline in energy metabolism. New York: Plenum Press; 1981.

Obungu VH, Kiaira JK, Olembo NK, Njobu MR. Pathways of glucose catabolism on procyclic Trypanosoma congolense. Indian J Biochem Biophys. 1999;36: 305–11. PubMed

Spitznagel D, Ebikeme C, Biran M, Nic A. Bhaird N, Bringaud F, Henehan GT, et al.. Alanine aminotransferase of Trypanosoma brucei—a key role in proline metabolism in procyclic life forms. FEBS J. 2009;276: 7187–7199. EJB7432 [pii] 10.1111/j.1742-4658.2009.07432.x PubMed DOI

Duschak VG, Cazzulo JJ. Subcellular localization of glutamate dehydrogenases and alanine aminotransferase in epimastigotes of Trypanosoma cruzi. FEMS Microbiol Lett. 1991;67: 131–5. 10.1016/0378-1097(91)90343-9 PubMed DOI

Allmann S, Morand P, Ebikeme C, Gales L, Biran M, Hubert J, et al.. Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux. J Biol Chem. 2013;288: 18494–505. M113.462978 [pii] 10.1074/jbc.M113.462978 PubMed DOI PMC

Millerioux Y, Morand P, Biran M, Mazet M, Moreau P, Wargnies M, et al.. ATP synthesis-coupled and -uncoupled acetate production from acetyl-CoA by the mitochondrial acetate:succinate CoA-transferase and acetyl-CoA thioesterase in Trypanosoma. J Biol Chem. 2012;287: 17186–17197. M112.355404 [pii] 10.1074/jbc.M112.355404 PubMed DOI PMC

Van Weelden SW, Fast B, Vogt A, Van Der Meer P, Saas J, Van Hellemond JJ, et al.. Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation. J Biol Chem. 2003;278: 12854–12863. 10.1074/jbc.M213190200 PubMed DOI

van Hellemond JJ, Opperdoes FR, Tielens AG. The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei. Biochem Soc Trans. 2005;33: 967–71. 10.1042/BST20050967 PubMed DOI

Welburn SC, Arnold K, Maudlin I, Gooday GW. Rickettsia-like organisms and chitinase production in relation to transmission of trypanosomes by tsetse flies. Parasitology. 1993;107 (Pt 2): 141–5. 10.1017/s003118200006724x PubMed DOI

Hall RJ, Flanagan LA, Bottery MJ, Springthorpe V, Thorpe S, Darby AC, et al.. A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet. MBio. 2019;10. 10.1128/mBio.02106-18 PubMed DOI PMC

Ong HB, Lee WS, Patterson S, Wyllie S, Fairlamb AH. Homoserine and quorum-sensing acyl homoserine lactones as alternative sources of threonine: a potential role for homoserine kinase in insect-stage Trypanosoma brucei. Mol Microbiol. 2014;95: 143–156. 10.1111/mmi.12853 PubMed DOI PMC

Balogun RA. Studies on the amino acids of the tsetse fly, Glossina morsitans, maintained on in vitro and in vivo feeding systems. Comp Biochem Physiol Comp Physiol. 1974;49: 215–22. 10.1016/0300-9629(74)90110-8 PubMed DOI

Millerioux Y, Ebikeme C, Biran M, Morand P, Bouyssou G, Vincent IM, et al.. The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control. Mol Microbiol. 2013;90: 114–129. 10.1111/mmi.12351 PubMed DOI PMC

Bringaud F, Biran M, Millerioux Y, Wargnies M, Allmann S, Mazet M. Combining reverse genetics and NMR-based metabolomics unravels trypanosome-specific metabolic pathways. Mol Microbiol. 2015;96: 917–926. 10.1111/mmi.12990 PubMed DOI

Wargnies M, Bertiaux E, Cahoreau E, Ziebart N, Crouzols A, Morand P, et al.. Gluconeogenesis is essential for trypanosome development in the tsetse fly vector. PLoS Pathog. 2018;14: e1007502. 10.1371/journal.ppat.1007502 PubMed DOI PMC

Coustou V, Besteiro S, Riviere L, Biran M, Biteau N, Franconi JM, et al.. A mitochondrial NADH-dependent fumarate reductase involved in the production of succinate excreted by procyclic Trypanosoma brucei. J Biol Chem. 2005;280: 16559–70. 10.1074/jbc.M500343200 PubMed DOI

Coustou V, Biran M, Besteiro S, Riviere L, Baltz T, Franconi JM, et al.. Fumarate is an essential intermediary metabolite produced by the procyclic Trypanosoma brucei. J Biol Chem. 2006;281: 26832–46. 10.1074/jbc.M601377200 PubMed DOI

Bringaud F, Ebikeme CE, Boshart M. Acetate and succinate production in amoebae, helminths, diplomonads, trichomonads and trypanosomatids: common and diverse metabolic strategies used by parasitic lower eukaryotes. Parasitology. 2010;137: 1315–1331. 10.1017/S0031182009991843 PubMed DOI

Muller M, Mentel M, van Hellemond JJ, Henze K, Woehle C, Gould SB, et al.. Biochemistry and evolution of anaerobic energy metabolism in eukaryotes. Microbiol Mol Biol Rev. 2012;76: 444–95. 76/2/444 [pii] 10.1128/MMBR.05024-11 PubMed DOI PMC

Engqvist MK, Esser C, Maier A, Lercher MJ, Maurino VG. Mitochondrial 2-hydroxyglutarate metabolism. Mitochondrion. 2014;19 Pt B: 275–81. 10.1016/j.mito.2014.02.009 PubMed DOI

Intlekofer AM, Wang B, Liu H, Shah H, Carmona-Fontaine C, Rustenburg AS, et al.. L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH. Nat Chem Biol. 2017;13: 494–500. 10.1038/nchembio.2307 PubMed DOI PMC

Johnston K, Kim DH, Kerkhoven EJ, Burchmore R, Barrett MP, Achcar F. Mapping the metabolism of five amino acids in bloodstream form Trypanosoma brucei using U-(13)C-labelled substrates and LC-MS. Biosci Rep. 2019;39. 10.1042/BSR20181601 PubMed DOI PMC

Bringaud F, Stripecke R, Frech GC, Freedland S, Turck C, Byrne EM, et al.. Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein. Mol Cell Biol. 1997;17: 3915–23. 10.1128/mcb.17.7.3915 PubMed DOI PMC

Kolev NG, Ramey-Butler K, Cross GA, Ullu E, Tschudi C. Developmental progression to infectivity in Trypanosoma brucei triggered by an RNA-binding protein. Science. 2012;338: 1352–3. 338/6112/1352 [pii] 10.1126/science.1229641 PubMed DOI PMC

Urwyler S, Studer E, Renggli CK, Roditi I. A family of stage-specific alanine-rich proteins on the surface of epimastigote forms of Trypanosoma brucei. Mol Microbiol. 2007;63: 218–228. 10.1111/j.1365-2958.2006.05492.x PubMed DOI

Dolezelova E, Kunzova M, Dejung M, Levin M, Panicucci B, Regnault C, et al.. Cell-based and multi-omics profiling reveals dynamic metabolic repurposing of mitochondria to drive developmental progression of Trypanosoma brucei. PLoS Biol. 2020;18: e3000741. 10.1371/journal.pbio.3000741 PubMed DOI PMC

Emmer BT, Daniels MD, Taylor JM, Epting CL, Engman DM. Calflagin inhibition prolongs host survival and suppresses parasitemia in Trypanosoma brucei infection. Eukaryot Cell. 2010;9: 934–42. 10.1128/EC.00086-10 PubMed DOI PMC

Ryley JF. Studies on the metabolism of protozoa. 9. Comparative metabolism of bloodstream and culture forms of Trypanosoma rhodesiense. Biochem J. 1962;85: 211–223. 10.1042/bj0850211 PubMed DOI PMC

Vickerman K. Polymorphism and mitochondrial activity in sleeping sickness trypanosomes. Nature. 1965;208: 762–6. 10.1038/208762a0 PubMed DOI

Flynn IW, Bowman IB. The metabolism of carbohydrate by pleomorphic African trypanosomes. Comp Biochem Physiol B. 1973;45: 25–42. 10.1016/0305-0491(73)90281-2 PubMed DOI

Bienen EJ, Maturi RK, Pollakis G, Clarkson AB. Non-cytochrome mediated mitochondrial ATP production in bloodstream form Trypanosoma brucei brucei. Eur J Biochem. 1993;216: 75–80. 10.1111/j.1432-1033.1993.tb18118.x PubMed DOI

Dewar CE, MacGregor P, Cooper S, Gould MK, Matthews KR, Savill NJ, et al.. Mitochondrial DNA is critical for longevity and metabolism of transmission stage Trypanosoma brucei. PLoS Pathog. 2018;14: e1007195. 10.1371/journal.ppat.1007195 PubMed DOI PMC

Wang X, Inaoka DK, Shiba T, Balogun EO, Allmann S, Watanabe YI, et al.. Expression, purification, and crystallization of type 1 isocitrate dehydrogenase from Trypanosoma brucei brucei. Protein Expr Purif. 2017;138: 56–62. 10.1016/j.pep.2017.06.011 PubMed DOI

Riviere L, Moreau P, Allmann S, Hahn M, Biran M, Plazolles N, et al.. Acetate produced in the mitochondrion is the essential precursor of lipid biosynthesis in procyclic trypanosomes. Proc Natl Aca Sci USA. 2009;106: 12694–12699. 10.1073/pnas.0903355106 PubMed DOI PMC

Forchhammer K, Selim KA. Carbon/Nitrogen Homeostasis Control in Cyanobacteria. FEMS Microbiol Rev. 2019. 10.1093/femsre/fuz025 PubMed DOI PMC

Li F, Xu W, Zhao S. Regulatory roles of metabolites in cell signaling networks. J Genet Genomics. 2013;40: 367–74. 10.1016/j.jgg.2013.05.002 PubMed DOI

Huergo LF, Dixon R. The Emergence of 2-Oxoglutarate as a Master Regulator Metabolite. Microbiol Mol Biol Rev. 2015;79: 419–35. 10.1128/MMBR.00038-15 PubMed DOI PMC

Ryan DG, Murphy MP, Frezza C, Prag HA, Chouchani ET, O’Neill LA, et al.. Coupling Krebs cycle metabolites to signalling in immunity and cancer. Nat Metab. 2019;1: 16–33. 10.1038/s42255-018-0014-7 PubMed DOI PMC

Rzem R, Vincent MF, Van Schaftingen E, Veiga-da-Cunha M. L-2-hydroxyglutaric aciduria, a defect of metabolite repair. J Inherit Metab Dis. 2007;30: 681–9. 10.1007/s10545-007-0487-0 PubMed DOI

Girard R, Crispim M, Alencar MB, Silber AM. Uptake of l-Alanine and Its Distinct Roles in the Bioenergetics of Trypanosoma cruzi. mSphere. 2018;3. 10.1128/mSphereDirect.00338-18 PubMed DOI PMC

Wolfe AJ. The acetate switch. Microbiol Mol Biol Rev. 2005;69: 12–50. 10.1128/MMBR.69.1.12-50.2005 PubMed DOI PMC

Ghozlane A, Bringaud F, Souedan H, Dutour I, Jourdan F, Thébault P. Flux analysis of the Trypanosoma brucei glycolysis based on a multiobjective-criteria bioinformatic approach. Adv Bioinforma. 2012;2012: 159423. 10.1155/2012/159423 PubMed DOI PMC

Brun R, Schonenberger M. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Acta Trop. 1979;36: 289–92. PubMed

Wirtz E, Leal S, Ochatt C, Cross GA. A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. Mol Biochem Parasitol. 1999;99: 89–101. 10.1016/s0166-6851(99)00002-x PubMed DOI

Wickstead B, Ersfeld K, Gull K. Targeting of a tetracycline-inducible expression system to the transcriptionally silent minichromosomes of Trypanosoma brucei. Mol Biochem Parasitol. 2002;125: 211–6. 10.1016/s0166-6851(02)00238-4 PubMed DOI

Bringaud F, Baltz D, Baltz T. Functional and molecular characterization of a glycosomal PPi-dependent enzyme in trypanosomatids: pyruvate, phosphate dikinase. Proc Natl Acad Sci USA. 1998;95: 7963–8. 10.1073/pnas.95.14.7963 PubMed DOI PMC

Harlow E, Lane D. Antibodies: a laboratory manual. Cold Spring Harbor Laboratory Press; 1988.

Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press; 1989.

Riviere L, van Weelden SW, Glass P, Vegh P, Coustou V, Biran M, et al.. Acetyl:succinate CoA-transferase in procyclic Trypanosoma brucei. Gene identification and role in carbohydrate metabolism. J Biol Chem. 2004;279: 45337–46. 10.1074/jbc.M407513200 M407513200 [pii] PubMed DOI

Clayton CE. Import of fructose bisphosphate aldolase into the glycosomes of Trypanosoma brucei. J Cell Biol. 1987;105: 2649–54. 10.1083/jcb.105.6.2649 PubMed DOI PMC

Bringaud F, Peyruchaud S, Baltz D, Giroud C, Simpson L, Baltz T. Molecular characterization of the mitochondrial heat shock protein 60 gene from Trypanosoma brucei. Mol Biochem Parasitol. 1995;74: 119–23. 10.1016/0166-6851(95)02486-7 PubMed DOI

Chaudhuri M, Ajayi W, Hill GC. Biochemical and molecular properties of the Trypanosoma brucei alternative oxidase. Mol Biochem Parasitol. 1998;95: 53–68. 10.1016/s0166-6851(98)00091-7 PubMed DOI

Brown ED, Wood JM. Conformational change and membrane association of the PutA protein are coincident with reduction of its FAD cofactor by proline. J Biol Chem. 1993;268: 8972–9. PubMed

Bergmeyer HU, Bergmeyer J, Grassi M. Methods of enzymatic analysis. Wiley; 1983. 10.1016/0003-2697(83)90607-3 DOI

Aranda A, Maugeri D, Uttaro AD, Opperdoes F, Cazzulo JJ, Nowicki C. The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms. Int J Parasitol. 2006;36: 295–307. 10.1016/j.ijpara.2005.09.013 PubMed DOI

Distribution and Functional Analysis of Isocitrate Dehydrogenases across Kinetoplastids

In silico prediction of the metabolism of Blastocrithidia nonstop, a trypanosomatid with non-canonical genetic code

Mitochondrion of the Trypanosoma brucei long slender bloodstream form is capable of ATP production by substrate-level phosphorylation