Revisiting the Central Metabolism of the Bloodstream Forms of Trypanosoma brucei: Production of Acetate in the Mitochondrion Is Essential for Parasite Viability

Abstract

BackgroundThe bloodstream forms of Trypanosoma brucei, the causative agent of sleeping sickness, rely solely on glycolysis for ATP production. It is generally accepted that pyruvate is the major end-product excreted from glucose metabolism by the proliferative long-slender bloodstream forms of the parasite, with virtually no production of succinate and acetate, the main end-products excreted from glycolysis by all the other trypanosomatid adaptative forms, including the procyclic insect form of T. brucei.Methodology/Principal FindingsA comparative NMR analysis showed that the bloodstream long-slender and procyclic trypanosomes excreted equivalent amounts of acetate and succinate from glucose metabolism. Key enzymes of acetate production from glucose-derived pyruvate and threonine are expressed in the mitochondrion of the long-slender forms, which produces 1.4-times more acetate from glucose than from threonine in the presence of an equal amount of both carbon sources. By using a combination of reverse genetics and NMR analyses, we showed that mitochondrial production of acetate is essential for the long-slender forms, since blocking of acetate biosynthesis from both carbon sources induces cell death. This was confirmed in the absence of threonine by the lethal phenotype of RNAi-mediated depletion of the pyruvate dehydrogenase, which is involved in glucose-derived acetate production. In addition, we showed that de novo fatty acid biosynthesis from acetate is essential for this parasite, as demonstrated by a lethal phenotype and metabolic analyses of RNAi-mediated depletion of acetyl-CoA synthetase, catalyzing the first cytosolic step of this pathway.Conclusions/SignificanceAcetate produced in the mitochondrion from glucose and threonine is synthetically essential for the long-slender mammalian forms of T. brucei to feed the essential fatty acid biosynthesis through the “acetate shuttle” that was recently described in the procyclic insect form of the parasite. Consequently, key enzymatic steps of this pathway, particularly acetyl-CoA synthetase, constitute new attractive drug targets against trypanosomiasis.

Contribution of Pyruvate Phosphate Dikinase in the Maintenance of the Glycosomal ATP/ADP Balance in the Trypanosoma brucei Procyclic Form

“…Indeed, a recently developed metabolite profiling assay showed that threonine is the main acetate source of procyclic trypanosomes (11,46). This approach is based on the ability of 1 H NMR spectrometry to distinguish 13 Fig.…”

“…For 1 H NMR analyses of glucose and threonine metabolism, 10 8 T. brucei procyclic cells were collected by centrifugation at 1,400 ϫ g for 10 min, washed once with PBS, and incubated for 6 h at 27°C in 5 ml of incubation buffer (PBS supplemented with 5 g/liter of NaHCO 3 , pH 7.4) containing D-[U- 13 C]glucose (4 mM) and threonine (4 mM). 50 l of maleate (20 mM) were added as an internal reference to a 500-l aliquot of the collected supernatant, and 1 H NMR spectra were performed at 500.13 MHz on a Bruker DPX500 spectrometer equipped with a 5-mm broadband probe head as described before (11,46 . In both NMR analyses, the integrity of the cells during the incubation was checked by microscopic observation.…”

Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum

“…PrPta II 1 activity in the acetyl-CoA-forming direction was measured by monitoring the increase in absorbance at 233 nm due to thioester bond formation (ε 233 ϭ 5.55 mM Ϫ1 cm Ϫ1 ) (20,21). The reaction mixture contained 50 mM Tris (pH 7.5), 20 mM KCl, 20 mM NH 4 Cl, and 1 mM dithiothreitol (DTT), and the concentrations of acetyl phosphate and CoA were varied. Activity in the acetyl phosphate-forming direction was measured with two different assays.…”

“…A cetate production has been studied for many years in bacteria but has received less attention in eukaryotic microbes, even though acetate is produced as an important end product of energy metabolism in yeasts (1-3) and protists (4)(5)(6). Four different pathways for production of acetate from acetyl-coenzyme A (CoA) have been identified in eukaryotic microbes (7).…”

“…Acetyl-CoA ϩ H 2 O ↔ acetate ϩ CoA (3) Phosphotransacetylase (Pta) (EC 2.3.1.8) (equation 4) and acetate kinase (Ack) (EC 2.7.2.1) (equation 5) form a pathway for the interconversion of acetate and acetyl-CoA that was previously thought to be limited to bacteria and one genus of archaea but has now been shown to be present in eukaryotes, such as green algae and Phytophthora (13,14). Acetyl-CoA ϩ P i ↔ acetyl phosphate ϩ CoA (4) Acetyl phosphate ϩ ADP ↔ acetate ϩ ATP (5) The Pta-Ack pathway is best understood in its roles in both acetate production and assimilation in Escherichia coli and other bacteria. The pathway is responsible for the production of acetate during mixed-acid fermentation under hypoxic conditions and in a metabolic overflow mechanism in which acetyl-CoA is diverted from the tricarboxylic acid (TCA) cycle when there is an imbalance between the rapid uptake of glucose and its conversion into products (15).…”