Muriel Mazet

Naphthoquinone Derivatives Exert Their Antitrypanosomal Activity via a Multi-Target Mechanism

Background and Methodology Recently, we reported on a new class of naphthoquinone derivatives showing a promising anti-trypanosomatid profile in cell-based experiments. The lead of this series (B6, 2-phenoxy-1,4-naphthoquinone) showed an ED50 of 80 nM against Trypanosoma brucei rhodesiense, and a selectivity index of 74 with respect to mammalian cells. A multitarget profile for this compound is easily conceivable, because quinones, as natural products, serve plants as potent defense chemicals with an intrinsic multifunctional mechanism of action. To disclose such a multitarget profile of B6, we exploited a chemical proteomics approach. Principal Findings A functionalized congener of B6 was immobilized on a solid matrix and used to isolate target proteins from Trypanosoma brucei lysates. Mass analysis delivered two enzymes, i.e. glycosomal glycerol kinase and glycosomal glyceraldehyde-3-phosphate dehydrogenase, as potential molecular targets for B6. Both enzymes were recombinantly expressed and purified, and used for chemical validation. Indeed, B6 was able to inhibit both enzymes with IC50 values in the micromolar range. The multifunctional profile was further characterized in experiments using permeabilized Trypanosoma brucei cells and mitochondrial cell fractions. It turned out that B6 was also able to generate oxygen radicals, a mechanism that may additionally contribute to its observed potent trypanocidal activity. Conclusions and Significance Overall, B6 showed a multitarget mechanism of action, which provides a molecular explanation of its promising anti-trypanosomatid activity. Furthermore, the forward chemical genetics approach here applied may be viable in the molecular characterization of novel multitarget ligands.

Probing the metabolic network in bloodstream-form Trypanosoma brucei using untargeted metabolomics with stable isotope labelled glucose

Metabolomics coupled with heavy-atom isotope-labelled glucose has been used to probe the metabolic pathways active in cultured bloodstream form trypomastigotes of Trypanosoma brucei, a parasite responsible for human African trypanosomiasis. Glucose enters many branches of metabolism beyond glycolysis, which has been widely held to be the sole route of glucose metabolism. Whilst pyruvate is the major end-product of glucose catabolism, its transamination product, alanine, is also produced in significant quantities. The oxidative branch of the pentose phosphate pathway is operative, although the non-oxidative branch is not. Ribose 5-phosphate generated through this pathway distributes widely into nucleotide synthesis and other branches of metabolism. Acetate, derived from glucose, is found associated with a range of acetylated amino acids and, to a lesser extent, fatty acids; while labelled glycerol is found in many glycerophospholipids. Glucose also enters inositol and several sugar nucleotides that serve as precursors to macromolecule biosynthesis. Although a Krebs cycle is not operative, malate, fumarate and succinate, primarily labelled in three carbons, were present, indicating an origin from phosphoenolpyruvate via oxaloacetate. Interestingly, the enzyme responsible for conversion of phosphoenolpyruvate to oxaloacetate, phosphoenolpyruvate carboxykinase, was shown to be essential to the bloodstream form trypanosomes, as demonstrated by the lethal phenotype induced by RNAi-mediated downregulation of its expression. In addition, glucose derivatives enter pyrimidine biosynthesis via oxaloacetate as a precursor to aspartate and orotate.

Revisiting the central metabolism of the bloodstream forms of Trypanosoma brucei: production of acetate in the mitochondrion is essential for parasite viability.

Background The 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 Findings A 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/Significance Acetate 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.

Cytosolic NADPH homeostasis in glucose-starved procyclic Trypanosoma brucei relies on malic enzyme and the pentose phosphate pathway fed by gluconeogenic flux.

Background: NADPH production is critical for growth and oxidative stress management. Results: Redundancy of the pentose phosphate pathway and the cytosolic malic enzyme for NADPH synthesis is carbon source-independent in procyclic trypanosomes. Conclusion: The parasite has gluconeogenic capacity from proline. Significance: This work illustrates the flexible carbon source-dependent flux changes for essential NADPH supply. All living organisms depend on NADPH production to feed essential biosyntheses and for oxidative stress defense. Protozoan parasites such as the sleeping sickness pathogen Trypanosoma brucei adapt to different host environments, carbon sources, and oxidative stresses during their infectious life cycle. The procyclic stage develops in the midgut of the tsetse insect vector, where they rely on proline as carbon source, although they prefer glucose when grown in rich media. Here, we investigate the flexible and carbon source-dependent use of NADPH synthesis pathways in the cytosol of the procyclic stage. The T. brucei genome encodes two cytosolic NADPH-producing pathways, the pentose phosphate pathway (PPP) and the NADP-dependent malic enzyme (MEc). Reverse genetic blocking of those pathways and a specific inhibitor (dehydroepiandrosterone) of glucose-6-phosphate dehydrogenase together established redundancy with respect to H2O2 stress management and parasite growth. Blocking both pathways resulted in ∼10-fold increase of susceptibility to H2O2 stress and cell death. Unexpectedly, the same pathway redundancy was observed in glucose-rich and glucose-depleted conditions, suggesting that gluconeogenesis can feed the PPP to provide NADPH. This was confirmed by (i) a lethal phenotype of RNAi-mediated depletion of glucose-6-phosphate isomerase (PGI) in the glucose-depleted Δmec/Δmec null background, (ii) an ∼10-fold increase of susceptibility to H2O2 stress observed for the Δmec/Δmec/RNAiPGI double mutant when compared with the single mutants, and (iii) the 13C enrichment of glycolytic and PPP intermediates from cells incubated with [U-13C]proline, in the absence of glucose. Gluconeogenesis-supported NADPH supply may also be important for nucleotide and glycoconjugate syntheses in the insect host.

The threonine degradation pathway of the Trypanosoma brucei procyclic form: the main carbon source for lipid biosynthesis is under metabolic control

The Trypanosoma brucei procyclic form resides within the digestive tract of its insect vector, where it exploits amino acids as carbon sources. Threonine is the amino acid most rapidly consumed by this parasite, however its role is poorly understood. Here, we show that the procyclic trypanosomes grown in rich medium only use glucose and threonine for lipid biosynthesis, with threonines contribution being ∼ 2.5 times higher than that of glucose. A combination of reverse genetics and NMR analysis of excreted end‐products from threonine and glucose metabolism, shows that acetate, which feeds lipid biosynthesis, is also produced primarily from threonine. Interestingly, the first enzymatic step of the threonine degradation pathway, threonine dehydrogenase (TDH, EC 1.1.1.103), is under metabolic control and plays a key role in the rate of catabolism. Indeed, a trypanosome mutant deleted for the phosphoenolpyruvate decarboxylase gene (PEPCK, EC 4.1.1.49) shows a 1.7‐fold and twofold decrease of TDH protein level and activity, respectively, associated with a 1.8‐fold reduction in threonine‐derived acetate production. We conclude that TDH expression is under control and can be downregulated in response to metabolic perturbations, such as in the PEPCK mutant in which the glycolytic metabolic flux was redirected towards acetate production.

Glycosomal ABC transporters of Trypanosoma brucei : characterisation of their expression, topology and substrate specificity.

Metabolism in trypanosomatids is compartmentalised with major pathways, notably glycolysis, present in peroxisome-like organelles called glycosomes. To date, little information is available about the transport of metabolites through the glycosomal membrane. Previously, three ATP-binding cassette (ABC) transporters, called GAT1-3 for Glycosomal ABC Transporters 1 to 3, have been identified in the glycosomal membrane of Trypanosoma brucei. Here we report that GAT1 and GAT3 are expressed both in bloodstream and procyclic form trypanosomes, whereas GAT2 is mainly or exclusively expressed in bloodstream-form cells. Protease protection experiments showed that the nucleotide-binding domain of GAT1 and GAT3 is exposed to the cytosol, indicating that these transporters mediate the ATP-dependent uptake of solutes from the cytosol into the glycosomal lumen. Depletion of GAT1 and GAT3 by RNA interference in procyclic cells grown in glucose-containing medium did not affect growth. Surprisingly, GAT1 depletion enhanced the expression of the very different GAT3 protein. Expression knockdown of GAT1, but not GAT3, in procyclic cells cultured in glucose-free medium was lethal. Depletion of GAT1 in glucose-grown procyclic cells caused a modification of the total cellular fatty-acid composition. No or only minor changes were observed in the levels of most fatty acids, including oleate (C18:1), nevertheless the linoleate (C18:2) abundance was significantly increased upon GAT1 silencing. Furthermore, glycosomes purified from procyclic wild-type cells incorporate oleoyl-CoA in a concentration- and ATP-dependent manner, whilst this incorporation was severely reduced in glycosomes from cells in which GAT1 levels had been decreased. Together, these results strongly suggest that GAT1 serves to transport primarily oleoyl-CoA, but possibly also other fatty acids, from the cytosol into the glycosomal lumen and that its depletion results in a cellular linoleate accumulation, probably due to the presence of an active oleate desaturase. The role of intraglycosomal oleoyl-CoA and its essentiality when the trypanosomes are grown in the absence of glucose, are discussed.

ATP Synthesis-coupled and -uncoupled Acetate Production from Acetyl-CoA by Mitochondrial Acetate:Succinate CoA-transferase and Acetyl-CoA Thioesterase in Trypanosoma

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. Insect stage trypanosomes use an “acetate shuttle” to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate:succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is ∼1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F0/F1-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F0/F1-ATP synthase F1β subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F0/F1-ATP synthase, for ATP production in the mitochondrion.

Contribution of pyruvate phosphate dikinase in the maintenance of the glycosomal ATP/ADP balance in the Trypanosoma brucei procyclic form*

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. Trypanosoma brucei belongs to a group of protists that sequester the first six or seven glycolytic steps inside specialized peroxisomes, named glycosomes. Because of the glycosomal membrane impermeability to nucleotides, ATP molecules consumed by the first glycolytic steps need to be regenerated in the glycosomes by kinases, such as phosphoenolpyruvate carboxykinase (PEPCK). The glycosomal pyruvate phosphate dikinase (PPDK), which reversibly converts phosphoenolpyruvate into pyruvate, could also be involved in this process. To address this question, we analyzed the metabolism of the main carbon sources used by the procyclic trypanosomes (glucose, proline, and threonine) after deletion of the PPDK gene in the wild-type (Δppdk) and PEPCK null (Δppdk/Δpepck) backgrounds. The rate of acetate production from glucose is 30% reduced in the Δppdk mutant, whereas threonine-derived acetate production is not affected, showing that PPDK function in the glycolytic direction with production of ATP in the glycosomes. The Δppdk/Δpepck mutant incubated in glucose as the only carbon source showed a 3.8-fold reduction of the glycolytic rate compared with the Δpepck mutant, as a consequence of the imbalanced glycosomal ATP/ADP ratio. The role of PPDK in maintenance of the ATP/ADP balance was confirmed by expressing the glycosomal phosphoglycerate kinase (PGKC) in the Δppdk/Δpepck cell line, which restored the glycolytic flux. We also observed that expression of PGKC is lethal for procyclic trypanosomes, as a consequence of ATP depletion, due to glycosomal relocation of cytosolic ATP production. This illustrates the key roles played by glycosomal and cytosolic kinases, including PPDK, to maintain the cellular ATP/ADP homeostasis.

The characterization and evolutionary relationships of a trypanosomal thiolase.

Thiolases are enzymes that remove an acetyl-coenzyme A group from acyl-CoA in the catabolic β-oxidation of fatty acids, or catalyse the reverse condensation reaction for anabolic processes such as the biosynthesis of sterols and ketone bodies. In humans, six homologous isoforms of thiolase have been described, differing from each other in sequence, oligomeric state, substrate specificity and subcellular localization. A bioinformatics analysis of parasite genomes, being (i) different species of African trypanosomes, (ii) Trypanosoma cruzi and (iii) Leishmania spp., using the six human sequences as queries, showed that the distribution of thiolases in human and each of the studied Trypanosomatidae is completely different. Only one of these isoforms, called SCP2-thiolase, was found in each of the Trypanosomatidae, whereas the TFE-thiolase was also found in T. cruzi and Leishmania spp., and the AB-thiolase only in T. cruzi. Each of the trypanosomatid thiolases clusters with its orthologues from other organisms in a phylogenetic analysis and shares with them the isoform-specific sequence fingerprints. The single T. brucei SCP2-thiolase has been expressed in Escherichia coli and characterized. It shows activity in both the degradative and synthetic directions. Transcripts of this thiolase were detected in both bloodstream- and procyclic-form trypanosomes, but the protein was found only in the procyclic form. The encoded protein has both a predicted N-terminal mitochondrial signal peptide and a C-terminal candidate type 1 peroxisomal-targeting signal for sorting it into glycosomes. However experimentally, only a mitochondrial localization was found for both procyclic trypanosomes grown with glucose and cells cultured with amino acids as an energy source. When the thiolase expression in procyclic cells was knocked down by RNA interference, no important change in growth rate occurred, irrespective of whether the cells were grown with or without glucose, indicating that the metabolic pathway(s) involving this enzyme is/are not essential for the parasite under either of these growth conditions.

Combining reverse genetics and nuclear magnetic resonance-based metabolomics unravels trypanosome-specific metabolic pathways

Numerous 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 mammalian‐specific 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 succinyl‐CoA 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.