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

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