Abstract
We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol, but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part of the Krebs cycle consisting of alpha-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.
Highlights
Trypanosoma brucei is a unicellular eukaryote that causes sleeping sickness in humans and nagana in livestock
Because significant production of labeled CO2 from [6-14C]glucose can occur only when pyruvate is degraded by the Krebs cycle, this result confirms that the activity of a complete Krebs cycle is negligible in procyclic cells [7]
As could be expected from the Influences of Substrate Availability—The absence of glucose and/or glycerol in SDM-79 medium did result in decreased growth, but did not result in halted proliferation or cell death of procyclic cells of this pleomorphic T. brucei strain
Summary
Trypanosome Culture Conditions—The pleomorphic procyclic T. brucei brucei TREU 927 strain (a gift from Jeremy Mottram, Wellcome Centre for Molecular Parasitology, Glasgow, Scotland, United Kingdom) was grown at 27 °C in the presence of 5% CO2 in SDM-79 medium with different glucose and/or glycerol concentrations. Standard culturing was carried out in SDM-79 medium containing 10 mM glucose, and changes to other media were performed by dilution (1:5) in the new medium for at least 5 consecutive days before the metabolic studies were performed. This procedure allows possible adaptations to take place. To investigate whether radioactive carbons were incorporated into saturated or unsaturated acyl chains, fatty acids obtained after petroleum ether extraction were either analyzed directly or first dissolved in methanol and hydrogenated by hydrogen gas in the presence of platinum(IV) oxide for 2 h before conversion into phenylacyl derivatives
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