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Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.

ABSTRACT: Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain the glycosomal NAD(+)/NADH balance as follows: (i) the glycosomal succinic fermentation branch; (ii) the glycerol 3-phosphate (Gly-3-P)/dihydroxyacetone phosphate (DHAP) shuttle that transfers reducing equivalents to the mitochondrion; and (iii) the glycosomal glycerol production pathway. We showed a hierarchy in the use of these glycosomal NADH-consuming pathways by determining metabolic perturbations and adaptations in single and double mutant cell lines using a combination of NMR, ion chromatography-MS/MS, and HPLC approaches. Although functional, the Gly-3-P/DHAP shuttle is primarily used when the preferred succinate fermentation pathway is abolished in the ?pepck knock-out mutant cell line. In the absence of these two pathways (?pepck/(RNAi)FAD-GPDH.i mutant), glycerol production is used but with a 16-fold reduced glycolytic flux. In addition, the ?pepck mutant cell line shows a 3.3-fold reduced glycolytic flux compensated by an increase of proline metabolism. The inability of the ?pepck mutant to maintain a high glycolytic flux demonstrates that the Gly-3-P/DHAP shuttle is not adapted to the procyclic trypanosome context. In contrast, this shuttle was shown earlier to be the only way used by the bloodstream forms of T. brucei to sustain their high glycolytic flux.

SUBMITTER: Ebikeme C

PROVIDER: S-EPMC2952232 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.

Ebikeme Charles C Hubert Jane J Biran Marc M Gouspillou Gilles G Morand Pauline P Plazolles Nicolas N Guegan Fabien F Diolez Philippe P Franconi Jean-Michel JM Portais Jean-Charles JC Bringaud Frédéric F

The Journal of biological chemistry 20100811 42

Trypanosoma brucei is a parasitic protist that undergoes a complex life cycle during transmission from its mammalian host (bloodstream forms) to the midgut of its insect vector (procyclic form). In both parasitic forms, most glycolytic steps take place within specialized peroxisomes, called glycosomes. Here, we studied metabolic adaptations in procyclic trypanosome mutants affected in their maintenance of the glycosomal redox balance. T. brucei can theoretically use three strategies to maintain ...[more]

PMID: 20702405

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Project description:The glycerol-3-phosphate (G-3-P) shuttle is an important pathway for delivery of cytosolic reducing equivalents into mitochondrial oxidative phosphorylation, and plays essential physiological roles in yeast, plants, and animals. However, its role has been unclear in filamentous and pathogenic fungi. Here, we characterize the function of the G-3-P shuttle in Pyricularia oryzae by genetic and molecular analyses. In P. oryzae, a glycerol-3-phosphate dehydrogenase 1 (PoGpd1) is involved in NO production, conidiation, and utilization of several carbon sources (pyruvate, sodium acetate, glutamate, and glutamine). A glycerol-3-phosphate dehydrogenase 2 (PoGpd2) is essential for glycerol utilization and fungal development. Deletion of PoGPD2 led to delayed aerial hyphal formation, accelerated aerial hyphal collapse, and reduced conidiation on complete medium (CM) under a light-dark cycle. Aerial mycelial surface hydrophobicity to water and Tween 20 was decreased in ?Pogpd2. Melanin synthesis genes required for cell wall construction and two transcription factor genes (COS1 and CONx2) required for conidiation and/or aerial hyphal differentiation were down-regulated in the aerial mycelia of ?Pogpd2 and ?Pogpd1. Culturing under continuous dark could complement the defects of aerial hyphal differentiation of ?Pogpd2 observed in a light-dark cycle. Two light-sensitive protein genes (PoSIR2 encoding an NAD+-dependent deacetylase and TRX2 encoding a thioredoxin 2) were up-regulated in ?Pogpd2 cultured on CM medium in a light-dark cycle. ?Pogpd2 showed an increased intracellular NAD+/NADH ratio and total NAD content, and alteration of intracellular ATP production. Culturing on minimal medium also could restore aerial hyphal differentiation of ?Pogpd2, which is deficient on CM medium in a light-dark cycle. Two glutamate synthesis genes, GDH1 and PoGLT1, which synthesize glutamate coupled with oxidation of NADH to NAD+, were significantly up-regulated in ?Pogpd2 in a light-dark cycle. Moreover, deletion of PoGpd1 or PoGpd2 led to reduced virulence of conidia or hyphae on rice. The glycerol-3-phosphate shuttle is involved in cellular redox, fungal development, and virulence in P. oryzae.

Project description:1. Rat liver slices were employed to study the relative rates of incorporation of a mixture of [2-(3)H]- or [1,3-(3)H]-glycerol and [1-(14)C]glycerol into lipids. 2. With 0.1mm-glycerol approx. 82% of the newly synthesized lipid, calculated from (14)C incorporation, was present as neutral lipid, 13% as phosphatidylcholine and 5% as phosphatidylethanolamine. Increasing the glycerol concentration to 40mm caused a decrease in the percentage of neutral lipid to 59% and a corresponding increase in the percentage of phosphatidylcholine to 36% of the newly synthesized lipid. 3. The (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in glycerolipid was considerably higher than that in precursor glycerol throughout the range of experimental conditions. In contrast the incorporation of a mixture of [1,3-(3)H]glycerol and [1-(14)C]glycerol into lipid occurred with little or no change in the (3)H/(14)C ratio. 4. Respiring rat liver mitochondria were found to oxidize a mixture of sn-[2-(3)H]- and sn-[1-(14)C]-glycerol 3-phosphate with a resultant increase in the (3)H/(14)C ratio of the remaining sn-glycerol 3-phosphate. This increase is due to a (3)H isotope effect of the mitochondrial sn-glycerol 3-phosphate dehydrogenase (EC 1.1.99.5), which discriminates against sn-[2-(3)H]glycerol 3-phosphate during oxidation. 5. A method is described for the simultaneous determination of the relative contributions of the glycerol phosphate and dihydroxyacetone phosphate pathways of glycerolipid biosynthesis in rat liver slices. The method involves measurement of the (d.p.m. of 2-(3)H)/(d.p.m. of 1-(14)C) ratio in both sn-glycerol 3-phosphate and glycerolipid after incubation of rat liver slices with a mixture of [2-(3)H]glycerol and [1-(14)C]glycerol for various times. 6. By using this method it was shown that 40-50% of the glycerol incorporated into lipid by rat liver slices proceeded via the sn-glycerol 3-phosphate pathway and 50-60% was incorporated via dihydroxyacetone phosphate.

Dataset Information

Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.

Publications

Ablation of succinate production from glucose metabolism in the procyclic trypanosomes induces metabolic switches to the glycerol 3-phosphate/dihydroxyacetone phosphate shuttle and to proline metabolism.

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