title: Procyclic Trypanosoma brucei do not use Krebs cycle activity for energy generation.
abstract: The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitro differentiation from wild type and mutant (Delta aco::NEO/Delta aco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle to CO(2). Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly, there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
authors.affiliation: Department of Biochemistry and Cell Biology, Faculty of Veterinary Medicine, Utrecht University, NL-3508 TD Utrecht, The Netherlands.
authors.forename: Susanne W H
authors.initials: SW
authors.lastname: van Weelden
authors.forename: Beate
authors.initials: B
authors.lastname: Fast
authors.forename: Achim
authors.initials: A
authors.lastname: Vogt
authors.forename: Pieter
authors.initials: P
authors.lastname: van der Meer
authors.forename: Joachim
authors.initials: J
authors.lastname: Saas
authors.forename: Jaap J
authors.initials: JJ
authors.lastname: van Hellemond
authors.forename: Aloysius G M
authors.initials: AG
authors.lastname: Tielens
authors.forename: Michael
authors.initials: M
authors.lastname: Boshart
mesh_terms: D000154:Aconitate Hydratase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002952:Citric Acid Cycle; D004734:Energy Metabolism / Q000502:physiology*; D017353:Gene Deletion; D005947:Glucose / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007521:Isocitrate Dehydrogenase / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D007655:Ketoglutarate Dehydrogenase Complex / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D011190:Potassium Cyanide / Q000494:pharmacology; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
chemical_list: D007521:Isocitrate Dehydrogenase; D007655:Ketoglutarate Dehydrogenase Complex; D005969:Glutamate Dehydrogenase; D000154:Aconitate Hydratase; D005947:Glucose; D011190:Potassium Cyanide