First comprehensive untargeted metabolomics study of suramin-treated Trypanosoma brucei: an integrated data analysis workflow from multifactor data modelling to functional analysis.

INTRODUCTION: Human African trypanosomiasis, commonly known as sleeping sickness, is a vector-borne parasitic disease prevalent in sub-Saharan Africa and transmitted by the tsetse fly. Suramin, a medication with a long history of clinical use, has demonstrated varied modes of action against Trypanosoma brucei. This study employs a comprehensive workflow to investigate the metabolic effects of suramin on T. brucei, utilizing a multimodal metabolomics approach. OBJECTIVES: The primary aim of this study is to comprehensively analyze the metabolic impact of suramin on T. brucei using a combined liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) approach. Statistical analyses, encompassing multivariate analysis and pathway enrichment analysis, are applied to elucidate significant variations and metabolic changes resulting from suramin treatment. METHODS: A detailed methodology involving the integration of high-resolution data from LC-MS and NMR techniques is presented. The study conducts a thorough analysis of metabolite profiles in both suramin-treated and control T. brucei brucei samples. Statistical techniques, including ANOVA-simultaneous component analysis (ASCA), principal component analysis (PCA), ANOVA 2 analysis, and bootstrap tests, are employed to discern the effects of suramin treatment on the metabolomics outcomes. RESULTS: Our investigation reveals substantial differences in metabolic profiles between the control and suramin-treated groups. ASCA and PCA analysis confirm distinct separation between these groups in both MS-negative and NMR analyses. Furthermore, ANOVA 2 analysis and bootstrap tests confirmed the significance of treatment, time, and interaction effects on the metabolomics outcomes. Functional analysis of the data from LC-MS highlighted the impact of treatment on amino-acid, and amino-sugar and nucleotide-sugar metabolism, while time effects were observed on carbon intermediary metabolism (notably glycolysis and di- and tricarboxylic acids of the succinate production pathway and tricarboxylic acid (TCA) cycle). CONCLUSION: Through the integration of LC-MS and NMR techniques coupled with advanced statistical analyses, this study identifies distinctive metabolic signatures and pathways associated with suramin treatment in T. brucei. These findings contribute to a deeper understanding of the pharmacological impact of suramin and have the potential to inform the development of more efficacious therapeutic strategies against African trypanosomiasis.

How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

A Trypanosoma cruzi phosphoglycerate kinase isoform with a Per-Arnt-Sim domain acts as a possible sensor for intracellular conditions.

Per-ARNT-Sim (PAS) domains constitute a family of domains present in a wide variety of prokaryotic and eukaryotic organisms. They form part of the structure of various proteins involved in diverse cellular processes. Regulation of enzymatic activity and adaptation to environmental conditions, by binding small ligands, are the main functions attributed to PAS-containing proteins. Recently, genes for a diverse set of proteins with a PAS domain were identified in the genomes of several protists belonging to the group of kinetoplastids, however, until now few of these proteins have been characterized. In this work, we characterize a phosphoglycerate kinase containing a PAS domain present in Trypanosoma cruzi (TcPAS-PGK). This PGK isoform is an active enzyme of 58 kDa with a PAS domain located at its N-terminal end. We identified the protein's localization within glycosomes of the epimastigote form of the parasite by differential centrifugation and selective permeabilization of its membranes with digitonin, as well as in an enriched mitochondrial fraction. Heterologous expression systems were developed for the protein with the N-terminal PAS domain (PAS-PGKc) and without it (PAS-PGKt), and the substrate affinities of both forms of the protein were determined. The enzyme does not exhibit standard Michaelis-Menten kinetics. When evaluating the dependence of the specific activity of the recombinant PAS-PGK on the concentration of its substrates 3-phosphoglycerate (3PGA) and ATP, two peaks of maximal activity were found for the complete enzyme with the PAS domain and a single peak for the enzyme without the domain. Km values measured for 3PGA were 219 ± 26 and 8.8 ± 1.3 µM, and for ATP 291 ± 15 and 38 ± 2.2 µM, for the first peak of PAS-PGKc and for PAS-PGKt, respectively, whereas for the second PAS-PGKc peak values of approximately 1.1-1.2 mM were estimated for both substrates. Both recombinant proteins show inhibition by high concentrations of their substrates, ATP and 3PGA. The presence of hemin and FAD exerts a stimulatory effect on PAS-PGKc, increasing the specific activity by up to 55%. This stimulation is not observed in the absence of the PAS domain. It strongly suggests that the PAS domain has an important function in vivo in T. cruzi in the modulation of the catalytic activity of this PGK isoform. In addition, the PAS-PGK through its PAS and PGK domains could act as a sensor for intracellular conditions in the parasite to adjust its intermediary metabolism.

Antimicrobial Peptides (AMPs): Potential Therapeutic Strategy against Trypanosomiases?

Trypanosomiases are a group of tropical diseases that have devastating health and socio-economic effects worldwide. In humans, these diseases are caused by the pathogenic kinetoplastids Trypanosoma brucei, causing African trypanosomiasis or sleeping sickness, and Trypanosoma cruzi, causing American trypanosomiasis or Chagas disease. Currently, these diseases lack effective treatment. This is attributed to the high toxicity and limited trypanocidal activity of registered drugs, as well as resistance development and difficulties in their administration. All this has prompted the search for new compounds that can serve as the basis for the development of treatment of these diseases. Antimicrobial peptides (AMPs) are small peptides synthesized by both prokaryotes and (unicellular and multicellular) eukaryotes, where they fulfill functions related to competition strategy with other organisms and immune defense. These AMPs can bind and induce perturbation in cell membranes, leading to permeation of molecules, alteration of morphology, disruption of cellular homeostasis, and activation of cell death. These peptides have activity against various pathogenic microorganisms, including parasitic protists. Therefore, they are being considered for new therapeutic strategies to treat some parasitic diseases. In this review, we analyze AMPs as therapeutic alternatives for the treatment of trypanosomiases, emphasizing their possible application as possible candidates for the development of future natural anti-trypanosome drugs.

Metabolic insights into phosphofructokinase inhibition in bloodstream-form trypanosomes.

Previously, we reported the development of novel small molecules that are potent inhibitors of the glycolytic enzyme phosphofructokinase (PFK) of Trypanosoma brucei and related protists responsible for serious diseases in humans and domestic animals. Cultured bloodstream-form trypanosomes, which are fully reliant on glycolysis for their ATP production, are rapidly killed at submicromolar concentrations of these compounds, which have no effect on the activity of human PFKs and human cells. Single-day oral dosing cures stage 1 human trypanosomiasis in an animal model. Here we analyze changes in the metabolome of cultured trypanosomes during the first hour after addition of a selected PFK inhibitor, CTCB405. The ATP level of T. brucei drops quickly followed by a partial increase. Already within the first five minutes after dosing, an increase is observed in the amount of fructose 6-phosphate, the metabolite just upstream of the PFK reaction, while intracellular levels of the downstream glycolytic metabolites phosphoenolpyruvate and pyruvate show an increase and decrease, respectively. Intriguingly, a decrease in the level of O-acetylcarnitine and an increase in the amount of L-carnitine were observed. Likely explanations for these metabolomic changes are provided based on existing knowledge of the trypanosome's compartmentalized metabolic network and kinetic properties of its enzymes. Other major changes in the metabolome concerned glycerophospholipids, however, there was no consistent pattern of increase or decrease upon treatment. CTCB405 treatment caused less prominent changes in the metabolome of bloodstream-form Trypanosoma congolense, a ruminant parasite. This agrees with the fact that it has a more elaborate glucose catabolic network with a considerably lower glucose consumption rate than bloodstream-form T. brucei.

Evolution: 'Millefoglie' origin of mitochondrial cristae.

Striated intracytoplasmic membranes in alphaproteobacteria are often reminiscent of millefoglie pastries. A new study reveals a protein complex homologous to that responsible for mitochondrial cristae formation drives intracytoplasmic membrane formation, thereby establishing bacterial ancestry for the biogenesis of mitochondrial cristae.

Anonaine from Annona crassiflora inhibits glutathione S-transferase and improves cypermethrin activity on Rhipicephalus (Boophilus) microplus (Canestrini, 1887).

Rhipicephalus (Boophilus) microplus (Canestrini, 1887) is one of the most important ectoparasites of cattle, causing severe economic losses in tropical and subtropical regions of the world. The selection of resistance to the most commonly used commercial acaricides has stimulated the search for new products for tick control. The identification and development of drugs that inhibit key tick enzymes, such as glutathione S-transferase (GST), is a rational approach that has already been applied to other parasites than ticks. In this context, alkaloids such as anonaine display several biological activities, including an acaricidal effect. This study aimed to assess the specific inhibition of the R. microplus GST by anonaine, and analyze the effect on ticks when anonaine is combined with cypermethrin. For this purpose, a molecular docking analysis was performed using an R. microplus GST three-dimensional structure model with anonaine and compared with a human GST-anonaine complex. The absorption, distribution, metabolism, excretion, and toxicity properties of anonaine were also predicted. Then, for in vitro analyses, anonaine was isolated from Annona crassiflora (Martius, 1841) leaves. The inhibition of purified recombinant R. microplus GST (rRmGST) by anonaine and the effect of this alkaloid on cypermethrin efficacy towards R. microplus were assessed. Anonaine has a higher affinity to the tick enzyme than to the human enzyme in silico and has moderate toxicity, being able to inhibit, in vitro, rRmGST up to 37.5% in a dose-dependent manner. Although anonaine alone has no activity against R. microplus, it increased the cypermethrin effect on larvae, reducing the LC50 from 44 to 22 µg/mL. In conclusion, anonaine is a natural compound that can increase the effect of cypermethrin against R. microplus.

Delineating transitions during the evolution of specialised peroxisomes: Glycosome formation in kinetoplastid and diplonemid protists.

One peculiarity of protists belonging to classes Kinetoplastea and Diplonemea within the phylum Euglenozoa is compartmentalisation of most glycolytic enzymes within peroxisomes that are hence called glycosomes. This pathway is not sequestered in peroxisomes of the third Euglenozoan class, Euglenida. Previous analysis of well-studied kinetoplastids, the 'TriTryps' parasites Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp., identified within glycosomes other metabolic processes usually not present in peroxisomes. In addition, trypanosomatid peroxins, i.e. proteins involved in biogenesis of these organelles, are divergent from human and yeast orthologues. In recent years, genomes, transcriptomes and proteomes for a variety of euglenozoans have become available. Here, we track the possible evolution of glycosomes by querying these databases, as well as the genome of Naegleria gruberi, a non-euglenozoan, which belongs to the same protist supergroup Discoba. We searched for orthologues of TriTryps proteins involved in glycosomal metabolism and biogenesis. Predicted cellular location(s) of each metabolic enzyme identified was inferred from presence or absence of peroxisomal-targeting signals. Combined with a survey of relevant literature, we refine extensively our previously postulated hypothesis about glycosome evolution. The data agree glycolysis was compartmentalised in a common ancestor of the kinetoplastids and diplonemids, yet additionally indicates most other processes found in glycosomes of extant trypanosomatids, but not in peroxisomes of other eukaryotes were either sequestered in this ancestor or shortly after separation of the two lineages. In contrast, peroxin divergence is evident in all euglenozoans. Following their gain of pathway complexity, subsequent evolution of peroxisome/glycosome function is complex. We hypothesize compartmentalisation in glycosomes of glycolytic enzymes, their cofactors and subsequently other metabolic enzymes provided selective advantage to kinetoplastids and diplonemids during their evolution in changing marine environments. We contend two specific properties derived from the ancestral peroxisomes were key: existence of nonselective pores for small solutes and the possibility of high turnover by pexophagy. Critically, such pores and pexophagy are characterised in extant trypanosomatids. Increasing amenability of free-living kinetoplastids and recently isolated diplonemids to experimental study means our hypothesis and interpretation of bioinformatic data are suited to experimental interrogation.

Biogenesis and metabolic homeostasis of trypanosomatid glycosomes: New insights and new questions.

Kinetoplastea and Diplonemea possess peroxisome-related organelles that, uniquely, contain most of the enzymes of the glycolytic pathway and are hence called glycosomes. Enzymes of several other core metabolic pathways have also been located in glycosomes, in addition to some characteristic peroxisomal systems such as pathways of lipid metabolism. A considerable amount of research has been performed on glycosomes of trypanosomes since their discovery four decades ago. Not only the role of the glycosomal enzyme systems in the overall cell metabolism appeared to be unique, but also the organelles display remarkable features regarding their biogenesis and structural properties. These features are similar to those of the well-studied peroxisomes of mammalian and plant cells and yeasts yet exhibit also differences reflecting the large evolutionary distance between these protists and the representatives of other major eukaryotic lineages. Despite all research performed, many questions remain about various properties and the biological roles of glycosomes and peroxisomes. Here, we review the current knowledge about glycosomes, often comparing it with information about peroxisomes. Furthermore, we highlight particularly many questions that remain about the biogenesis, and the heterogeneity in structure and content of these enigmatic organelles, and the properties of their boundary membrane.

Carbohydrate metabolism in trypanosomatids: New insights revealing novel complexity, diversity and species-unique features.

The human pathogenic trypanosomatid species collectively called the "TriTryp parasites" - Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp. - have complex life cycles, with each of these parasitic protists residing in a different niche during their successive developmental stages where they encounter diverse nutrients. Consequently, they adapt their metabolic network accordingly. Yet, throughout the life cycles, carbohydrate metabolism - involving the glycolytic, gluconeogenic and pentose-phosphate pathways - always plays a central role in the biology of these parasites, whether the available carbon and free energy sources are saccharides, amino acids or lipids. In this paper, we provide an updated review of the carbohydrate metabolism of the TriTryps, highlighting new data about this metabolic network, the interconnection of its pathways and the compartmentalisation of its enzymes within glycosomes, cytosol and mitochondrion. Differences in the expression of the branches of the metabolic network between the successive life-cycle stages of each of these parasitic trypanosomatids are discussed, as well as differences between them. Recent structural and kinetic studies have revealed unique regulatory mechanisms for some of the network's key enzymes with important species-specific variations. Furthermore, reports of multiple post-translational modifications of trypanosomal glycolytic enzymes suggest that additional mechanisms for stage- and/or environmental cues that regulate activity are operational in the parasites. The detailed comparison of the carbohydrate metabolism of the TriTryps has thus revealed multiple differences and a greater complexity, including for the reduced metabolic network in bloodstream-form T. brucei, than previously appreciated. Although these parasites are related, share many cytological and metabolic features and are grouped within a single taxonomic family, the differences highlighted in this review reflect their separate evolutionary tracks from a common ancestor to the extant organisms. These differences are indicative of their adaptation to the different insect vectors and niches occupied in their mammalian hosts.

Fast acting allosteric phosphofructokinase inhibitors block trypanosome glycolysis and cure acute African trypanosomiasis in mice.

The parasitic protist Trypanosoma brucei is the causative agent of Human African Trypanosomiasis, also known as sleeping sickness. The parasite enters the blood via the bite of the tsetse fly where it is wholly reliant on glycolysis for the production of ATP. Glycolytic enzymes have been regarded as challenging drug targets because of their highly conserved active sites and phosphorylated substrates. We describe the development of novel small molecule allosteric inhibitors of trypanosome phosphofructokinase (PFK) that block the glycolytic pathway resulting in very fast parasite kill times with no inhibition of human PFKs. The compounds cross the blood brain barrier and single day oral dosing cures parasitaemia in a stage 1 animal model of human African trypanosomiasis. This study demonstrates that it is possible to target glycolysis and additionally shows how differences in allosteric mechanisms may allow the development of species-specific inhibitors to tackle a range of proliferative or infectious diseases.

Biochemical and transcript level differences between the three human phosphofructokinases show optimisation of each isoform for specific metabolic niches.

6-Phosphofructokinase-1-kinase (PFK) tetramers catalyse the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F16BP). Vertebrates have three PFK isoforms (PFK-M, PFK-L, and PFK-P). This study is the first to compare the kinetics, structures, and transcript levels of recombinant human PFK isoforms. Under the conditions tested PFK-M has the highest affinities for F6P and ATP (K0.5ATP 152 µM; K0.5F6P 147 µM), PFK-P the lowest affinities (K0.5ATP 276 µM; K0.5F6P 1333 µM), and PFK-L demonstrates a mixed picture of high ATP affinity and low F6P affinity (K0.5ATP 160 µM; K0.5F6P 1360 µM). PFK-M is more resistant to ATP inhibition compared with PFK-L and PFK-P (respectively, 23%, 31%, 50% decreases in specificity constants). GTP is an alternate phospho donor. Interface 2, which regulates the inactive dimer to active tetramer equilibrium, differs between isoforms, resulting in varying tetrameric stability. Under the conditions tested PFK-M is less sensitive to fructose 2,6-bisphosphate (F26BP) allosteric modulation than PFK-L or PFK-P (allosteric constants [K0.5ATP+F26BP/K0.5ATP] 1.10, 0.92, 0.54, respectively). Structural analysis of two allosteric sites reveals one may be specialised for AMP/ADP and the other for smaller/flexible regulators (citrate or phosphoenolpyruvate). Correlations between PFK-L and PFK-P transcript levels indicate that simultaneous expression may expand metabolic capacity for F16BP production whilst preserving regulatory capabilities. Analysis of cancer samples reveals intriguing parallels between PFK-P and PKM2 (pyruvate kinase M2), and simultaneous increases in PFK-P and PFKFB3 (responsible for F26BP production) transcript levels, suggesting prioritisation of metabolic flexibility in cancers. Our results describe the kinetic and transcript level differences between the three PFK isoforms, explaining how each isoform may be optimised for distinct roles.

Pyruvate kinase from Plasmodium falciparum: Structural and kinetic insights into the allosteric mechanism.

During its intra-erythrocytic growth phase, the malaria parasite Plasmodium falciparum relies heavily on glycolysis for its energy requirements. Pyruvate kinase (PYK) is essential for regulating glycolytic flux and for ATP production, yet the allosteric mechanism of P. falciparum PYK (PfPYK) remains poorly understood. Here we report the first crystal structure of PfPYK in complex with substrate analogues oxalate and the ATP product. Comparisons of PfPYK structures in the active R-state and inactive T-state reveal a 'rock-and-lock' allosteric mechanism regulated by rigid-body rotations of each subunit in the tetramer. Kinetic data and structural analysis indicate glucose 6-phosphate is an activator by increasing the apparent maximal velocity of the enzyme. Intriguingly, the trypanosome drug suramin inhibits PfPYK, which points to glycolysis as a set of potential therapeutic targets against malaria.

Structure, Properties, and Function of Glycosomes in Trypanosoma cruzi.

Glycosomes are peroxisome-related organelles that have been identified in kinetoplastids and diplonemids. The hallmark of glycosomes is their harboring of the majority of the glycolytic enzymes. Our biochemical studies and proteome analysis of Trypanosoma cruzi glycosomes have located, in addition to enzymes of the glycolytic pathway, enzymes of several other metabolic processes in the organelles. These analyses revealed many aspects in common with glycosomes from other trypanosomatids as well as features that seem specific for T. cruzi. Their enzyme content indicates that T. cruzi glycosomes are multifunctional organelles, involved in both several catabolic processes such as glycolysis and anabolic ones. Specifically discussed in this minireview are the cross-talk between glycosomal metabolism and metabolic processes occurring in other cell compartments, and the importance of metabolite translocation systems in the glycosomal membrane to enable the coordination between the spatially separated processes. Possible mechanisms for metabolite translocation across the membrane are suggested by proteins identified in the organelle's membrane-homologs of the ABC and MCF transporter families-and the presence of channels as inferred previously from the detection of channel-forming proteins in glycosomal membrane preparations from the related parasite T. brucei. Together, these data provide insight in the way in which different parts of T. cruzi metabolism, although uniquely distributed over different compartments, are integrated and regulated. Moreover, this information reveals opportunities for the development of drugs against Chagas disease caused by these parasites and for which currently no adequate treatment is available.

Kinetic and structural studies of Trypanosoma and Leishmania phosphofructokinases show evolutionary divergence and identify AMP as a switch regulating glycolysis versus gluconeogenesis.

Trypanosomatids possess glycosome organelles that contain much of the glycolytic machinery, including phosphofructokinase (PFK). We present kinetic and structural data for PFK from three human pathogenic trypanosomatids, illustrating intriguing differences that may reflect evolutionary adaptations to differing ecological niches. The activity of Leishmania PFK - to a much larger extent than Trypanosoma PFK - is reliant on AMP for activity regulation, with 1 mm AMP increasing the L. infantum PFK (LiPFK) kcat/K0.5F6P value by 10-fold, compared to only a 1.3- and 1.4-fold increase for T. cruzi and T. brucei PFK, respectively. We also show that Leishmania PFK melts at a significantly lower (> 15 °C) temperature than Trypanosoma PFKs and that addition of either AMP or ATP results in a marked stabilization of the protein. Sequence comparisons of Trypanosoma spp. and Leishmania spp. show that divergence of the two genera involved amino acid substitutions that occur in the enzyme's 'reaching arms' and 'embracing arms' that determine tetramer stability. The dramatic effects of AMP on Leishmania activity compared with the Trypanosoma PFKs may be explained by differences between the T-to-R equilibria for the two families, with the low-melting Leishmania PFK favouring the flexible inactive T-state in the absence of AMP. Sequence comparisons along with the enzymatic and structural data presented here also suggest there was a loss of AMP-dependent regulation in Trypanosoma species rather than gain of this characteristic in Leishmania species and that AMP acts as a key regulator in Leishmania governing the balance between glycolysis and gluconeogenesis.

Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

African trypanosomiasis, sleeping sickness in humans or nagana in animals, is a potentially fatal neglected tropical disease and a threat to 65 million human lives and 100 million small and large livestock animals in sub-Saharan Africa. Available treatments for this devastating disease are few and have limited efficacy, prompting the search for new drug candidates. Simultaneous inhibition of the trypanosomal glycerol kinase (TGK) and trypanosomal alternative oxidase (TAO) is considered a validated strategy toward the development of new drugs. Our goal is to develop a TGK-specific inhibitor for coadministration with ascofuranone (AF), the most potent TAO inhibitor. Here, we report on the identification of novel compounds with inhibitory potency against TGK. Importantly, one of these compounds (compound 17) and its derivatives (17a and 17b) killed trypanosomes even in the absence of AF. Inhibition kinetics revealed that derivative 17b is a mixed-type and competitive inhibitor for TGK and TAO, respectively. Structural data revealed the molecular basis of this dual inhibitory action, which, in our opinion, will aid in the successful development of a promising drug to treat trypanosomiasis. Although the EC50 of compound 17b against trypanosome cells was 1.77 µM, it had no effect on cultured human cells, even at 50 µM.-Balogun, E. O., Inaoka, D. K., Shiba, T., Tsuge, C., May, B., Sato, T., Kido, Y., Nara, T., Aoki, T., Honma, T., Tanaka, A., Inoue, M., Matsuoka, S., Michels, P. A. M., Watanabe, Y.-I., Moore, A. L., Harada, S., Kita, K. Discovery of trypanocidal coumarins with dual inhibition of both the glycerol kinase and alternative oxidase of Trypanosoma brucei brucei.

Pyruvate Kinase Regulates the Pentose-Phosphate Pathway in Response to Hypoxia in Mycobacterium tuberculosis.

In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate-pyruvate-oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb.

Gamma-glutamylcysteine synthetase and tryparedoxin 1 exert high control on the antioxidant system in Trypanosoma cruzi contributing to drug resistance and infectivity.

Trypanothione (T(SH)2) is the main antioxidant metabolite for peroxide reduction in Trypanosoma cruzi; therefore, its metabolism has attracted attention for therapeutic intervention against Chagas disease. To validate drug targets within the T(SH)2 metabolism, the strategies and methods of Metabolic Control Analysis and kinetic modeling of the metabolic pathway were used here, to identify the steps that mainly control the pathway fluxes and which could be appropriate sites for therapeutic intervention. For that purpose, gamma-glutamylcysteine synthetase (γECS), trypanothione synthetase (TryS), trypanothione reductase (TryR) and the tryparedoxin cytosolic isoform 1 (TXN1) were separately overexpressed to different levels in T. cruzi epimastigotes and their degrees of control on the pathway flux as well as their effect on drug resistance and infectivity determined. Both experimental in vivo as well as in silico analyses indicated that γECS and TryS control T(SH)2 synthesis by 60-74% and 15-31%, respectively. γECS overexpression prompted up to a 3.5-fold increase in T(SH)2 concentration, whereas TryS overexpression did not render an increase in T(SH)2 levels as a consequence of high T(SH)2 degradation. The peroxide reduction flux was controlled for 64-73% by TXN1, 17-20% by TXNPx and 11-16% by TryR. TXN1 and TryR overexpression increased H2O2 resistance, whereas TXN1 overexpression increased resistance to the benznidazole plus buthionine sulfoximine combination. γECS overexpression led to an increase in infectivity capacity whereas that of TXN increased trypomastigote bursting. The present data suggested that inhibition of high controlling enzymes such as γECS and TXN1 in the T(SH)2 antioxidant pathway may compromise the parasite's viability and infectivity.

Proteomic analysis of glycosomes from Trypanosoma cruzi epimastigotes.

In Trypanosoma cruzi, the causal agent of Chagas disease, the first seven steps of glycolysis are compartmentalized in glycosomes, which are authentic but specialized peroxisomes. Besides glycolysis, activity of enzymes of other metabolic processes have been reported to be present in glycosomes, such as ß-oxidation of fatty acids, purine salvage, pentose-phosphate pathway, gluconeogenesis and biosynthesis of ether-lipids, isoprenoids, sterols and pyrimidines. In this study, we have purified glycosomes from T. cruzi epimastigotes, collected the soluble and membrane fractions of these organelles, and separated peripheral and integral membrane proteins by Na2CO3 treatment and osmotic shock. Proteomic analysis was performed on each of these fractions, allowing us to confirm the presence of enzymes involved in various metabolic pathways as well as identify new components of this parasite's glycosomes.

Control and regulation of the pyrophosphate-dependent glucose metabolism in Entamoeba histolytica.

Entamoeba histolytica has neither Krebs cycle nor oxidative phosphorylation activities; therefore, glycolysis is the main pathway for ATP supply and provision of carbon skeleton precursors for the synthesis of macromolecules. Glucose is metabolized through fermentative glycolysis, producing ethanol as its main end-product as well as some acetate. Amoebal glycolysis markedly differs from the typical Embden-Meyerhof-Parnas pathway present in human cells: (i) by the use of inorganic pyrophosphate, instead of ATP, as the high-energy phospho group donor; (ii) with one exception, the pathway enzymes can catalyze reversible reactions under physiological conditions; (iii) there is no allosteric regulation and sigmoidal kinetic behavior of key enzymes; and (iv) the presence of some glycolytic and fermentation enzymes similar to those of anaerobic bacteria. These peculiarities bring about alternative mechanisms of control and regulation of the PPi-dependent fermentative glycolysis in the parasite in comparison to the ATP-dependent and allosterically regulated glycolysis in many other eukaryotic cells. In this review, the current knowledge of the carbohydrate metabolism enzymes in E. histolytica is analyzed. Thermodynamics and stoichiometric analyses indicate 2 to 3.5 ATP yield per glucose metabolized, instead of the often presumed 5 ATP/glucose ratio. PPi derived from anabolism seems insufficient for PPi-glycolysis; hence, alternative ways of PPi supply are also discussed. Furthermore, the underlying mechanisms of control and regulation of the E. histolytica carbohydrate metabolism, analyzed by applying integral and systemic approaches such as Metabolic Control Analysis and kinetic modeling, contribute to unveiling alternative and promising drug targets.