Background: Mitochondrial acetate production is essential for viability of the procyclic trypanosomes and probably many other protists. Results:We identified an acetyl-CoA thioesterase (ACH) contributing to acetate production from acetyl-CoA. Conclusion: Acetate production by ASCT, but not by ACH, is involved in ATP production. Significance: In trypanosomes and probably other protists, ASCT/SCoAS cycle-derived ATP production can substitute for oxidative phosphorylation.
Background:The role of pyruvate phosphate dikinase (PPDK), which catalyzes a reversible reaction, is unknown in many eukaryotes. Results: Deletion of the trypanosomal PPDK gene affects glycolysis. Conclusion:In trypanosomes, PPDK works in the glycolytic direction and participates in the maintenance of the glycosomal ATP/ADP balance. Significance: The glycosomal PPDK provides a metabolic flexibility by producing 2 ATP per phosphoenolpyruvate consumed.
In the glucose-free environment that is the midgut of the tsetse fly vector, the procyclic form of Trypanosoma brucei primarily uses proline to feed its central carbon and energy metabolism. In these conditions, the parasite needs to produce glucose 6-phosphate (G6P) through gluconeogenesis from metabolism of non-glycolytic carbon source(s). We showed here that two phosphoenolpyruvate-producing enzymes, PEP carboxykinase (PEPCK) and pyruvate phosphate dikinase (PPDK) have a redundant function for the essential gluconeogenesis from proline. Indeed, incorporation of 13C-enriched proline into G6P was abolished in the PEPCK/PPDK null double mutant (Δppdk/Δpepck), but not in the single Δppdk and Δpepck mutant cell lines. The procyclic trypanosome also uses the glycerol conversion pathway to feed gluconeogenesis, since the death of the Δppdk/Δpepck double null mutant in glucose-free conditions is only observed after RNAi-mediated down-regulation of the expression of the glycerol kinase, the first enzyme of the glycerol conversion pathways. Deletion of the gene encoding fructose-1,6-bisphosphatase (Δfbpase), a key gluconeogenic enzyme irreversibly producing fructose 6-phosphate from fructose 1,6-bisphosphate, considerably reduced, but not abolished, incorporation of 13C-enriched proline into G6P. In addition, the Δfbpase cell line is viable in glucose-free conditions, suggesting that an alternative pathway can be used for G6P production in vitro. However, FBPase is essential in vivo, as shown by the incapacity of the Δfbpase null mutant to colonise the fly vector salivary glands, while the parental phenotype is restored in the Δfbpase rescued cell line re-expressing FBPase. The essential role of FBPase for the development of T. brucei in the tsetse was confirmed by taking advantage of an in vitro differentiation assay based on the RNA-binding protein 6 over-expression, in which the procyclic forms differentiate into epimastigote forms but not into mammalian-infective metacyclic parasites. In total, morphology, immunofluorescence and cytometry analyses showed that the differentiation of the epimastigote stages into the metacyclic forms is abolished in the Δfbpase mutant.
SummaryNumerous eukaryotes have developed specific metabolic traits that are not present in extensively studied model organisms. For instance, the procyclic insect form of Trypanosoma brucei, a parasite responsible for sleeping sickness in its mammalianspecific bloodstream form, metabolizes glucose into excreted succinate and acetate through pathways with unique features. Succinate is primarily produced from glucose-derived phosphoenolpyruvate in peroxisome-like organelles, also known as glycosomes, by a soluble NADH-dependent fumarate reductase only described in trypanosomes so far. Acetate is produced in the mitochondrion of the parasite from acetyl-CoA by a CoA-transferase, which forms an ATP-producing cycle with succinylCoA synthetase. The role of this cycle in ATP production was recently demonstrated in procyclic trypanosomes and has only been proposed so far for anaerobic organisms, in addition to trypanosomatids. We review how nuclear magnetic resonance spectrometry can be used to analyze the metabolic network perturbed by deletion (knockout) or downregulation (RNAi) of the candidate genes involved in these two particular metabolic pathways of procyclic trypanosomes. The role of succinate and acetate production in trypanosomes is discussed, as well as the connections between the succinate and acetate branches, which increase the metabolic flexibility probably required by the parasite to deal with environmental changes such as oxidative stress.
Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.
The genome of trypanosomatids rearranges by using repeated sequences as platforms for amplification or deletion of genomic segments. These stochastic recombination events have a direct impact on gene dosage and foster the selection of adaptive traits in response to environmental pressure. We provide here such an example by showing that the phosphoenolpyruvate carboxykinase ( PEPCK ) gene knockout (Δ pepck ) leads to the selection of a deletion event between two tandemly arranged fumarate reductase ( FRDg and FRDm2 ) genes to produce a chimeric FRDg-m2 gene in the Δ pepck∗ cell line. FRDg is expressed in peroxisome-related organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is nonfunctional and cytosolic. Re-expression of FRDg significantly impaired growth of the Δ pepck∗ cells, but FRD enzyme activity was not required for this negative effect. Instead, glycosomal localization as well as the covalent flavinylation motif of FRD is required to confer growth retardation and intracellular accumulation of reactive oxygen species (ROS). The data suggest that FRDg, similar to Escherichia coli FRD, can generate ROS in a flavin-dependent process by transfer of electrons from NADH to molecular oxygen instead of fumarate when the latter is unavailable, as in the Δ pepck background. Hence, growth retardation is interpreted as a consequence of increased production of ROS, and rearrangement of the FRD locus liberates Δ pepck∗ cells from this obstacle. Interestingly, intracellular production of ROS has been shown to be required to complete the parasitic cycle in the insect vector, suggesting that FRDg may play a role in this process.
The genome of trypanosomatids is rearranged at the level of repeated sequences, where serve as platforms for amplification or deletion of genomic segments. We report here that the PEPCK gene knockout (pepck) leads to the selection of such a deletion event between the FRDg and FRDm2 genes to produce a chimeric FRDg-m2 gene in the pepck* cell line. FRDg is expressed in peroxisome-like organelles, named glycosomes, expression of FRDm2 has not been detected to date, and FRDg-m2 is a non-functional cytosolic FRD. Re-expression of FRDg significantly impaired growth of the pepck* cells, while inhibition of FRDg-m2 expression had no effect, which indicated that this recombination event has been selected in the pepck* cells to eliminate FRDg. FRD activity was not involved in the FRDg-mediated negative effect, while its auto-flavinylation motif is required to impair growth. Considering that (i) FRDs are known to generate reactive oxygen species (ROS) by transferring electrons from their flavin moiety(ies) to oxygen, (ii) intracellular ROS production is essential for the differentiation of procyclic to epimastigote forms of the parasite and (iii) the fumarate reductase activity is not essential for the parasite, we propose that the main role of FRD is to produce part of the ROS necessary to complete the parasitic cycle in the tsetse fly. In this context, the negative effect of FRDg expression in the PEPCK null background is interpreted as an increased production of ROS from oxygen since fumarate, the natural electron acceptor of FRDg, is no longer produced in glycosomes.
SUMMARYMicroorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, allowing to redirect cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism, named “metabolic contest”, for regulating the use of carbon sources without nutrient sensing and signaling. In contrast to most microorganisms, trypanosomes show a glycerol-to-glucose preference that depends on the combination of three conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-like organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways being both ATP-dependent (glycerol kinase and hexokinase, respectively) and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being ~80-fold higher than the hexokinase activity.
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