No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Surprising variety in energy metabolism within Trypanosomatidae
Abstract
The metabolism of Trypanosomatidae differs significantly between distinct species and can even be completely different between various life-cycle stages of the same species. It has been proposed that differences in energy metabolism are related to differences in nutrient supply in the environments of the various trypanosomatids. However, the literature shows that availability of substrates does not dictate the type of energy metabolism of trypanosomatids, as Trypanosoma theileri, Trypanosoma lewisi and African trypanosomes all live in the bloodstream of their mammalian host, but have surprisingly large differences in metabolism. Furthermore, in trypanosomatids no obvious relationship exists between energy metabolism and phylogeny or mode of transmission. We provide an overview of the metabolic capacities in the energy metabolism of distinct trypanosomatids, and suggest that these can be divided into four different metabolic categories of increasing complexity.
... Fundamental biochemical and gene expression pathways of the disease-causing, insect transmitted trypanosomatids are often deciphered in the model organism Trypanosoma brucei. Gene expression and flux through major metabolic pathways are known to dramatically differ between T. brucei proliferative insect and mammalian life stages [1][2][3][4] trypanosomatids differ from T. brucei in important aspects of their life cycle. Trypanosoma cruzi, causative agent of Chagas disease, undergoes starvation, pH changes, and passage through the entire insect digestive tract in its transition to its mammalian infective stage [5], a transition very different than that of T. brucei. ...
... Trypanosoma cruzi, causative agent of Chagas disease, undergoes starvation, pH changes, and passage through the entire insect digestive tract in its transition to its mammalian infective stage [5], a transition very different than that of T. brucei. Furthermore, once in the mammalian host, T. cruzi replicates intracellularly rather than in the bloodstream as does T. brucei [1,6]. Utilization of metabolic pathways, particularly across the pathogen life cycle, must therefore be studied for each trypanosomatid individually. ...
... In these conditions, a number of pathways and parameters may be differentially utilized or regulated among T. cruzi strains to result in a differential release of acidic byproducts. These include substrate-level phosphorylation of amino acids, oxidative phosphorylation, and the specific amino acids and/or lipids preferred as substrates [1,31,53]. ...
The insect-transmitted protozoan parasite Trypanosoma cruzi experiences changes in nutrient availability and rate of flux through different metabolic pathways across its life cycle. The species encompasses much genetic diversity of both the nuclear and mitochondrial genomes among isolated strains. The genetic or expression variation of both genomes are likely to impact metabolic responses to environmental stimuli, and even steady state metabolic function, among strains. To begin formal characterization these differences, we compared aspects of metabolism between genetically similar strains CL Brener and Tulahuen with less similar Esmeraldo and Sylvio X10 strains in a culture environment. Epimastigotes of all strains took up glucose at similar rates. However, the degree of medium acidification that could be observed when glucose was absent from the medium varied by strain, indicating potential differences in excreted metabolic byproducts. Our main focus was differences related to electron transport chain function. We observed differences in ATP-coupled respiration and maximal respiratory capacity, mitochondrial membrane potential, and mitochondrial morphology between strains, despite the fact that abundances of two nuclear-encoded proteins of the electron transport chain are similar between strains. RNA sequencing reveals strain-specific differences in abundances of mRNAs encoding proteins of the respiratory chain but also other metabolic processes. From these differences in metabolism and mitochondrial phenotypes we have generated tentative models for the differential metabolic fluxes or differences in gene expression that may underlie these results.
... The pathways involved in energy metabolism in T. brucei are also strikingly distinct along parasite development, in a way that insect procyclic stages exhibit an active mitochondrion, TCA cycle, and OXPHOS, while in human bloodstream forms, mitochondria are hardly functional, prevailing glucose fermentation. The differences seem to represent important adaptations to distinct nutrient availabilities between the insect midgut, which is an aminoacidrich but glucose-poor environment, and glucose-rich human blood (van Hellemond et al., 2005;Tielens and van Hellemond, 2009). For a comprehensive description of metabolic pathways and regulatory mechanisms operating in T. brucei mitochondrion, the reader is referred to excellent reviews in this topic (van Hellemond et al., 2005;Tielens and van Hellemond, 2009;Verner et al., 2015). ...
... The differences seem to represent important adaptations to distinct nutrient availabilities between the insect midgut, which is an aminoacidrich but glucose-poor environment, and glucose-rich human blood (van Hellemond et al., 2005;Tielens and van Hellemond, 2009). For a comprehensive description of metabolic pathways and regulatory mechanisms operating in T. brucei mitochondrion, the reader is referred to excellent reviews in this topic (van Hellemond et al., 2005;Tielens and van Hellemond, 2009;Verner et al., 2015). ...
... Studies on fundamental aspects on Leishmania energy metabolism revealed the existent similarities with other trypanosomatids, especially T. cruzi (Tielens and van Hellemond, 2009). For example, both parasite species exhibit active glycolysis, OXPHOS, amino acid metabolism, while produce acetate and succinate as major end-products (Berm udez et al., 1997;Tielens and van Hellemond, 2009). ...
Hematophagous organisms undergo remarkable metabolic changes during the blood digestion process, increasing fermentative glucose metabolism and reducing respiratory rates, both consequence of functional mitochondrial remodeling. Here, we review the pathways involved in energy metabolism and mitochondrial functionality in a comparative framework across different hematophagous species, and consider how these processes regulate redox homeostasis during blood digestion. The trend across distinct species indicate that a switch in energy metabolism might represent an important defensive mechanism to avoid the potential harmful interaction of oxidants generated from aerobic energy metabolism with products derived from blood digestion. Indeed, in insect vectors, blood feeding transiently reduces respiratory rates and oxidant production, irrespective of tissue and insect model. On the other hand, a different scenario is observed in several unrelated parasite species when exposed to blood digestion products, as respiratory rates reduce and mitochondrial oxidant production increase. The emerging picture indicates that re-wiring of energy metabolism, through reduced mitochondrial function, culminates in improved tolerance to redox insults and seems to represent a key step for hematophagous organisms to cope with the overwhelming and potentially toxic blood meal.
... There are multiple changes in biochemical and metabolite pathways in the different species of Trypanosomatidae, including L. tropica, L. major and C. fasciculata (8,9). The biochemical dissimilarities in the different parasites can play an important role not only in the prevention and treatment of leishmaniasis but also in the identification of different species. ...
... Indeed, the result of 1 H-NMR and metabolic analysis confirmed the result of PCR-RFLP based on SL mini-exon and genotyping based on sequencing. Our study confirmed that the metabolic models of different species of Leishmania were exclusive and species-specific (8,(23)(24)(25)(26)(27)(28). The species-specific metabolic model can lead to different clinical manifestations and cause the resistance to treatment; therefore, it is very important to identify the species of parasite accurately. ...
Background
We aimed to evaluate the accuracy of genotyping of Leishmania species by the spliced leader mini-exon gene.
Methods
Suspected leishmaniasis patients, referred to Masieh Daneshvary Hospital, Tehran, Iran were included from May 2017 to September 2021. The Leishmania species were genotyped by PCRRFLP based on the SL mini-exon gene and the ITS1 region of SSU-rRNA gene and compared with the sequencing results. The expressed metabolites of metacyclic promastigotes were evaluated by Proton nuclear magnetic resonance (¹H-NMR).
Results
Out of 66 suspected cases, 36 (54.4%) were positive for Leishmania species based on the PCR assays. In 21 (31.8%) cases, promastigotes grew on culture tubes. Based on the RFLP of SL RNA profile, 13 (19.7%) L. tropica, 9 (13.6%) L. major, 3 (4.5%) L. infantum, and 8 (12.1%) C. fasciculata isolates, isolated from culture media, were identified; however, 3 (4.5%) cases were unidentifiable due to the low number of parasites. Seventeen metabolites were expressed by the metacyclic forms of L. major, L. tropica and C. fasciculata isolates. The top differential metabolites expressed more in C. fasciculata were FAD, p-Methoxybenzyl alcohol and S-b-G-5, 5-G-b-S (A = CH2) (P<0.005) whereas Veratryl glycerols and D-(+)-Mannose were significantly increased in L. major and Betulin, LTyrosine in L. tropica (P<0.01).
Conclusion
The invaluable techniques such as sequencing and 1H-NMR confirmed the results of genotyping of Leishmania species based on the SL mini-exon gene. SL mini exon gene can be used as a diagnostic tool to differentiate various Leishmania genotypes and detect contamination of culture media with C. fasciculata.
... Glycolytic compartmentalization presumably allows T. brucei to utilize this process to achieve rapid adaptation. A complicating factor for this explanation of why glycosomal contents are maintained throughout the kinetoplastid lineage is the fact that the metabolic remodeling of T. brucei is very extreme between its replicative stages in its mammalian and insect hosts [43]. Metabolic studies demonstrate that dixenous mammalian kinetoplastids that replicate intracellularly, rather than in the bloodstream as T. brucei does, do not have as radical a metabolic transformation, [43]. ...
... A complicating factor for this explanation of why glycosomal contents are maintained throughout the kinetoplastid lineage is the fact that the metabolic remodeling of T. brucei is very extreme between its replicative stages in its mammalian and insect hosts [43]. Metabolic studies demonstrate that dixenous mammalian kinetoplastids that replicate intracellularly, rather than in the bloodstream as T. brucei does, do not have as radical a metabolic transformation, [43]. Even T. brucei may experience alternative metabolic states when in fatty tissues rather than in the blood [44]. ...
In kinetoplastids, the first seven steps of glycolysis are compartmentalized into a glycosome along with parts of other metabolic pathways. This organelle shares a common ancestor with the better-understood eukaryotic peroxisome. Much of our understanding of the emergence, evolution, and maintenance of glycosomes is limited to explorations of the dixenous parasites, including the enzymatic contents of the organelle. Our objective was to determine the extent that we could leverage existing studies in model kinetoplastids to determine the composition of glycosomes in species lacking evidence of experimental localization. These include diverse monoxenous species and dixenous species with very different hosts. For many of these, genome or transcriptome sequences are available. Our approach initiated with a meta-analysis of existing studies to generate a subset of enzymes with highest evidence of glycosome localization. From this dataset we extracted the best possible glycosome signal peptide identification scheme for in silico identification of glycosomal proteins from any kinetoplastid species. Validation suggested that a high glycosome localization score from our algorithm would be indicative of a glycosomal protein. We found that while metabolic pathways were consistently represented across kinetoplastids, individual proteins within those pathways may not universally exhibit evidence of glycosome localization.
... However, the trypanosomatid Krebs cycle does not function as a classical cycle. Pyruvate is converted mainly to acetate in a reaction linked with ATP synthesis (Tielens & van Hellemond, 2009;Bringaud et al., 2015). A similar reaction series has been described in a number of unicellular and multicellular species, but trypanosomatids are among the few species requiring oxygen (Müller et al., 2012). ...
... A similar reaction series has been described in a number of unicellular and multicellular species, but trypanosomatids are among the few species requiring oxygen (Müller et al., 2012). Thus, trypanosomatid mitochondrial energy metabolism is an evolutionary intermediate between the metabolism of aerobic and anaerobic mitochondria (Tielens & van Hellemond, 2009). ...
Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (β-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.
... There are five predicted malate dehydrogenases -one mitochondrial, one glycosomal, and three cytosolic isoenzymes (Table 1; Table S5). As in most trypanosomatids, the single mitochondrial isocitrate dehydrogenase is predicted to be an NADP + -dependent enzyme suggesting that this enzyme in B. nonstop is also involved in the TCA cycle and enables reductive carboxylation rather than the complete oxidation of pyruvate to CO 2 and H 2 O [55,56]. ...
Background Almost all extant organisms use the same, so‐called canonical, genetic code with departures from it being very rare. Even more exceptional are the instances when a eukaryote with non‐canonical code can be easily cultivated and has its whole genome and transcriptome sequenced. This is the case of Blastocrithidia nonstop, a trypa‐ nosomatid flagellate that reassigned all three stop codons to encode amino acids.
Results We in silico predicted the metabolism of B. nonstop and compared it with that of the well‐studied human parasites Trypanosoma brucei and Leishmania major. The mapped mitochondrial, glycosomal and cytosolic metabo‐ lism contains all typical features of these diverse and important parasites. We also provided experimental valida‐ tion for some of the predicted observations, concerning, specifically presence of glycosomes, cellular respiration, and assembly of the respiratory complexes.
Conclusions In an unusual comparison of metabolism between a parasitic protist with a massively altered genetic code and its close relatives that rely on a canonical code we showed that the dramatic differences on the level
of nucleic acids do not seem to be reflected in the metabolisms. Moreover, although the genome of B. nonstop
is extremely AT‐rich, we could not find any alterations of its pyrimidine synthesis pathway when compared to other trypanosomatids. Hence, we conclude that the dramatic alteration of the genetic code of B. nonstop has no significant repercussions on the metabolism of this flagellate.
... Trypanosomatids prefer carbohydrates for energy; however, they can proliferate using amino acids, fatty acids, and glycerol [5,6,34,35]. L-threonine is the most consumed carbon source in T. brucei grown in glucose-depleted medium at the insect stage. ...
Chagas disease is caused by the protozoan parasite, Trypanosoma cruzi . This parasite alternates between an insect vector and a mammalian host. T . cruzi epimastigotes reside in the insect vector and coexist with the blood components of the vertebrate host. The metabolic profile of T . cruzi has been extensively studied; however, changes in its metabolism in response to signaling molecules present in the vector are poorly understood. Heme acts as a physiological oxidant that triggers intense epimastigote proliferation and upregulates the expression of genes related to glycolysis and aerobic fermentation in vitro . Here, heme-cultured epimastigotes increased D-glucose consumption. In fact, heme-cultured parasites secreted more succinate (the end product of the so-called succinic fermentation) followed by glucose intake. Increased succinate levels reduced the extracellular pH, leading to acidification of the supernatant. However, the acidification and proliferation stimulated by heme was impaired when glycolysis was inhibited. Otherwise, when glucose amount is enhanced in supernatant, heme-cultured parasites increased its growth whereas the glucose depletion caused a delay in proliferation. Heme supplementation increased epimastigote electron transport system-related O 2 consumption rates, while glucose addition reduced both the electron transport system-related O 2 consumption rates and spare respiratory capacity, indicating a Crabtree- like effect. These results show that glycolysis predominated in heme-cultured epimastigotes over oxidative phosphorylation for energy supply when glucose is present to sustain its high proliferation in vitro . Furthermore, it provided an insight into the parasite biology in the vector environment that supply glucose and the digestion of blood generates free heme that can lead to the growth of T . cruzi epimastigotes.
... Leishmania lacks the versatility of African trypanosomes to switch between glycolysis and oxidative phosphorylation to fulfil their ATP needs [11][12][13]. The electron leakage from the respiratory chain in mitochondria is the main source of ROS production [14]. ...
Leishmaniases have a broad spectrum of clinical manifestations, ranging from a cutaneous to a progressive and fatal visceral disease. Chemotherapy is nowadays the almost exclusive way to fight the disease but limited by its scarce therapeutic arsenal, on its own compromised by adverse side effects and clinical resistance. Cyclobenzaprine (CBP), an FDA-approved oral muscle relaxant drug has previously demonstrated in vitro and in vivo activity against Leishmania sp., but its targets were not fully unveiled. This study aimed to define the role of energy metabolism as a target for the leishmanicidal mechanisms of CBP. Methodology to assess CBP leishmanicidal mechanism variation of intracellular ATP levels using living Leishmania transfected with a cytoplasmic luciferase. Induction of plasma membrane permeability by assessing depolarization with DiSBAC(2)3 and entrance of the vital dye SYTOX® Green. Mitochondrial depolarization by rhodamine 123 accumulation. Mapping target site within the respiratory chain by oxygen consumption rate. Reactive oxygen species (ROS) production using MitoSOX. Morphological changes by transmission electron microscopy. CBP caused on L. infantum promastigotes a decrease of intracellular ATP levels, with irreversible depolarization of plasma membrane, the collapse of the mitochondrial electrochemical potential, mild uncoupling of the respiratory chain, and ROS production, with ensuing intracellular Ca2+ imbalance and DNA fragmentation. Electron microscopy supported autophagic features but not a massive plasma membrane disruption. The severe and irreversible mitochondrial damage induced by CBP endorsed the bioenergetics metabolism as a relevant target within the lethal programme induced by CBP in Leishmania. This, together with the mild-side effects of this oral drug, endorses CBP as an appealing novel candidate as a leishmanicidal drug under a drug repurposing strategy.
... In Leishmania, especially in the promastigote, oxidative phosphorylation is the main source for ATP biosynthesis [75], dependent on the maintenance of the electrochemical potential (∆Ψ m ) created by the respiratory chain. ...
More than 1 billion people live in areas endemic for leishmaniasis, which is a relevant threat for public health worldwide. Due to the inadequate treatments, there is an urgent need to develop novel alternative drugs and to validate new targets to fight this disease. One appealing approach is the selective inhibition of protein kinases (PKs), enzymes involved in a wide range of processes along the life cycle of Leishmania. Several PKs, including glycogen synthase kinase 3 (GSK-3), have been validated as essential for this parasite by genetic or pharmacological methods. Recently, novel chemical scaffolds have been uncovered as Leishmania GSK-3 inhibitors with antiparasitic activity. In order to find new inhibitors of this enzyme, a virtual screening of our in-house chemical library was carried out on the structure of the Leishmania GSK-3. The virtual hits identified were experimentally assayed both for leishmanicidal activity and for in vitro inhibition of the enzyme. The best hits have a quinone scaffold. Their optimization through a medicinal chemistry approach led to a set of new compounds, provided a frame to establish biochemical and antiparasitic structure–activity relationships, and delivered molecules with an improved selectivity index. Altogether, this study paves the way for a systemic search of this class of inhibitors for further development as potential leishmanicidal drugs.
... Glucose uptake was decreased (mean reduction of 37% in uptake compared to uninduced controls after 72 h; Fig 4F), suggesting that either lower levels of glucose are sufficient for energy generation in T. congolense, or the parasite can utilize other carbon sources for ATP production. These alternative sources could include medium or serum components such as amino acids [75]. ...
Animal African Trypanosomiasis (AAT) is a debilitating livestock disease prevalent across sub-Saharan Africa, a main cause of which is the protozoan parasite Trypanosoma congolense. In comparison to the well-studied T. brucei, there is a major paucity of knowledge regarding the biology of T. congolense. Here, we use a combination of omics technologies and novel genetic tools to characterise core metabolism in T. congolense mammalian-infective bloodstream-form parasites, and test whether metabolic differences compared to T. brucei impact upon sensitivity to metabolic inhibition. Like the bloodstream stage of T. brucei, glycolysis plays a major part in T. congolense energy metabolism. However, the rate of glucose uptake is significantly lower in bloodstream stage T. congolense, with cells remaining viable when cultured in concentrations as low as 2 mM. Instead of pyruvate, the primary glycolytic endpoints are succinate, malate and acetate. Transcriptomics analysis showed higher levels of transcripts associated with the mitochondrial pyruvate dehydrogenase complex, acetate generation, and the glycosomal succinate shunt in T. congolense, compared to T. brucei. Stable-isotope labelling of glucose enabled the comparison of carbon usage between T. brucei and T. congolense, highlighting differences in nucleotide and saturated fatty acid metabolism. To validate the metabolic similarities and differences, both species were treated with metabolic inhibitors, confirming that electron transport chain activity is not essential in T. congolense. However, the parasite exhibits increased sensitivity to inhibition of mitochondrial pyruvate import, compared to T. brucei. Strikingly, T. congolense exhibited significant resistance to inhibitors of fatty acid synthesis, including a 780-fold higher EC50 for the lipase and fatty acid synthase inhibitor Orlistat, compared to T. brucei. These data highlight that bloodstream form T. congolense diverges from T. brucei in key areas of metabolism, with several features that are intermediate between bloodstream- and insect-stage T. brucei. These results have implications for drug development, mechanisms of drug resistance and host-pathogen interactions.
... Amastigote forms that have been taken up by the sand fly vector transform directly into promastigotes in the gut of the insect. In addition to polymorphism, trypanosomatid parasites adapt their metabolism to the mammalian and insect vector environments [15,16]. In this respect, the best-studied example is T. brucei. ...
In this article, we review the disease, biology, and biochemistry of kinetoplastids, as well as the new drugs and drug candidates that have entered the clinic in the last decade. We also describe examples of the preclinical exploration of small molecules against various protein targets (e.g. cysteine proteases, the proteasome, and tubulin), as well as cutting‐edge molecular and computational strategies and technologies being brought to bear to discover and develop new antitrypanosomal drugs. For comprehensive descriptions of the disease, biology, and drug therapies prior to 2011, the reader is encouraged to review the article by P.M. Woster that appeared in 2010 in the seventh edition of Burger's Medicinal Chemistry, Drug Discovery, and Development, entitled Antiprotozoal/Antiparasitic Agents.
... The energy and carbon metabolism of PCF and BSF has been compared in a number of reviews Michels et al., 2006;Tielens and van Hellemond, 2009;Creek et al., 2012;Smith et al., 2017). The next few lines highlight the main features of energy metabolism in these two parasitic forms of T. ...
Trypanosoma brucei, un protiste responsable de la Trypanosomose Humaine Africaine, également connue sous le nom de la maladie du sommeil, est transmis par la mouche tsé-tsé (Glossina sp.). La découverte d'organites de type peroxysome spécialisés dans la glycolyse, appelés glycosomes, a soulevé un certain nombre de questions sur le rôle de cet organite dans la biologie des trypanosomes. Plusieurs voies métaboliques présentes dans le cytosol d'autres eucaryotes, comme la glycolyse et la biosynthèse des sucres nucléotidiques, sont compartimentées dans les glycosomes. Les raisons et les avantages de la présence des enzymes glycolytiques dans l'organite ont été largement discutés, mais la fonctionnalité et le rôle des voies de biosynthèse des sucres nucléotidiques glycosomales ne sont pas connus. Notre étude s'est focalisée sur l'UDP-glucose pyrophosphorylase (UGP), une enzyme impliquée dans la synthèse de l'UDP-glucose (UDP-Glc). Sur la base de la double localisation glycosomale et cytosolique de l'UGP mise en évidence ici à l'aide de plusieurs techniques de localisation subcellulaire, nous avons abordé deux questions en utilisant comme modèle les formes procycliques de T. brucei présentes dans l'insecte vecteur. La première est liée au mécanisme d'import de l'UGP dans les glycosomes, car cette protéine ne possède aucun signal d'adressage aux peroxysomes de type PTS1 ou PTS2. Nous avons montré que l'UGP est importée dans les glycosomes par "piggybacking" en s'associant à la phosphoénolpyruvate décarboxylase (PEPCK) possédant un signal d’adressage PTS1. Les interactions entre l'UGP et la PEPCK ont été montrées in situ et l'identification les régions impliquées dans ces interactions ont été identifiées. Nos résultats suggèrent que le complexe UGP-PEPCK est formé de manière transitoire lors de son import dans les glycosomes nouvellement produits et compétents pour l'import des protéines. La seconde question concerne le rôle de l'UGP dans les glycosomes. Nous avons montré que l'UGP est essentielle à la croissance des trypanosomes et que les voies métaboliques glycosomales et cytosoliques dont l'UGP fait partie sont fonctionnelles. En effet, des mutants viables contenant l'UGP exclusivement dans les glycosomes ou dans le cytosol sont viables et produisent des quantités similaires d'UDP-Glc. La raison d'être de la production glycosomale d'UDP-Glc par l'UGP reste inconnue, mais n'est probablement pas liée aux réactions de glycosylation, étant donné qu'aucune glycosyltransférase n'a été détectée dans l'organite.Un autre aspect de ce travail concerne le rôle des intermédiaires du cycle de l'acide tricarboxylique (TCA) dans le métabolisme mitochondrial des formes procycliques. Dans le tractus digestif de son insecte vecteur, les trypanosomes dépendent de la proline pour alimenter leur métabolisme énergétique. Cependant, la disponibilité d'éventuelles autres sources de carbone pouvant être utilisées par le parasite est actuellement inconnue. Nous avons montré que les intermédiaires du cycle TCA, i.e. succinate, malate et a-cétoglutarate, stimulent la croissance des formes procycliques incubées dans un milieu contenant 2 mM de proline, concentration se situant dans la gamme des quantités mesurées dans l'intestin de la mouche. De plus, le développement de nouvelles approches ont permis d'étudier une branche peu explorée du cycle TCA convertissant le malate en a-cétoglutarate, précédemment décrite comme peu ou pas utilisée par le parasite, quellles que soient les quantités de glucose disponibles. L'activité de cette branche suggère qu'un cycle TCA complet peut être mis en œuvre dans les formes procycliques et probablement dans les autres formes parasitaires de l'insecte. Nos données élargissent le potentiel métabolique des trypanosomes et ouvrent la voie vers une meilleure compréhension du métabolisme de ce parasite dans divers organes de la mouche tsé-tsé, où il évolue.
... . . . GCUAGG |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } templated UUUAAAAAAAAAAAAAAAAAA zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl }|ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl { in-tail UAAUUAAUAAAAUUAAUAUAU zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl }|ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl { ex-tail |ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl {zffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl ffl } untemplated tail In T. brucei the respiratory pathway that is in part encoded in the mitochondrial genome is essential for its survival in the insect but shut off when T. brucei is in the glucose-rich bloodstream [15][16][17]. Remodeling of mitochondrial gene expression occurs as part of this transition [18]. At least some regulation of the mitochondrial transcriptome occurs at the RNA level [13], and we have previously analyzed the variation of content (nucleotide length and composition) in the 3' tail additions between life stages. ...
In this study, hierarchies of probabilistic models are evaluated for their ability to characterize the untemplated addition of adenine and uracil to the 3’ ends of mitochondrial mRNAs of the human pathogen Trypanosoma brucei , and for their generative abilities to reproduce populations of these untemplated adenine/uridine “tails”. We determined the most ideal Hidden Markov Models (HMMs) for this biological system. While our HMMs were not able to generatively reproduce the length distribution of the tails, they fared better in reproducing nucleotide composition aspects of the tail populations. The HMMs robustly identified distinct states of nucleotide addition that correlate to experimentally verified tail nucleotide composition differences. However they also identified a surprising subclass of tails among the ND1 gene transcript populations that is unexpected given the current idea of sequential enzymatic action of untemplated tail addition in this system. Therefore, these models can not only be utilized to reflect biological states that we already know about, they can also identify hypotheses to be experimentally tested. Finally, our HMMs supplied a way to correct a portion of the sequencing errors present in our data. Importantly, these models constitute rare simple pedagogical examples of applied bioinformatic HMMs, due to their binary emissions.
... Leishmania uses these phosphorylated sugars for generation of energy (ATP and reducing sugars) and several biosynthetic processes. Leishmania possesses all the glycolytic enzymes which are localized in glycosomes (first seven) and cytosol (188,189). In glucose rich condition, phosphorylated sugars in the glycosome (such as glucose-6-phospahte) are catabolized into pyruvate via glycolysis which then enters into the mitochondria and gets converted to acetyl-CoA for further metabolism. ...
... 30,31 As a result, the mitochondrion differs morphologically and metabolically between these life cycle stages. [32][33][34][35][36] In the present study, we have exploited the unusual role of the T brucei bloodstream form mitochondrion to study the effects of CL depletion in a cell where a canonical respiratory chain is absent and the F o F 1 -ATP synthase works in reverse direction compared to most other eukaryotes. We generated bloodstream form TbCls conditional knock-out parasites and examined time-dependent changes in protein levels and metabolites during CL depletion using quantitative comparative mass spectrometry. ...
The mitochondrial inner membrane glycerophospholipid cardiolipin (CL) associates with mitochondrial proteins to regulate their activities and facilitate protein complex and supercomplex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated. Trypanosoma brucei bloodstream forms use an unconventional ETC composed of glycerol‐3‐phosphate dehydrogenase and alternative oxidase (AOX), while the mitochondrial membrane potential (ΔΨm) is generated by the hydrolytic action of the FoF1‐ATP synthase (aka FoF1‐ATPase). We now report that the inducible depletion of cardiolipin synthase (TbCls) is essential for survival of T brucei bloodstream forms. Loss of CL caused a rapid drop in ATP levels and a decline in the ΔΨm. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of FoF1‐ATPase subunits and AOX, resulting in a strong decline of glycerol‐3‐phosphate‐stimulated oxygen consumption. The changes in cellular respiration preceded the observed decrease in FoF1‐ATPase stability, suggesting that the AOX‐mediated ETC is the first pathway responding to the decline in CL. Select proteins and pathways involved in glucose and amino acid metabolism were upregulated to counteract the CL depletion‐induced drop in cellular ATP.
... Recent metabolomic analyses of trypanosomatid parasites, T. brucei and Leishmania spp. have further highlighted the extraordinary metabolic diversity of these parasites 12 . In one of the most comprehensive analyses of its type, the uptake and catabolism of six different 13 C-carbon sources by Leishmania parasites was assessed using GC-MS and 13 C-NMR 6 . ...
Protozoan parasites cause a number of important diseases in humans, including malaria, African trypanosomiasis, Chagas disease and the leishmaniases. Current therapeutics for these diseases are limited and their effectiveness is being further undermined by the emergence of drug-resistant parasite strains. Parasite genome sequencing projects have provided new insights into the metabolic capacity of these pathogens and have highlighted potential drug targets. However, these genome-based reconstructions of metabolic networks are incomplete and we still have only a limited understanding of the metabolic requirements of these pathogens during infection. Metabolomics has emerged as a powerful new tool for investigating parasite metabolism and host responses, complementing more established omics technologies as well as being useful as a stand-alone technique.
... 30,31 As a result, the mitochondrion differs morphologically and metabolically between these life cycle stages. [32][33][34][35][36] In the present study, we have exploited the unusual role of the T brucei bloodstream form mitochondrion to study the effects of CL depletion in a cell where a canonical respiratory chain is absent and the F o F 1 -ATP synthase works in reverse direction compared to most other eukaryotes. We generated bloodstream form TbCls conditional knock-out parasites and examined time-dependent changes in protein levels and metabolites during CL depletion using quantitative comparative mass spectrometry. ...
Cardiolipin (CL) is a mitochondrial inner membrane glycerophospholipid that associates with mitochondrial proteins to promote their activities and to facilitate protein complex and super-complex formation. Loss of CL leads to destabilized respiratory complexes and mitochondrial dysfunction. The role of CL in an organism lacking a conventional electron transport chain (ETC) has not been elucidated so far. We now report that in Trypanosoma brucei bloodstream forms, in which the ETC is truncated and composed of alternative oxidase and glycerol-3-phosphate dehydrogenase, and the mitochondrial membrane potential is generated by the hydrolytic action of the FoF1-ATP synthase, the inducible depletion of cardiolipin synthase (TbCls) is essential for parasite survival. Loss of TbCls and CL caused a rapid drop in ATP levels and a decline in the mitochondrial membrane potential. Unbiased proteomic analyses revealed a reduction in the levels of many mitochondrial proteins, most notably of FoF1-ATP synthase subunits and of the alternative oxidase, resulting in a strong decline of glycerol-3-phosphate-stimulated oxygen consumption. Interestingly, the changes in cellular respiration preceded the observed decrease in FoF1-ATPase stability, suggesting that the truncated ETC is the first pathway responding to the decline in CL. In addition, proteomic and metabolomic analyses revealed that select proteins and pathways involved in glucose and amino acid transport and metabolism are up-regulated during CL depletion, possibly as a stress response to restore cellular ATP levels.
... The metabolite α-ketoglutarate can be metabolized via pyruvate to alanine or acetate or just partially oxidized to malate, which is then diverted to feed gluconeogenesis, a process essential for in vivo development in the tsetse fly [53]. Four canonical TCA cycle enzymes, malate dehydrogenase, CS, aconitase, and IDH seem to have no metabolic role in trypanosomes grown either in glucose or proline/threonine [54,55]. Strikingly, all of these enzymes were strongly up-regulated during the RBP6 overexpression, with CS being the most affected (increased in expression by 3.5 to 6.5 times during the differentiation). ...
Mitochondrial metabolic remodeling is a hallmark of the Trypanosoma brucei digenetic life cycle because the insect stage utilizes a cost-effective oxidative phosphorylation (OxPhos) to generate ATP, while bloodstream cells switch to aerobic glycolysis. Due to difficulties in acquiring enough parasites from the tsetse fly vector, the dynamics of the parasite’s metabolic rewiring in the vector have remained obscure. Here, we took advantage of in vitro–induced differentiation to follow changes at the RNA, protein, and metabolite levels. This multi-omics and cell-based profiling showed an immediate redirection of electron flow from the cytochrome-mediated pathway to an alternative oxidase (AOX), an increase in proline consumption, elevated activity of complex II, and certain tricarboxylic acid (TCA) cycle enzymes, which led to mitochondrial membrane hyperpolarization and increased reactive oxygen species (ROS) levels. Interestingly, these ROS molecules appear to act as signaling molecules driving developmental progression because ectopic expression of catalase, a ROS scavenger, halted the in vitro–induced differentiation. Our results provide insights into the mechanisms of the parasite’s mitochondrial rewiring and reinforce the emerging concept that mitochondria act as signaling organelles through release of ROS to drive cellular differentiation.
... In eukaryotes, including many single-celled pathogens, expression of a mitochondrial genome must be coordinated with nuclear gene expression because subunits of mitochondrial electron transport chain (ETC) The best studied trypanosome, Trypanosoma brucei, utilizes its ETC to generate adenosine triphosphate (ATP) when replicating in its insect host, but when replicating in the glucose-rich mammalian bloodstream environment is entirely reliant on glycolysis for energy, primarily excreting the pyruvate product of glycolysis rather than extracting additional energy from this metabolic intermediate in its single mitochondrion (Tielens & van Hellemond, 2009). Not surprisingly, mature mRNAs of subunits of ETC complexes III and IV that are derived from the mitochondrial genome are not present at this life stage as they are in T. brucei's insect stage (Bhat, Souza, Feagin, & Stuart, 1992;Feagin & Stuart, 1985;Feagin, Abraham, & Stuart, 1988;Read, Stankey, Fish, Muthiani, & Stuart, 1994). ...
The protozoan Trypanosoma cruzi has a complicated dual‐host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian‐infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation‐induced changes in gene expression. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation in insect‐stage cells, despite the previously‐observed increases in mitochondrial mRNAs encoding electron transport chain components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short‐flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet‐unknown signaling mechanism. Cell lengths are analyzed in Trypanosoma cruzi overexpressing the mitochondrial ribonuclease TcRND.
... Trypanosomatids have a high rate of glucose consumption and this is associated with aerobic fermentation of glucose [35]. The mitochondrial energy metabolism of trypanosomatids can be considered an intermediate between classical aerobically functioning mitochondria and true anaerobically functioning mitochondria, similar to most parasitic helminths and several marine organisms [36]. ...
Chagas disease, also known as American trypanosomiasis, is a potentially life-threatening illness caused by the protozoan parasite, Trypanosoma cruzi, and is transmitted by triatomine insects during its blood meal. Proliferative epimastigotes forms thrive inside the insects in the presence of heme (iron protoporphyrin IX), an abundant product of blood digestion, however little is known about the metabolic outcome of this signaling molecule in the parasite. Trypanosomatids exhibit unusual gene transcription employing a polycistronic transcription mechanism through trans-splicing that regulates its life cycle. Using the Deep Seq transcriptome sequencing we characterized the heme induced transcriptome of epimastigotes and determined that most of the upregulated genes were related to glucose metabolism inside the glycosomes. These results were supported by the upregulation of glycosomal isoforms of PEPCK and fumarate reductase of heme-treated parasites, implying that the fermentation process was favored. Moreover, the downregulation of mitochondrial gene enzymes in the presence of heme also supported the hypothesis that heme shifts the parasite glycosomal glucose metabolism towards aerobic fermentation. These results are examples of the environmental metabolic plasticity inside the vector supporting ATP production, promoting epimastigotes proliferation and survival.
... The energy metabolism of trypanosomatids is largely dependent on the available carbon sources present in their hosts [41,42]. In addition to their crucial role in ATP production [43], mitochondria are also the main sources of ROS, as a consequence of electron leakage during oxidative phosphorylation [44]. ...
During their life cycle, trypanosomatids are exposed to stress conditions and adapt their energy and antioxidant metabolism to colonize their hosts. Strigomonas culicis is a monoxenous protist found in invertebrates with an endosymbiotic bacterium that completes essential biosynthetic pathways for the trypanosomatid. Our research group previously generated a wild-type H2O2-resistant (WTR) strain that showed improved mitochondrial metabolism and antioxidant defenses, which led to higher rates of Aedes aegypti infection. Here, we assess the biological contribution of the S. culicis endosymbiont and reactive oxygen species (ROS) resistance to oxidative and energy metabolism processes. Using high-throughput proteomics, several proteins involved in glycolysis and gluconeogenesis, the pentose phosphate pathway and glutathione metabolism were identified. The results suggest that ROS resistance decreases glucose consumption and indicate that the metabolic products from gluconeogenesis are key to supplying the protist with high-energy and reducing intermediates. Our hypothesis was confirmed by biochemical assays showing opposite profiles for glucose uptake and hexokinase and pyruvate kinase activity levels in the WTR and aposymbiotic strains, while the enzyme glucose-6P 1-dehydrogenase was more active in both strains. Regarding the antioxidant system, ascorbate peroxidase has an important role in H2O2 resistance and may be responsible for the high infection rates previously described for A. aegypti. In conclusion, our data indicate that the energy-related and antioxidant metabolic processes of S. culicis are modulated in response to oxidative stress conditions, providing new perspectives on the biology of the trypanosomatid-insect interaction as well as on the possible impact of resistant parasites in accidental human infection.
... Article Bearing in mind that ΔΨ m is essential for mitochondrial ATP synthesis, 27 we analyzed the levels of ATP in parasites treated with 0.02, 0.1, and 1 μM of 1 after 30, 60, 120 min, and 24 h using the bioluminescence assay CellTiter-Glo. Compound 1 elicited a rapid time and concentrationdependent depletion of ATP in the treated parasites ( Figure 2B). ...
We report the discovery of new 4-hydroxyphenyl phosphonium salt derivatives active in the submicromolar range (EC50 from 0.04 to 0.28 µM, SI > 10) against the protozoan parasite Leishmania donovani. The pharmacokinetics and in vivo oral efficacy of compound 1 [(16-(2,4-dihydroxyphenyl)-16-oxohexadecyl)triphenylphosphonium bromide] in a mouse model of visceral leishmaniasis were established. Compound 1 reduced the parasite load in spleen (98.9%) and liver (95.3%) of infected mice after an oral dosage of 4 daily doses of 1.5 mg/kg. Mode of action studies showed that compound 1 diffuses across the plasma membrane, as designed, and targets the mitochondrion of Leishmania parasites. Disruption of the energetic metabolism, with a decrease of intracellular ATP levels as well as mitochondrial depolarization together with a significant ROS production, contributes to the leishmanicidal effect of 1. Importantly, this compound was equally effective against antimonials and miltefosine-resistant clinical isolates of Leishmania infantum indicating its potential as antileishmanial lead.
... Proper nuclear-mitochondrial crosstalk is expected to be particularly cycle. The best studied trypanosome, Trypanosoma brucei, actively respires when replicating in 47 its insect host, but when replicating in the glucose-rich mammalian bloodstream environment is 48 entirely reliant on glycolysis for energy, primarily excreting the pyruvate product of glycolysis 49 rather than extracting additional energy from this metabolic intermediate in its single 50 mitochondrion (Tielens and van Hellemond, 2009). Not surprisingly, mature mRNAs of subunits 51 of ETC complexes III and IV that are derived from the mitochondrial genome are not present at 52 this life stage as they are in T. brucei's insect stage (Feagin and Stuart, 1985;Feagin et al., 1988;53 We performed our previous study in culture conditions of both replete nutrients and 67 starvation, as starvation is a major signal for differentiation of epimastigotes to trypomastigotes. ...
The protozoan Trypanosoma cruzi has a complicated dual-host life cycle, and starvation can trigger transition from the replicating insect stage to the mammalian-infectious nonreplicating insect stage (epimastigote to trypomastigote differentiation). Abundance of some mature RNAs derived from its mitochondrial genome increase during culture starvation of T. cruzi for unknown reasons. Here we examine T. cruzi mitochondrial gene expression in the mammalian intracellular replicating life stage (amastigote), and uncover implications of starvation-induced changes in gene expression in insect-stage cells. Mitochondrial RNA levels in general were found to be lowest in actively replicating amastigotes. We discovered that mitochondrial respiration decreases during starvation, despite the previously-observed increases in mitochondrial mRNAs encoding electron transport chain components. Surprisingly, T. cruzi epimastigotes in replete medium grow at normal rates when we genetically compromised their ability to perform insertion/deletion editing and thereby generate mature forms of some mitochondrial mRNAs. However, these cells, when starved, were impeded in the epimastigote to trypomastigote transition. Further, they experience a short-flagella phenotype that may also be linked to differentiation. We hypothesize a scenario where levels of mature RNA species or editing in the single T. cruzi mitochondrion are linked to differentiation by a yet-unknown signaling mechanism.
... First, we studied if the energetic metabolism of L. infantum parasites was modified in parasites after the treatment with 1. In Leishmania, ATP is mainly synthesized by mitochondrial oxidative phosphorylation [18]. Thus, we analyzed the levels of ATP in parasites treated with 0.5, 1 and 5 mM of compound 1 at the timepoints of 30, 60, 120 min and 24 h. ...
Among neglected tropical diseases, leishmaniasis is one of the most relevant with an estimated 30,000 deaths annually. Existing therapies have serious drawbacks in safety, drug resistance, field-adapted application and cost; therefore, new safer and shorter treatments are needed for this disease. Here we report on the synthesis of novel 4-amino-7-chloroquinoline-based compounds with leishmanicidal activity, together with deeper insight into the mechanism of action of our previously published hit compound 1. New derivatives showed comparable activity to 1 against both promastigote and intracellular amastigote forms of Leishmania infantum, with IC50 < 1 μM. Furthermore, we have determined that compound 1 induced a decrease of intracellular ATP levels, as well as a mitochondrial depolarization, together with an alteration of plasma membrane permeability and a significant ROS production. The inhibition of the energy metabolism of Leishmania plays an important role in the leishmanicidal mechanism of this compound. In all, these results support the consideration of compound 1 for the future development of new leishmanicidal drugs.
... L. major promastigotes experience glucoserich conditions in the digestive tract of their sandfly vector, as the sugar meals consumed from the plant sap were digested by amylase, kinase and sucrose enzymes (30). ...
Abstract
Background: Xanthium strumarium L. is extensively used as a traditional herb to
treat many diseases and is also known as a source of phytochemicals. It has been
used traditionally to treat trypanosomiasis, malaria fever, eczema, cancer, ulcer, fever,
herpes headache, and skin lesion such as leishmaniasis. In this preliminary
study, nuclear magnetic resonance (NMR)-metabolomics approaches was used to
evaluate the inhibitory effects and metabolic alterations caused by leaf extract of X.
strumarium on the stationary phases of promastigotes in Leishmania major.
Methods: The promastigotes were cultured in Biochemistry Laboratory at Pasteur
Institute of Iran in 2017, stationary phases were obtained from 5 to 6 day-old cultures
and treated with different concentrations of the plant’s extract. Antileishmanial
activity was assayed by MTT method and cell metabolites were extracted. 1H
NMR spectroscopy was applied, and outliers were separated using multivariate statistical
analysis.
Results: The most affected metabolic pathways in the experimental groups were
limited to amino sugar and nucleotide sugar metabolism, cyanoamino acid metabolism,
starch and sucrose metabolism, butanoate metabolism, and galactose metabolism.
Conclusion: The ethanolic leaf extract of X. strumarium is a potent growth inhibitor
of Leishmania major and can affect vital metabolic pathways of Leishmania promastigotes.
The assay provided new perspectives on the development of novel
treatment strategies for leishmanial activity derived from natural products.
... One of the most intriguing features of Trypanosoma brucei, a unicellular kinetoplastid parasite, is its unique way of producing energy in different life cycle stages 1 . In the insect vector, the single mitochondrion in T. brucei undergoes extensive morphological alterations and changes in gene expression. ...
RNA-binding proteins play a particularly important role in regulating gene expression in trypanosomes. A map of the network of protein complexes in Trypanosoma brucei uncovered an essential protein (Tb927.10.7910) that is postulated to be an RNA-binding protein implicated in the regulation of the mitochondrial post-transcriptional gene regulatory network by its association with proteins that participate in a multi-protein RNA editing complex. However, the mechanism by which this protein interacts with its multiple target transcripts remained unknown. Using sensitive database searches and experimental data, we identify Z-DNA-binding domains in T. brucei in the N- and C-terminal regions of Tb927.10.7910. RNA-binding studies of the wild-type protein, now referred to as RBP7910 (RNA binding protein 7910), and site-directed mutagenesis of residues important for the Z-DNA binding domains show that it preferentially interacts with RNA molecules containing poly(U) and poly(AU)-rich sequences. The interaction of RBP7910 with these regions may be involved in regulation of RNA editing of mitochondrial transcripts.
... Trypanosoma brucei (TB) does not have a fully functional complex I in the procyclic life stage (TB (PF)) [23], which dramatically lowers the activity of the respiratory chain. Reduced ubiquinone is regenerated only by TAO in the blood stream form (TB(BF)) because activity of the rest of the respiratory chain is reduced in this cell cycle stage (for review see [21]). Leishmania tarentolae has no TAO and no measurable NADH dehydrogenase activity [16,18,24]. ...
The measurement of respiratory chain enzyme activities is an integral part of basic research as well as for specialized examinations in clinical biochemistry. Most of the enzymes use ubiquinone as one of their substrates. For current in vitro measurements, several hydrophilic analogues of native ubiquinone are used depending on the enzyme and the workplace. We tested five readily available commercial analogues and we showed that Coenzyme Q2 is the most suitable for the measurement of all tested enzyme activities. Use of a single substrate in all laboratories for several respiratory chain enzymes will improve our ability to compare data, in addition to simplifying the stock of chemicals required for this type of research.
... In order to test the mitochondrial tropism of our compounds, we decided to use Leishmania promastigotes as a model system. At this stage of the parasite life cycle, mitochondrial synthesis accounts for nearly 70% of the total production of ATP [21,24,35], and therefore the study of promastigote forms facilitates the identification of drugs affecting mitochondrial ATP production. Thus, the activity of compounds 11e23 was evaluated against Leishmania donovani promastigotes (strain MHOM/SD/00/1S-2D) in a phenotypic screening. ...
A new class of quinoline derivatives, bearing amino chains at C-4 and a styryl group at C-2, were tested on Leishmania donovani promastigotes and axenic and intracellular Leishmania pifanoi amastigotes. The introduction of the C-4 substituent improves the activity, which is due to interference with the mitochondrial activity of the parasite and its concomitant bioenergetic collapse by ATP exhaustion. Some compounds show a promising antileishmanial profile, with low micromolar or submicromolar activity on promastigote and amastigote forms and a good selectivity index.
... For instance, in the tsetse fly, TAO is expressed at a low level to allow respiratory plasticity, which helps the parasite survive the very harsh environment in the midgut of the fly 14 ; however, these procyclic forms have a fully functional electron transport chain, and express TAO together with COX and all the tricarboxylic acid (TCA) cycle proteins. Conversely, the BSFs present in the mammalian hosts do not express COX or most TCA cycle proteins, 50,64 and their mitochondria do not contain the succinate dehydrogenase complex (complex II) 28 or use the classical electron transport chain to generate the proton-motive force over the inner mitochondrial membrane. 65,66 Instead, TAO is upregulated through a posttranscriptional mechanism and the parasite relies exclusively on TAO as the sole terminal oxidase in its mitochondrial electron transport chain. ...
The alternative oxidase (AOX) is a ubiquitous terminal oxidase of plants and many fungi, catalyzing the four electron reduction of oxygen to water alongside the cytochrome‐based electron transfer chain. Unlike the classical electron transfer chain, however, the activity of AOX does not generate adenosine triphosphate but has functions such as thermogenesis and stress response. As it lacks a mammalian counterpart, it has been investigated intensely in pathogenic fungi. However, it is in African trypanosomes, which lack cytochrome‐based respiration in their infective stages, that trypanosome alternative oxidase
(TAO) plays the central and essential role in their energy metabolism. TAO was validated as a drug target decades ago and among the first inhibitors to be identified was salicylhydroxamic acid (SHAM), which produced the expected trypanocidal effects, especially when potentiated by coadministration with glycerol to inhibit anaerobic energy metabolism as well. However, the efficacy of this combination was too low to be of practical clinical use. The antibiotic ascofuranone (AF) proved a much stronger TAO inhibitor and was able to cure Trypanosoma vivax infections in mice without glycerol and at much lower doses, providing an
important proof of concept milestone. Systematic efforts to improve the SHAM and AF scaffolds, aided with the elucidation of the TAO crystal structure, provided detailed structure‐activity relationship information and reinvigorated the drug discovery effort. Recently, the coupling of
mitochondrion‐targeting lipophilic cations to TAO inhibitors has dramatically improved drug targeting and trypanocidal activity while retaining target protein potency. These developments appear to have finally signposted the way to preclinical development of TAO inhibitors.
... Interestingly, a strong pattern of preferential enrichment of G-tracts in some transcripts was observed, particularly with respect to the U-insertion/deletion editing state of the transcripts. Specifically, we divided pre-edited transcripts into nine that undergo extensive editing, with more than 50% of the final sequence consisting of inserted uridines ("pan-edited" henceforth); three that undergo editing events 4 6.55 ± 0.10 to a much less dramatic extent and exclusively to the 5′ end (Cyb and Murf II) or correction of an internal frameshift (COII) ("limited" editing); and the six mitochondrial transcripts that undergo no insertion/deletion editing ("never-edited"). The final versions of the twelve pan-and limited-editing transcripts after all editing events are complete are denoted "post-editing". ...
Pentatricopeptide repeat (PPR) proteins, a helical repeat family of organellar RNA binding proteins, play essential roles in post-transcriptional RNA processing. In Trypanosoma brucei, an expanded family of PPR proteins localize to the parasite’s single mitochondrion, where they are believed to perform important roles in both RNA processing and translation. We studied the RNA binding specificity of the simplest T. brucei PPR protein (KRIPP11) using electrophoretic mobility shift assays, fluorescence anisotropy, circular dichroism spectroscopy, and in vitro selection. We found KRIPP11 to be an RNA binding protein with specificity for sequences of four or more consecutive guanosine residues (G-tracts). Such G-tracts are dramatically enriched in T. brucei mitochondrial transcripts that are destined for extensive uridine insertion/deletion editing but are not present in mRNAs following editing. We further found that the quadruplex oligoguanosine RNA conformation is preferentially recognized by KRIPP11 over other conformational forms, and is bound without disruption of the quadruplex structure. In combination with prior data demonstrating association of KRIPP11 with the small ribosomal subunit, these results suggest possible roles for KRIPP11 in bridging mRNA maturation and translation or in facilitating translation of unusual dual-coded open reading frames.
... For instance, T. equiperdum, as well as T. evansi and the trypomastigote slender and stumpy stages of T. brucei, live in the bloodstream of their mammalian host, which warrants a permanent source of glucose. Parasite trypanosomes living in carbohydrate-rich environments seem to depend on carbohydrates for their energy metabolism, and do not require a cytochrome-containing respiratory chain or an elaborate mitochondrial metabolism (Tielens and van Hellemond, 2009). Since glucose deprivation activates the T. equiperdum protein kinase described here, this enzyme may perhaps participate in a signalling cascade triggered by nutritional and/ or energy stress. ...
Kemptide (sequence: LRRASLG) is a synthetic peptide holding the consensus recognition site for the catalytic subunit of the cAMP-dependent protein kinase (PKA). cAMP-independent protein kinases that phosphorylate kemptide were stimulated in Trypanosoma equiperdum following glucose deprivation. An enriched kemptide kinase-containing fraction was isolated from glucose-starved parasites using sedimentation throughout a sucrose gradient, followed by sequential chromatography on diethylaminoethyl-Sepharose and Sephacryl S-300. The trypanosome protein possesses a molecular mass of 39.07–51.73 kDa, a Stokes radius of 27.4 Ǻ, a sedimentation coefficient of 4.06 S and a globular shape with a frictional ratio f/fo = 1.22–1.25. Optimal enzymatic activity was achieved at 37 °C and pH 8.0, and kinetic studies showed Km values for ATP and kemptide of 11.8 ± 4.1 and 24.7 ± 3.8 µm , respectively. The parasite enzyme uses ATP and Mg ²⁺ and was inhibited by other nucleotides and/or analogues of ATP, such as cAMP, AMP, ADP, GMP, GDP, GTP, CTP, β , γ -imidoadenosine 5′-triphosphate and 5′-[ p -(fluorosulfonyl)benzoyl] adenosine, and by other divalent cations, such as Zn ²⁺ , Mn ²⁺ , Co ²⁺ , Cu ²⁺ , Ca ²⁺ and Fe ²⁺ . Additionally, the trypanosome kinase was inhibited by the PKA-specific heat-stable peptide inhibitor PKI- α . This study is the first biochemical and enzymatic characterization of a protein kinase from T. equiperdum .
... It has a complex life cycle including two main proliferative stages, the procyclic form (PF) in the tsetse fly (Glossina sp.) and the bloodstream form (BF) in the final mammalian host [19]. As these two stages occur in entirely different environments, they differ fundamentally in cell morphology and physiology, including their mechanisms of energy conversion [20]. In the PF, ATP is produced mostly by oxidative phosphorylation where the F-ATPase generates ATP [21]. ...
Hydrolysis of ATP by the mitochondrial F‐ATPase is inhibited by a protein called IF1. In the parasitic flagellate, Trypanosoma brucei, this protein, known as TbIF1, is expressed exclusively in the procyclic stage, where the F‐ATPase is synthesizing ATP. In the bloodstream stage, where TbIF1 is absent, the F‐ATPase hydrolyzes ATP made by glycolysis and compensates for the absence of a proton pumping respiratory chain by translocating protons into the intermembrane space, thereby maintaining the essential mitochondrial membrane potential. We have defined regions and amino acid residues of TbIF1 that are required for its inhibitory activity by analyzing the binding of several modified recombinant inhibitors to F1‐ATPase isolated from the procyclic stage of T. brucei. Kinetic measurements revealed that the C‐terminal portion of TbIF1 facilitates homodimerization, but it is not required for the inhibitory activity, similar to the bovine and yeast orthologs. However, in contrast to bovine IF1, the inhibitory capacity of the C‐terminally truncated TbIF1 diminishes with decreasing pH, similar to full length TbIF1. This effect does not involve the dimerization of active dimers to form inactive tetramers. Over a wide pH range, the full length and C‐terminally truncated TbIF1 form dimers and monomers, respectively. TbIF1 has no effect on bovine F1‐ATPase, and this difference in the mechanism of regulation of the F‐ATPase between the host and the parasite could be exploited in the design of drugs to combat human and animal African trypanosomiases. This article is protected by copyright. All rights reserved.
... T. brucei and related trypanosomatid flagellates contain a single canonical mitochondrion that generates ATP via oxidative phosphorylation, with oxygen being the terminal electron acceptor (Tielens and van Hellemond 2009;Škodová-Sveráková et al. 2015). It is likely that in terms of main metabolic setup, Diplonema papillatum ( Fig. 6.1) and other diplonemids have a rather similar organelle (our unpublished data). ...
RNA editing and processing in the mitochondrion of Diplonema papillatum and other diplonemids are arguably the most complex processes of their kind described in any organelle so far. Prior to translation, each transcript has to be accurately trans-spliced from gene fragments encoded on different circular chromosomes. About half of the transcripts are massively edited by several types of substitution editing and addition of blocks of uridines. Comparative analysis of mitochondrial RNA processing among the three euglenozoan groups, diplonemids, kinetoplastids, and euglenids, highlights major differences between these lineages. Diplonemids remain poorly studied, yet they were recently shown to be extremely diverse and abundant in the ocean and hence are rapidly attracting increasing attention. It is therefore important to turn them into genetically tractable organisms, and we report here that they indeed have the potential to become such.
... Amino acids are available in both mammalian and insect hosts of TriTryps at different concentrations and these metabolites can be consumed as energy source, what occurs mainly when they colonize insect vectors [29][30][31]. Amino acid transporters with a broad range of affinities and specificities have been described for TriTryps, probably reflecting the broad range of concentrations in which amino acids are present in the different hosts. ...
Trypanosoma brucei
, as well asTrypanosoma cruziand more than 20 species of the genusLeishmania, form a group of flagellated protists that threaten human health. These organisms are transmitted by insects that, together with mammals, are their natural hosts. This implies that during their life cycles each of them faces environments with different physical, chemical, biochemical, and biological characteristics. In this work we review how amino acids are obtained from such environments, how they are metabolized, and how they and some of their intermediate metabolites are used as a survival toolbox to cope with the different conditions in which these parasites should establish the infections in the insects and mammalian hosts.
... Remarkably, due to their complex mt gene expression, several hundreds of proteins are required to synthe- size the only two mt encoded proteins (RPS12 and F o F 1 -ATPase subunit A6) deemed to be essential for the extracellular pathogen to reside in the bloodstream of its mammalian host 7,21 . While the insect stage (procyclic form, PF) of the parasite depends on the oxidative phosphorylation pathway to generate sufficient quantities of cellular ATP, the bloodstream form (BF) exploits the high glucose content of its surroundings to synthesize ATP through glycolysis 22 . This bioenergetics adaptation to the varied nutrients available throughout its life cycle results in a dramatic remodeling of the singular mitochondrion. ...
Trypanosoma brucei is an extracellular parasite that alternates between an insect vector (procyclic form) and the bloodstream of a mammalian host (bloodstream form). While it was previously reported that mitochondrial release factor 1 (TbMrf1) is essential in cultured procyclic form cells, we demonstrate here that in vitro bloodstream form cells can tolerate the elimination of TbMrf1. Therefore, we explored if this discrepancy is due to the unique bioenergetics of the parasite since procyclic form cells rely on oxidative phosphorylation; whereas bloodstream form cells utilize glycolysis for ATP production and FoF1-ATPase to maintain the essential mitochondrial membrane potential. The observed disruption of intact bloodstream form FoF1-ATPases serves as a proxy to indicate that the translation of its mitochondrially encoded subunit A6 is impaired without TbMrf1. While these null mutants have a decreased mitochondrial membrane potential, they have adapted by increasing their dependence on the electrogenic contributions of the ADP/ATP carrier to maintain the mitochondrial membrane potential above the minimum threshold required for T. brucei viability in vitro. However, this inefficient compensatory mechanism results in avirulent mutants in mice. Finally, the depletion of the codon-independent release factor TbPth4 in the TbMrf1 knockouts further exacerbates the characterized mitchondrial phenotypes.
... The proteomic studies published so far have been conducted using either L. donovani or L. infantum, probably due to the medical importance of the visceral forms arising from these parasites. In accordance with our studies it has been described [60][61][62] that resultant proteome can be divided into five groups of proteins with similar function: -(i) stress response (e.g., heat, oxidative stress); (ii) cytoskeleton and cell membrane; (iii) carbohydrate/ energy metabolism, stress response and phosphorylation; (iv) cell cycle and proliferation; and (v) amino acid metabolism.‖ Heat shock proteins (HSPs) 60 and 70, and HSP 70-related proteins are also overexpressed. ...
Purpose:
Although in trypanosomatids, monoxeny (Leptomonas) is ancestral to dixeny (Leishmania), however clinical cases of visceral leishmanisis with Leptomonas co-infection are increasingly being reported from India. Using a proteogenomic approach, we carried out a detailed proteome analysis of these two kinetoplastid parasites viz. Leishmania and its sister Leptomonas, to catalog the key proteins associated with and therefore possibly responsible for phenotype changes in Leptomonas evolution and domestication as co-infection with Leishmania.
Experimental design:
Liquid chromatography mass spectrometry (LC-MS/MS) was utilized for this proteomic purpose. One Leishmania donovani WHO reference strain and two Leptomonas seymouri isolates which were originally isolated from clinical cases of kala azar patients with different inherent drug sensitivity viz. responsive and unresponsive, were used in this study.
Results:
A network analysis, leveraging protein-protein interaction data helped to find the roles of the proteins in carbon metabolism and biosynthesis of secondary metabolites which was seen to be altered under stress conditions like drug resistance.
Conclusions and clinical relevance:
The information provided about the metabolic pathways modulated when contrasting these two phenotypes may lead to the development of new strategies to block parasite differentiation within the host and to also circumvent the problem of drug resistance. This proteomic study also offers new grounds for the investigation of novel hypothetical proteins potentially playing a role in evolutionary biology the knowledge of which is essential for treatment of patients co-infected with these two kinetoplastid parasites. This article is protected by copyright. All rights reserved.
... In-tails are fairly ubiquitous oligomer tails added to mtRNAs. The As in in-tails are 96 Read et al., 1992Read et al., , 1994Souza et al., 1992Souza et al., , 1993Corell et al., 1994), as its metabolism dramatically 97 changes in these two environments (Tielens and van Hellemond, 2009). A fascinating and plausible 98 possibility is that tails play a role in this developmental regulation. ...
The intricate life cycle of Trypanosoma brucei requires extensive regulation of gene expression levels of the mtRNAs for adaptation. Post-transcriptional gene regulatory programs, including unencoded mtRNA 3' tail additions, potentially play major roles in this adaptation process. Intriguingly, T. brucei mitochondrial transcripts possess two distinct unencoded 3' tails, each with a differing functional role; i.e., while one type is implicated in RNA stability (in-tails), the other type appears associated with translation (ex-tails). We examined the degree to which tail characteristics differ among cytochrome c oxidase subunits I and III (CO1 and CO3), and NADH dehydrogenase subunit 1 (ND1) transcripts, and to what extent these characteristics differ developmentally. We found that CO1, CO3 and ND1 transcripts possess longer in-tails in the mammalian life stage. By mathematically modeling states of in-tail and ex-tail addition, we determined that the typical length at which an in-tail is extended to become an ex-tail differs by transcript and, in the case of ND1, by life stage. To the best of our knowledge, we provide the first evidence that developmental differences exist in tail length distributions of mtRNAs, underscoring the potential involvement of in-tail and ex-tail populations in mitochondrial post-transcriptional regulation mechanisms.
... These algorithms however, cannot be used in the analysis of our edited transcripts as the two-component genetic system (mRNAs created by gRNA editing) introduces another layer of mutational constraint [32]. In addition, the edited sequence of the transcripts is known for only a limited number of kinetoplastids, and only the salivarian trypanosomes have the same general life cycle; other kinetoplastids, like Leishmania and T. cruzi, have evolved different infective cycles and are under very different selective pressures [5,33,34]. Fully edited sequences are known for T. vivax, the earliest branching salivarian trypanosome [35,36]. T. vivax differs from T. brucei in that they complete the insect phase of their life cycle entirely within the proboscis of the fly. ...
Trypanosoma brucei is transmitted between mammalian hosts by the tsetse fly. In the mammal, they are exclusively extracellular, continuously replicating within the bloodstream. During this stage, the mitochondrion lacks a functional electron transport chain (ETC). Successful transition to the fly, requires activation of the ETC and ATP synthesis via oxidative phosphorylation. This life cycle leads to a major problem: in the bloodstream, the mitochondrial genes are not under selection and are subject to genetic drift that endangers their integrity. Exacerbating this, T. brucei undergoes repeated population bottlenecks as they evade the host immune system that would create additional forces of genetic drift. These parasites possess several unique genetic features, including RNA editing of mitochondrial transcripts. RNA editing creates open reading frames by the guided insertion and deletion of U-residues within the mRNA. A major question in the field has been why this metabolically expensive system of RNA editing would evolve and persist. Here, we show that many of the edited mRNAs can alter the choice of start codon and the open reading frame by alternative editing of the 5’ end. Analyses of mutational bias indicate that six of the mitochondrial genes may be dual-coding and that RNA editing allows access to both reading frames. We hypothesize that dual-coding genes can protect genetic information by essentially hiding a non-selected gene within one that remains under selection. Thus, the complex RNA editing system found in the mitochondria of trypanosomes provides a unique molecular strategy to combat genetic drift in non-selective conditions.
... A short review by Schatz (2013), neatly presented how research on mitochondrial biogenesis, once considered inaccessible, initiated about 50 years back when powerful molecular techniques, like yeast genetics and electron microscopy were brought to address this problem. The role of anaerobically functioning mitochondria and its enzyme complexes in parasitic microorganisms, like Trypanosomatidae (Hellemond et al. 2007;Tielens et al. 2009), and Fasciola hepatica (Hellemond et al. 1996;Hellemond & Tielens 1997; van Grinsven et al. 2009), have been researched in fine details during the last two decades. Although our understanding has expanded, there remain speculations about origin of mitochondria. ...
... During the differ- ent life-cycle stages, the parasite adapts its metabolism to the nutrient availability inside the different hosts. It is postulated that T. cruzi's energy metabolism relies on the respiratory chain, at least in some stages of its life cycle [12][13][14]. It has also been proposed that a branched respiratory chain is functioning in the parasite mito- chondrion, with a type aa3 CcO as its main terminal oxidase, and with a contribution to the total oxygen con- sumption of a putative alternative oxidase, as found in Trypanosoma brucei (TAO), and/or an oxidase containing cytochrome o [15][16][17]. ...
Trypanosoma cruzi , the causative agent of Chagas disease, presents a complex life cycle and adapts its metabolism to nutrients availability. Although T. cruzi is an aerobic organism, it does not produce heme. This cofactor is acquired from the host, and is distributed and inserted into different heme-proteins such as respiratory complexes in the parasite mitochondrion. It has been proposed that T. cruzi's energy metabolism relies on a branched respiratory chain with a cytochrome c oxidase type aa 3 (C c O) as the main terminal oxidase. Heme A, the cofactor for all eukaryotic C c O, is synthesized via two sequential enzymatic reactions catalyzed by heme O synthase (HOS) and heme A synthase (HAS). Previously, TcCox10 and TcCox15 were identified in T. cruzi They presented HOS and HAS activity, respectively, when were expressed in yeast. Here, we present the first characterization of TcCox15 in T. cruzi , confirming its role as heme A synthase. It was differentially detected in the different T. cruzi stages, being more abundant in the replicative forms. This regulation could reflect the necessity of more heme A synthesis, and therefore more C c O activity at the replicative stages. Over-expression of a non-funtional mutant caused a reduction in heme A content. Moreover, our results clearly showed that this hindrance in the heme A synthesis provoked a reduction on C c O activity and, in consequence, an impairment on T. cruzi survival, proliferation and infectivity. These evidences support that T. cruzi depends on the respiratory chain activity along its life cycle, being C c O an essential terminal oxidase.
... This metabolic adjustment requires activation of the mitochondrion, with which PCF cells generate most cellular ATP via oxidative phosphorylation, thus making the organelle more essential for this life cycle PLOS stage [3]. Mitochondrial morphology is also dramatically altered during this inter-stagial transformation, changing from a simple tube-like appearance in BSF to an elaborate and highly branched structure with multiple cristae in PCF [4,5]. While the advantages of living in a nutrient-rich environment are obvious, the mammalian bloodstream also presents BSF parasites with a number of challenges. ...
Dynamins and dynamin-like proteins (DLPs) belong to a family of large GTPases involved in membrane remodelling events. These include both fusion and fission processes with different dynamin proteins often having a specialised function within the same organism. Trypanosoma brucei is thought to have only one multifunctional DLP (TbDLP). While this was initially reported to function in mitochondrial division only, an additional role in endocytosis and cytokinesis was later also proposed. Since there are two copies of TbDLP present in the trypanosome genome, we investigated potential functional differences between these two paralogs by re-expressing either protein in a TbDLP RNAi background. These paralogs, called TbDLP1 and TbDLP2, are almost identical bar a few amino acid substitutions. Our results, based on cell lines carrying tagged and RNAi-resistant versions of each protein, show that overexpression of TbDLP1 alone is able to rescue the observed endocytosis and growth defects in the mammalian bloodstream form (BSF) of the parasite. While TbDLP2 shows no rescue in our experiments in BSF, this might also be due to lower expression levels of the protein in this life stage. In contrast, both TbDLP proteins apparently play more complementary roles in the insect procyclic form (PCF) since neither TbDLP1 nor TbDLP2 alone can fully restore wildtype growth and morphology in TbDLP-depleted parasites.
... 15 T. cruzi energy metabolism changes according to the carbon source available in the different microenvironments the parasite finds itself during its life cycle. 16 In the infectious, nonreplicative bloodstream trypomastigote form, the parasite is able to utilize the abundant glucose supply found in its mammalian host's bloodstream. In contrast, the replicative intracellular amastigotes grow in almost free glucose medium, and are predicted to depend on amino acid catabolism for energy production. ...
Trypanosoma cruzi is the causative agent of Chagas disease. The lack of an efficient and safe treatment supports the research into novel metabolic targets, with the malic enzyme (ME) representing one such potential candidate. T. cruzi expresses a cytosolic (TcMEc) and a mitochondrial (TcMEm) ME isoform, with these activities functioning to generate NADPH, a key source of reducing equivalents that drives a range of anabolic and protective processes. To identify specific inhibitors that target TcMEs, two independent high-throughput screening strategies using a diversity library containing 30,000 compounds were employed. IC50 values of 262 molecules were determined for both TcMEs, as well as for three human ME isoforms, with the inhibitors clustered into six groups according to their chemical similarity. The most potent hits belonged to a sulfonamide group that specifically target TcMEc. Moreover, several selected inhibitors of both TcMEs showed a trypanocidal effect against the replicative forms of T. cruzi. The chemical diversity observed among those compounds that inhibit TcMEs activity emphasizes the druggability of these enzymes, with a sulfonamide-based subset of compounds readily able to block TcMEc function at a low nanomolar range.
Leishmaniasis is a parasitic and neglected tropical disease which majorly impacts poor and developing nations. One of the significant factors that impacts the severity of the pathological condition includes the socioeconomic background of the affected region. The rise of drug-resistant Leishmania is a serious concern for the effectiveness of the present treatment. As a result, the drug options need to be relooked immediately. Leishmania employs Krebs cycle intermediates for its needs after infection for establishing various defense mechanisms to escape the host immune responses. Nevertheless, a variety of immunological reactions are also seen during infection, which clear the parasites. One of the more promising strategies in this regard would involve combining targeted therapy and immunotherapy. The targeted treatments work by obstructing vital pathways that are required for Leishmania to grow and survive. The mechanism of action of immunotherapy is the control of the host immune response, which entails the blockage of molecular pathways essential for the growth and maintenance of the parasite. The Krebs cycle intermediates have important biochemical roles. Additionally, in macrophages and dendritic cells, they play roles as signalling molecules for controlling inflammatory responses. The review brings together the available literature about the importance of Krebs cycle metabolites as potential treatment targets for leishmaniasis.
A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE‐like domains are present in few eukaryotic taxa, for example the N‐terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) in vivo in the physiological and organellar context. Using direct in‐gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE‐like domain of FRDg has flavin transferase activity in vivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans‐flavinylation activity, the ApbE domain catalyses the intramolecular cis‐flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1‐37) as 4‐kDa heat‐stable, detergent‐resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.
Trypanosoma brucei ( Tb ) is a pathogenic protozoan causing sleeping sickness in humans. Despite little knowledge of how the produced reactive oxygen species (ROS) kills this protozoan, the research on the killing mechanism using chemical compounds and the phagosome in the macrophages has suggested that the protozoan is highly susceptible to the increased oxidative stress. Because the prescribed drug can react with various kinds of molecules and the second produced intermediate compounds, in this study, we clarified the immediate killing effect on Tb in the condition of increased oxidative stress using a low-temperature plasma at atmospheric pressure (LTP) equipment. Results Show the significant growth inhibition of Tb in the LTP-treated medium, the loss of morphological homeostasis with twisted to puffed appearance, and demonstrated the swelled changes on mitochondria and endoplasmic reticulum. In conclusion, this study revealed how the increased oxidative stress kills Tb using LTP technology.
Malaria, leishmaniasis and trypanosomiasis are arthropod-borne, parasitic diseases that constitute a major global health problem. They are generally found in developing countries, where lack of access to preventive tools and treatment hinders their management. Because these parasites share similar metabolic requirements between them and with cancer cells, six compounds used in anti-tumoral research were selected to be tested as antiparasitic agents in in vitro models of Leishmania infantum, Trypanosoma brucei, T. cruzi, and Plasmodium falciparum: dichloroacetic acid (DCA), 3-bromopyruvic acid (3BP), 2-deoxy-D-glucose (2DG), lonidamine (LND), metformin (MET), and sirolimus (SIR). No parasite-killing activity was found in L. infantum promastigotes, whereas DCA and 3BP reduced the burden of intra-macrophagic amastigotes. For T. brucei all selected compounds, but 2DG, decreased parasite survival. DCA, 2DG, LND and MET showed parasite-killing activity in T. cruzi. Finally, anti-plasmodial activity was found for DCA, 2DG, LND, MET and SIR. These results reinforce the hypothesis that drugs with proven efficacy in the treatment of cancer by interfering with energy production, proliferation, and survival cell strategies might be useful in treating threatening parasitic diseases and provide new opportunities for their repurposing.
Diseases caused by trypanosomatids include leishmaniasis (Leishmania spp.), Chagas disease (Trypanosoma cruzi), and sleeping sickness (Trypanosoma brucei) that affect millions of people, especially low-income populations, being classified as neglected tropical diseases. Limitations in the clinical treatment, associated with the huge number of cases, make these infections a health and socioeconomic problem worldwide. To complete their life cycle, trypanosomatids survive to environmental changes in different hosts, including oxidative stress. A paradoxal role of reactive oxygen species (ROS) has been proposed, such as signaling as a proliferation regulator or even presenting cytotoxic activity, depending on the concentration. Mitochondrial electron transport chain, especially complex III, is figured as one of the most important ROS resources in trypanosomatids. In relation to antioxidant defenses, trypanothione pathway plays a crucial role, being a peculiar thiol-redox system responsible for the maintenance of protozoa functions mediated by thiol-dependent processes. In this chapter, we discuss the biological aspects of oxidative stress in trypanosomatids and its implications for the success of the infection. The possible ROS resources in these protozoa and their consequent antioxidant machinery involved in detoxification were also focused in this review, including alternative strategies for the development of new drugs for these diseases based on oxidative stress modulation.
Lipoic acid (LA) is a cofactor of relevant enzymatic complexes including the glycine cleave system and 2-ketoacid dehydrogenases. Intervention on LA de novo synthesis or salvage could have pleiotropic deleterious effect in cells, making both pathways attractive for chemotherapy. We show that Trypanosoma cruzi was susceptible to treatment with LA analogues. 8-Bromo-octanic acid (BrO) inhibited the growth of epimastigote forms of both Dm28c and CL Brener strains, although only at high (chemotherapeutically irrelevant) concentrations. The methyl ester derivative MBrO, was much more effective, with EC50 values one order of magnitude lower (62-66 μM). LA did not bypass the toxic effect of its analogues. Small monocarboxylic acids appear to be poorly internalized by T. cruzi: [14C]-octanoic acid was taken up 12 fold less efficiently than [14C]-palmitic acid. Western blot analysis of lipoylated proteins allowed the detection of the E2 subunits of pyruvate dehydrogenase (PDH), branched chain 2-ketoacid dehydrogenase and 2-ketoglutarate dehydrogenase complexes. Growth of parasites in medium with 10 fold lower glucose content, notably increased PDH activity and the level of its lipoylated E2 subunit. Treatment with BrO (1 mM) and MBrO (0.1 mM) completely inhibited E2 lipoylation and all three dehydrogenases activities. These observations indicate the lack of specific transporters for octanoic acid and most probably also for BrO and LA, which is in agreement with the lack of a LA salvage pathway, as previously suggested for T. brucei. They also indicate that the LA synthesis/protein lipoylation pathway could be a valid target for drug intervention. Moreover, the free LA available in the host would not interfere with such chemotherapeutic treatments.
Since the discovery of the first examples of 2-oxoglutarate-dependent oxygenase-catalysed reactions in the 1960s, a remarkably broad diversity of alternate reactions and substrates has been revealed, and extensive advances have been achieved in our understanding of the structures and catalytic mechanisms. These enzymes are important agrochemical targets and are being pursued as therapeutic targets for a wide range of diseases including cancer and anemia.
This book provides a central source of information that summarizes the key features of the essential group of 2-oxoglutarate-dependent dioxygenases and related enzymes. Given the numerous recent advances and biomedical interest in the field, this book aims to unite the latest research for those already working in the field as well as to provide an introduction for those newly approaching the topic, and for those interested in translating the basic science into medicinal and agricultural benefits.
The book begins with four broad chapters that highlight critical aspects, including an overview of possible catalytic reactions, structures and mechanisms. The following seventeen chapters focus on carefully selected topics, each written by leading experts in the area. Readers will find explanations of rapidly evolving research, from the chemistry of isopenicillin N synthase to the oxidation mechanism of 5-methylcytosine in DNA by ten-eleven-translocase oxygenases.
Over the last fifty years, the metabolism of
Metabolic studies on Phytomonas sp. isolated from the lactiferous tubes of the latex-bearing spurge Euphorbia characias indicate that glucose is the preferred energy and carbon substrate during logarithmic growth. In stationary phase cells glucose consumption was dramatically reduced. Glucose consumption and end-product formation were measured on logarithmically growing cells, both under aerobic (air and 95% O2/5% CO2) and anerobic (95% N2/5% CO2 and 100% N2) conditions. The rate of glucose consumption slightly increased anerobic conditions indicating that Phytomonas facks a ‘reverse Pasteur’ effect contrary to the situation encountered in Leishmania major. Major end-products of glucose catabolism under aerobic conditions, detected by enzymatic and NMR measurements, were acetate, ethanol and carbon dioxide and under anaerobic conditions ethanol, glycerol and carbon dioxide. Smaller amounts of pyruvate, succinate, L-malate, L-lactate, phosphenolpyruvate, alanine and aspartate were also detected.
Hydrogenosome-containing anaerobic protists, such as the trichomonads, produce large amounts of acetate by an acetate:succinate CoA transferase (ASCT)/succinyl CoA synthetase cycle. The notion that mitochondria and hydrogenosomes may have originated from the same alpha-proteobacterial endosymbiont has led us to look for the presence of a similar metabolic pathway in trypanosomatids because these are the earliest-branching mitochondriate eukaryotes and because they also are known to produce acetate. The mechanism of acetate production in these organisms, however, has remained unknown. Four different members of the trypanosomatid family: promastigotes of Leishmania mexicana mexicana, L. infantum and Phytomonas sp., and procyclics of Trypanosoma brucei were analyzed as well as the parasitic helminth Fasciola hepatica. They all use a mitochondrial ASCT for the production of acetate from acetyl CoA. The succinyl CoA that is produced during acetate formation by ASCT is recycled presumably to succinate by a mitochondrial succinyl CoA synthetase, concomitantly producing ATP from ADP. The ASCT of L. mexicana mexicana promastigotes was further characterized after partial purification of the enzyme. It has a high affinity for acetyl CoA (Km 0.26 mM) and a low affinity for succinate (Km 6.9 mM), which shows that significant acetate production can occur only when high mitochondrial succinate concentrations prevail. This study identifies a metabolic pathway common to mitochondria and hydrogenosomes, which strongly supports a common origin for these two organelles.
Procyclic forms of Trypanosoma brucei isolated from the midguts of infected tsetse flies, or freshly transformed from a strain that is close to field isolates,
do not use a complete Krebs cycle. Furthermore, short stumpy bloodstream forms produce acetate and are apparently metabolically
preadapted to adequate functioning in the tsetse fly.
ATP generation by both glycolysis and glycerol catabolism is autocatalytic, because the first kinases of these pathways are fuelled by ATP produced downstream. Previous modeling studies predicted that either feedback inhibition or compartmentation of glycolysis can protect cells from accumulation of intermediates. The deadly parasite Trypanosoma brucei lacks feedback regulation of early steps in glycolysis yet sequesters the relevant enzymes within organelles called glycosomes, leading to the proposal that compartmentation prevents toxic accumulation of intermediates. Here, we show that glucose 6-phosphate indeed accumulates upon glucose addition to PEX14 deficient trypanosomes, which are impaired in glycosomal protein import. With glycerol catabolism, both in silico and in vivo, loss of glycosomal compartmentation led to dramatic increases of glycerol 3-phosphate upon addition of glycerol. As predicted by the model, depletion of glycerol kinase rescued PEX14-deficient cells of glycerol toxicity. This provides the first experimental support for our hypothesis that pathway compartmentation is an alternative to allosteric regulation.
• glycosome
• PEX14
• regulation of glycolysis
• systems biology
• Trypanosoma brucei
The amino acid compositions of several culture media have been analysed and compared. The utilization and excretion of amino acids and other metabolites have been followed during growth of Trypanosoma brucei S42 in a defined medium. All of the added L-threonine was metabolized by the cells, even when it was present at elevated concentrations. Glucose was consumed throughout the growth cycle: glutamine was consumed more rapidly than glutamic acid, which was itself used at about the same rate as proline. Threonine was cleaved to form glycine and acetate, both of which accumulated in the medium. Alanine and succinate were excreted together with a small amount of pyruvate, but these three products accounted for less than half of the glucose used. CO2 production from glucose was not measured, but insignificant amounts of CO2 were produced from threonine. Tetraethylthiuram disulphide blocked the cleavage of threonine and was a potent inhibitor of trypanosome growth.
Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.
In all trypanosomatids, including Trypanosoma brucei, glycolysis takes place in peroxisome-like organelles called glycosomes. These are closed compartments wherein the energy and redox (NAD(+)/NADH) balances need to be maintained. We have characterized a T. brucei gene called FRDg encoding a protein 35% identical to Saccharomyces cerevisiae fumarate reductases. Microsequencing of FRDg purified from glycosome preparations, immunofluorescence, and Western blot analyses clearly identified this enzyme as a glycosomal protein that is only expressed in the procyclic form of T. brucei but is present in all the other trypanosomatids studied, i.e. Trypanosoma congolense, Crithidia fasciculata and Leishmania amazonensis. The specific inactivation of FRDg gene expression by RNA interference showed that FRDg is responsible for the NADH-dependent fumarate reductase activity detected in glycosomal fractions and that at least 60% of the succinate secreted by the T. brucei procyclic form (in the presence of d-glucose as the sole carbon source) is produced in the glycosome by FRDg. We conclude that FRDg plays a key role in the energy metabolism by participating in the maintenance of the glycosomal NAD(+)/NADH balance. We have also detected a significant pyruvate kinase activity in the cytosol of the T. brucei procyclic cells that was not observed previously. Consequently, we propose a revised model of glucose metabolism in procyclic trypanosomes that may also be valid for all other trypanosomatids except the T. brucei bloodstream form. Interestingly, H. Gest has hypothesized previously (Gest, H. (1980) FEMS Microbiol. Lett. 7, 73-77) that a soluble NADH-dependent fumarate reductase has been present in primitive organisms and evolved into the present day fumarate reductases, which are quinol-dependent. FRDg may have the characteristics of such an ancestral enzyme and is the only NADH-dependent fumarate reductase characterized to date.
The importance of a functional Krebs cycle for energy generation in the procyclic stage of Trypanosoma brucei was investigated under physiological conditions during logarithmic phase growth of a pleomorphic parasite strain. Wild type
procyclic cells and mutants with targeted deletion of the gene coding for aconitase were derived by synchronous in vitrodifferentiation from wild type and mutant (Δaco::NEO/Δaco::HYG) bloodstream stage parasites, respectively, where aconitase is not expressed and is dispensable. No differences in intracellular
levels of glycolytic and Krebs cycle intermediates were found in procyclic wild type and mutant cells, except for citrate
that accumulated up to 90-fold in the mutants, confirming the absence of aconitase activity. Surprisingly, deletion of aconitase
did not change differentiation nor the growth rate or the intracellular ATP/ADP ratio in those cells. Metabolic studies using
radioactively labeled substrates and NMR analysis demonstrated that glucose and proline were not degraded via the Krebs cycle
to CO2. Instead, glucose was degraded to acetate, succinate, and alanine, whereas proline was degraded to succinate. Importantly,
there was absolutely no difference in the metabolic products released by wild type and aconitase knockout parasites, and both
were for survival strictly dependent on respiration via the mitochondrial electron transport chain. Hence, although the Krebs
cycle enzymes are present, procyclic T. brucei do not use Krebs cycle activity for energy generation, but the mitochondrial respiratory chain is essential for survival
and growth. We therefore propose a revised model of the energy metabolism of procyclic T. brucei.
Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.
In this review, we describe the unique compartmentation of metabolism in Kinetoplastida and the metabolic properties resulting from this compartmentation. We discuss the evidence for our recently proposed hypothesis that a common ancestor of Kinetoplastida and Euglenida acquired a photosynthetic alga as an endosymbiont, contrary to the earlier notion that this event occurred at a later stage of evolution, in the Euglenida lineage alone. The endosymbiont was subsequently lost from the kinetoplastid lineage but, during that process, some of its pathways of energy and carbohydrate metabolism were sequestered in the kinetoplastid peroxisomes, which consequently became glycosomes. The evolution of the kinetoplastid glycosomes and the possible selective advantages of these organelles for Kinetoplastida are discussed. We propose that the possession of glycosomes provided metabolic flexibility that has been important for the organisms to adapt easily to changing environmental conditions. It is likely that metabolic flexibility has been an important selective advantage for many kinetoplastid species during their evolution into the highly successful parasites today found in many divergent taxonomic groups.
Also addressed is the evolution of the kinetoplastid mitochondrion, from a supposedly pluripotent organelle, attributed to a single endosymbiotic event that resulted in all mitochondria and hydrogenosomes of extant eukaryotes. Furthermore, indications are presented that Kinetoplastida may have acquired other enzymes of energy and carbohydrate metabolism by various lateral gene transfer events different from those that involved the algal- and α-proteobacterial-like endosymbionts responsible for the respective formation of the glycosomes and mitochondria.
We investigated whether substrate availability influences the type of energy metabolism in procyclic Trypanosoma brucei. We show that absence of glycolytic substrates (glucose and glycerol) does not induce a shift from a fermentative metabolism
to complete oxidation of substrates. We also show that glucose (and even glycolysis) is not essential for normal functioning
and proliferation of pleomorphic procyclic T. brucei cells. Furthermore, absence of glucose did not result in increased degradation of amino acids. Variations in availability
of glucose and glycerol did result, however, in adaptations in metabolism in such a way that the glycosome was always in redox
balance. We argue that it is likely that, in procyclic cells, phosphoglycerate kinase is located not only in the cytosol,
but also inside glycosomes, as otherwise an ATP deficit would occur in this organelle. We demonstrate that procyclic T. brucei uses parts of the Krebs cycle for purposes other than complete degradation of mitochondrial substrates. We suggest that citrate
synthase plus pyruvate dehydrogenase and malate dehydrogenase are used to transport acetyl-CoA units from the mitochondrion
to the cytosol for the biosynthesis of fatty acids, a process we show to occur in proliferating procyclic cells. The part
of the Krebs cycle consisting of α-ketoglutarate dehydrogenase and succinyl-CoA synthetase was used for the degradation of
proline and glutamate to succinate. We also demonstrate that the subsequent enzymes of the Krebs cycle, succinate dehydrogenase
and fumarase, are most likely used for conversion of succinate into malate, which can then be used in gluconeogenesis.
Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic stage of T. brucei expresses a soluble NADH-dependent fumarate reductase (FRDg) in the peroxisome-like organelles called glycosomes. This enzyme is responsible for the production of about 70% of the excreted succinate, the major end product of glucose metabolism in this form of the parasite. Here we functionally characterize a new gene encoding FRD (FRDm1) expressed in the procyclic stage. FRDm1 is a mitochondrial protein, as evidenced by immunolocalization, fractionation of digitonin-permeabilized cells, and expression of EGFP-tagged FRDm1 in the parasite. RNA interference was used to deplete FRDm1, FRDg, or both together. The analysis of the resulting mutant cell lines showed that FRDm1 is responsible for 30% of the cellular NADH-FRD activity, which solves a long standing debate regarding the existence of a mitochondrial FRD in trypanosomatids. FRDg and FRDm1 together account for the total NADH-FRD activity in procyclics, because no activity was measured in the double mutant lacking expression of both proteins. Analysis of the end products of 13C-enriched glucose excreted by these mutant cell lines showed that FRDm1 contributes to the production of between 14 and 44% of the succinate excreted by the wild type cells. In addition, depletion of one or both FRD enzymes results in up to 2-fold reduction of the rate of glucose consumption. We propose that FRDm1 is involved in the maintenance of the redox balance in the mitochondrion, as proposed for the ancestral soluble FRD presumably present in primitive anaerobic cells.
To complement the sequencing of the three kinetoplastid genomes reported in this issue, we have undertaken a whole-organism,
proteomic analysis of the four life-cycle stages of Trypanosoma cruzi. Peptides mapping to 2784 proteins in 1168 protein groups from the annotated T. cruzi genome were identified across the four life-cycle stages. Protein products were identified from >1000 genes annotated as
“hypothetical” in the sequenced genome, including members of a newly defined gene family annotated as mucin-associated surface
proteins. The four parasite stages appear to use distinct energy sources, including histidine for stages present in the insect
vectors and fatty acids by intracellular amastigotes.
African trypanosomes cause human sleeping sickness and livestock trypanosomiasis in sub-Saharan Africa. We present the sequence and analysis of the 11 megabase-sized chromosomes of Trypanosoma brucei. The 26-megabase genome contains 9068 predicted genes, including approximately 900 pseudogenes and approximately 1700 T. brucei-specific genes. Large subtelomeric arrays contain an archive of 806 variant surface glycoprotein (VSG) genes used by the parasite to evade the mammalian immune system. Most VSG genes are pseudogenes, which may be used to generate expressed mosaic genes by ectopic recombination. Comparisons of the cytoskeleton and endocytic trafficking systems with those of humans and other eukaryotic organisms reveal major differences. A comparison of metabolic pathways encoded by the genomes of T. brucei, T. cruzi, and Leishmania major reveals the least overall metabolic capability in T. brucei and the greatest in L. major. Horizontal transfer of genes of bacterial origin has contributed to some of the metabolic differences in these parasites, and a number of novel potential drug targets have been identified.
African trypanosomes are parasitic protozoa that cause sleeping sickness and nagana. Trypanosomes are not only of scientific interest because of their clinical importance, but also because these protozoa contain several very unusual biological features, such as their specially adapted mitochondrion and the compartmentalization of glycolytic enzymes in glycosomes. The energy metabolism of Trypanosoma brucei differs significantly from that of their hosts and changes drastically during the life cycle. Despite the presence of all citric acid cycle enzymes in procyclic insect-stage T. brucei, citric acid cycle activity is not used for energy generation. Recent investigations on the influence of substrate availability on the type of energy metabolism showed that absence of glycolytic substrates did not induce a shift from a fermentative metabolism to complete oxidation of substrates. Apparently, insect-stage T. brucei use parts of the citric acid cycle for other purposes than for complete degradation of mitochondrial substrates. Parts of the cycle are suggested to be used for (i) transport of acetyl-CoA units from the mitochondrion to the cytosol for the biosynthesis of fatty acids, (ii) degradation of proline and glutamate to succinate, (iii) generation of malate, which can then be used for gluconeogenesis. Therefore the citric acid cycle in trypanosomes does not function as a cycle.
The complete analysis of the genomes of three major trypanosomatid parasites has facilitated comparison of the metabolic capabilities of each, as predicted from gene sequences. Not surprisingly, there are differences but is it possible to correlate these with the lives of the parasites themselves and make further predictions of the meaning and physiological importance of the apparently parasite-specific metabolism? In this article, we relate gene predictions with the results from experimental studies. We also speculate on the key metabolic adaptations of Leishmania and reasons why it differs from Trypanosoma brucei and Trypanosoma cruzi.
Bloodstream form Trypanosoma theileri degrades glucose to acetate (47%) and succinate (45%) and, therefore, does not solely rely on glycolysis for ATP production.
This trypanosomatid does not use amino acids for energy metabolism. These results refute the prevailing hypothesis that substrate
availability determines the type of energy metabolism of trypanosomatids.
The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.
The utilisation of substrates by Leishmania mexicana amastigotes and promastigotes differed significantly. The rates of uptake and catabolism of nonesterified fatty acids were up to 10-fold higher with amastigotes. Almost all the available exogenous fatty acids were consumed during amastigote transformation and by stationary phase of promastigote growth. The results suggest that fatty acids are important energy substrates for amastigotes, whereas promastigote utilisation may reflect the requirement for these substrates in anabolism. Glucose was utilised by amastigotes and promastigotes but the rate of catabolism was up to 10-fold higher in promastigotes. Uptake of glucose occurred throughout amastigote transformation and growth in vitro of promastigotes. High-subpassage promastigotes exhibited markedly lower glucose but higher amino acid utilisation than low-subpassage promastigotes. Asparagine, glutamine, glutamate, leucine, lysine, methionine, and threonine were consumed in large quantities by amastigotes and promastigotes, whereas alanine and glycine were excreted. Proline was catabolised to CO2 by amastigotes and promastigotes but only at a low rate, and it was excreted in large amounts throughout promastigote growth. The major end products of energy metabolism were found to be CO2 and succinate with both forms of the parasite and there was a secretion of up to 12 and 16% of the total protein synthesised by transforming amastigotes and growing promastigotes, respectively. Catabolism in amastigotes and promastigotes was found to be sensitive to cyanide and amytal, whereas 2-mercaptoacetate and 4-pentenoate primarily affected β-oxidation in the amastigote.
Bloodforms of Trypanosoma brucei lack complete cytochrome and Krebs cycle systems but a fully functional mitochondrial respiratory system is elaborated upon differentiation to procyclics. We previously found differential expression of some mitochondrial genes, at the level of transcript abundance, between these two life cycle stages. We report here that mitochondrial genes are also differentially expressed between the two morphological types of bloodforms. Some transcripts that are more abundant in procyclics than slender bloodforms are intermediate in abundance in stumpy bloodforms. Most major mitochondrial transcripts are more abundant in stumpy than slender bloodform RNA; some are also more abundant than in procyclic RNA. Transcripts from protein coding genes are increased in abundance to varying degrees. Treatment with difluoromethylornithine, which induces a stumpy morphology, produces transcript abundance patterns similar to those in naturally occurring stumpy bloodforms. Stumpy bloodforms also have a decrease in tubulin transcript abundance, consistent with their nondividing character and smaller flagellum. These studies suggest that molecular events associated with mitochondrial development during the differentiation from bloodforms to procyclics can be initiated in the bloodstream, and that maxicircle transcript abundances are individually modulated during the life cycle.
1.1. Monomorphic Trypanosoma brucei and T. rhodesiense metabolize glucose via the glycolytic pathway, producing pyruvate as the major metabolic end-product with a small amount of glycerol and no carbon dioxide, acetate or succinate.2.2. Under in vitro conditions, pleomorphic strains of T. rhodesiense produce significant quantities of succinate, acetate and carbon dioxide.3.3. The time course of metabolite production has been used to evaluate the significance of acetate, succinate and carbon dioxide formation in vivo and indicates that succinate production is induced by in vitro incubation.4.4. Oxidative decarboxylases for pyruvate and α-oxoglutarate are confined to the short stumpy forms of pleomorphic infections. However, the tricarboxylic acid cycle has minimal activity in these organisms due to limiting levels of citrate synthase (E.C. 4.1.3.7) and succinate dehydrogenase (E.C. 1.3.99.1).5.5. l-Glycerol-3-phosphate oxidase is the principal terminal oxidase of both long slender and short stumpy forms of T. rhodesiense.
Trypanosoma brucei, a protozoan parasite causing sleeping sickness, is transmitted by the tsetse fly and undergoes a complex lifecycle including several defined stages within the insect vector and its mammalian host. In the latter, differentiation from the long slender to the short stumpy form is induced by a yet unknown factor of trypanosomal origin. Here we describe that some thiazolidinediones are also able to induce differentiation. In higher eukaryotes, thiazolidinediones are involved in metabolism and differentiation processes mainly by binding to the intracellular receptor peroxisome proliferator activated receptor gamma. Our studies focus on the effects of troglitazone on bloodstream form trypanosomes. Differentiation was monitored using mitochondrial markers (membrane potential, succinate dehydrogenase activity, inhibition of oxygen uptake by KCN, amount of cytochrome transcripts), morphological changes (Transmission EM and light microscopy), and transformation experiments (loss of the Variant Surface Glycoprotein coat and increase of dihydroliponamide dehydrogenase activity). To further investigate the mechanisms responsible for these changes, microarray analyses were performed, showing an upregulation of expression site associated gene 8 (ESAG8), a potential differentiation regulator.
In a previous study, three in vitro methods for the assessment of drug sensitivity among Trypanosoma evansi isolates were compared--a direct counting method, pyruvate production method and uptake of radiolabelled hypoxanthine. The pyruvate assay system, which measures the amount of pyruvate in the supernatant of growing populations of trypanosomes by a spectrophotometric method, was selected for further investigation with regard to its suitability for field studies. The effect of initial seeding density and incubation time on the growth of three stocks of T. evansi--TREU 1840 and TREU 1981 (suramin sensitive) and TREU 2136 (suramin resistant)--and drug sensitivities revealed by the pyruvate assay and direct counting were examined to optimise assay conditions. Maximum densities and pyruvate production achieved were not affected by varying the initial seeding densities in the range of 5 x 10(4)-5 x 10(5)/ml and had been reached after 48 hours incubation with one exception: Pyruvate levels continued to increase up to 72 hours in the suramin resistant stock. However, inhibition curves were affected by initial seeding density and incubation period. Results suggested that an initial seeding density of 1 x 10(5)/ml and an incubation time of 48 hours are optimal for the assay. Using these assay conditions, the isolates were screened against suramin, quinapyramine sulphate and Cymelarsan, the trypanocides used most commonly against T. evansi. This assay proved to be a relatively simple and cheap technique applicable to screening large numbers of isolates of differing sensitivities to trypanocidal drugs.
The consumption of glucose by trypanosomatid protozoa such as Trypanosoma brucei, Trypanosoma cruzi, Leishmania spp., and Crithidia spp. is characterized by the excretion of reduced products such as succinate, pyruvate, ethanol, L-alanine, or lactate (depending on the species) not only in anaerobiosis, but also under aerobic conditions. The "aerobic fermentation" of glucose is accompanied by a complete lack, or even a reversal, of the Pasteur effect. This peculiar catabolism is mediated by a so-far unique compartmentation of the glycolytic enzymes, most of which are placed in an organelle called the glycosome; by an almost complete lack of inhibitory controls at the level of hexokinase and phosphofructokinase; and by a central role of CO2 fixation through the reaction catalyzed by phosphoenolpyruvate carboxykinase. The production of fermentative products seems to be due to a relative inefficiency of the respiratory chain, which lacks NADH dehydrogenase and the first phosphorylation site and preferentially uses succinate as substrate.
All four life cycle stages (bloodstream, procyclic, epimastigote, and metacyclic) of Trypanosoma congolense IL 3000 were assayed with an oxygen electrode (polarograph) for the presence of terminal oxidases and carbon-source preference. In addition, these stages were used for histochemical analysis of mitochondrial activity using rhodamine 123, nitroblue tetrazolium, and diaminobenzidine. Morphometry was used to compare mitochondrial volumes and surface area among the different life cycle stages. It was found that in contrast to epimastigote forms, which were metabolically almost identical to procyclic forms, metacyclic forms showed characteristics of, and seemed preadapted to, differentiation into the bloodstream stage. While mitochondrial NAD+ diaphorase activity and an electrochemical potential were detected in all life cycle stages, metacyclic metabolism was glucose-based and terminal oxidase activity was primarily dependent upon the trypanosome alternative oxidase with the contribution of cyanide-sensitive respiration accounting for only 20-30% of the total respiratory capacity.
The major products of glucose metabolism were determined for amastigotes and promastigotes of Leishmania (mexicana) pifanoi under aerobic and anaerobic conditions using carbon-13 nuclear magnetic resonance. Under aerobic conditions, the major products for both forms were carbon dioxide, succinate, malate, acetate and alanine. Succinate was the dominant metabolite of promastigotes, whereas acetate and alanine were most abundant with amastigotes. Under anaerobic conditions, promastigotes produced glycerol as the dominant metabolite, along with lesser amounts of succinate, acetate and alanine; acetate and alanine remained major metabolites in amastigotes, with an increase in the relative amount of succinate and the production of some glycerol. Promastigotes generated carbon dioxide at a 5-fold greater rate than amastigotes under aerobic conditions, but this rate was reduced by more than 95% in the absence of oxygen. Amastigotes were relatively less affected by lack of oxygen and produced carbon dioxide at a rate comparable to promastigotes under anaerobic conditions. The presence of carbohydrates with a possible role in storage was detected in both promastigotes and amastigotes.
A rapid switch from a fermentative to a primarily oxidative type of glucose utilization was observed during in vitro differentiation of Trypanosoma brucei STIB348 and EATRO1244 bloodstream to procyclic trypomastigotes. In accordance with previously published reports bloodstream populations produced pyruvate as the major end product of glucose catabolism, together with very small amounts of CO2, succinate and glycerol. During differentiation pyruvate excretion decreased within 48 h to the low levels produced by 28-day procyclic stages. Concomitant with the decline in pyruvate formation, acetate appeared as a new product and the rates of respiratory CO2 increased considerably. The amount of carbon released with these compounds could account for nearly all of the glucose carbon consumed. Rates of glucose utilization and formation of acetate and CO2 in cells differentiated for 48 h were essentially the same as those found in 28-day procyclics. Succinate and glycerol excretion remained low during the entire transformation process, and no significant difference in the pattern and quantities of end products were found between the two trypanosome strains. During trypanosome differentiation the changes in metabolism were associated with marked alterations in enzyme activity levels. Activities of the tricarboxylic acid (TCA) cycle enzymes citrate synthase, isocitrate dehydrogenase (NAD+), succinate dehydrogenase and fumarase were not detectable in bloodstream trypomastigotes but appeared upon differentiation for 24 h. An exception was citrate synthase whose activity was not demonstrable until 48 h postinoculation into culture. After 48 h the majority of the TCA cycle enzyme activities continued to increase steadily until day 28. Pyruvate kinase activity decreased in differentiating cells after 48 h to about 25% of the level found in bloodstream trypomastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)
Proton nuclear magnetic resonance (NMR) spectroscopy was used to follow glucose metabolism in Crithidia luciliae. Parasites were grown aerobically and anaerobically in culture, with glucose as the major carbon source and 1H NMR spectra were acquired for the cell free medium. The 1H NMR resonances of metabolites utilised and produced during cell growth were identified by difference spectroscopy, and quantitated from standard curves using 3-trimethylsilyl propionate-2,2,3,3-d4 sodium salt as an internal standard. The major metabolites produced by C. luciliae grown aerobically on 8 mM glucose were succinate, pyruvate, acetate and ethanol, in final concentrations in the media when the cells entered stationary phase of 8.5 +/- 0.5, 5.0 +/- 0.3, 2.1 +/- 0.2 and 2.5 +/- 0.6 mM, respectively. The production of succinate and pyruvate, but not acetate and ethanol, followed closely the growth curve of the parasites. Succinate was also measured enzymically and glucose using an autoanalyser. In both cases the results correlated well with the NMR data. The amounts of end products formed were greater than could be accounted for by the utilisation of glucose or any other metabolite observable in the 1H NMR spectra. There was approximately one extra atom of carbon for each molecule of succinate formed, supporting the view that succinate is produced via phosphoenolpyruvate carboxykinase and carbon dioxide fixation. Anaerobically the same major metabolites were produced, but with a decreased ratio of succinate to acetate and ethanol. The formation of glycerol from glucose was not observed under these conditions.
Fatty acid uptake, distribution, and beta-oxidation were investigated in Leishmania mexicana amastigotes. The uptake of radiolabeled palmitic, stearic, and oleic acids was similar, reaching 3-6 nmol/10(8) cells in 2 min and 8-12 nmol/10(8) cells in 60 min. The percent of radiolabeled fatty acid that was esterified in the form of triglycerides or phospholipids increased from less than 25% at 2 min to 65-86% at 60 min. The dehydrogenase(s) in an amastigote granule fraction were unusual in that the Vmax for long-chain substrates (0.95-1.6 delta Abs units/min-mg protein) approximated the Vmax for short-chain substrates (0.82-2.0 delta Abs units/min-mg protein), and the Km for long-chain substrates was high (approximately 250 microM), in contrast to data for a mammalian liver mitochondrial fraction. The high Vmax and Km for long-chain substrates suggest a biochemical mechanism for the postulated high utilization of fatty acids as an energy source for amastigotes. Although the primary anti-leishmanial agent, Sb in the form of Pentostam, inhibited oxidation of palmitic acid to CO2 by intact organisms, Sb did not significantly inhibit fatty acid uptake or esterification by organisms, or beta-oxidation by the granule fraction, and the mechanism of action of Sb remains unclear.
Trypanosoma cruzi (epimastigotes), Crithidia fasciculata and Leishmania mexicana (promastigotes) were grown in a brain-heart-tryptose medium supplemented with heat-inactivated fetal calf serum. T. cruzi and C. fasciculata utilized glucose completely during the log phase of growth, whereas L. mexicana used significant amounts of the carbohydrate only at the end of the log phase and at the beginning of the stationary phase. In all cases glucose consumption resulted in excretion of succinate, and much smaller amounts of acetate. C. fasciculata and L. mexicana produced very small amounts of pyruvate. C. fasciculata produced ethanol, which was taken up again and metabolysed after glucose was exhausted. Lactate and malate were not produced. The cells were disrupted by sonic disintegration, and the activities of some key enzymes of carbohydrate and amino acid catabolism were assayed in the whole homogenates. Phosphoenolpyruvate carboxykinase was present in the three organisms; L. mexicana presented the highest specific activity. The activity of this enzyme was maximal during glucose consumption, and slightly decreased after glucose was exhausted. This suggests that the role played by the enzyme is glycolytic and not gluconeogenic; the latter is the case in most higher organisms. Hexokinase and pyruvate kinase presented their highest levels in C. fasciculata and T. cruzi during glucose consumption. L. mexicana, which was in active glycolysis during the whole experimental period, presented the highest specific activities of both enzymes. Citrate synthase, on the other hand, increased in C. fasciculata and, to a lesser extent, in T. cruzi, after glucose was exhausted; the enzyme could not be detected in L. mexicana. The NAD-linked glutamate dehydrogenase increased considerably in C. fasciculata and T. cruzi after glucose was exhausted, suggesting a catabolic role for the enzyme. This increase coincided with an increase in NH3 production by both organisms after glucose consumption. The NADP-linked glutamate dehydrogenase, on the other hand, presented a maximum about the time when glucose was exhausted, and then decreased again, which suggests a catabolic role for the enzyme. Both glutamate dehydrogenases had low activities in L. mexicana; this fits in well with the low NH3 production throughout the culture of this organism. The results are in good agreement with current ideas on the mechanism of aerobic glucose fermentation by trypanosomatids, and suggest that, under the experimental conditions used, both T. cruzi and C. fasciculata used glucose perferentially over amino acids for growth.
SYNOPSIS
Exponentially dividing culture forms of Trypanosoma brucei did not utilize glucose provided in the culture medium. The inclusion of 2‐deoxyglucose in the medium had no effect on the growth of the trypanosomes. Glucose could be replaced by proline in the liquid phase of biphasic medium without affecting the doubling time of the organisms. Proline added to the culture medium in this way disappeared during the log phase of growth. Glucose in the culture medium was used by the trypanosomes only when the stationary growth phase had been reached. Lipid accumulated in stationary phase trypanosomes grown in glucose‐containing medium, but there was no lipid accumulation in log phase organisms or in those which had been grown in proline‐containing medium. Bloodstream trypanosomes transferred to liquid medium rapidly utilized glucose over the first 12 hr of culture, and this was accompanied by an accumulation of free pyruvate in the medium. The rate of glucose utilization fell off over the next 36 hr; this was accompanied by a lowering of free pyruvate in the medium and a rise in the proline oxidase activity of the trypanosomes. The possible biologic significance of proline to trypanosomes developing in the midgut of the tsetse vector is discussed.
Epimastigotes of Trypanosoma cruzi, the causative agent of Chagas disease, catabolize proteins and amino acids with production of MH3, and glucose with production of reduced catabolites, chiefly succinate and L-alanine, even under aerobic conditions. This "aerobic fermentation of glucose" is probably due to both the presence of low levels of some cytochromes, causing a relative inefficiency of the respiratory chain for NADH, reoxidation during active glucose catabolism, and the lack of NADH dehydrogenase and phosphorylation site I, resulting in the entry of reduction equivalents into the chain mostly as succinate. Phosphoenol pyruvate carboxykinase and pyruvate kinase may play an essential role in diverting glucose carbon to succinate or L-alanine, and L-malate seems to be the major metabolite for the transport of glucose carbon and reduction equivalents between glycosome and mitochondrion. The parasite contains proteinase and peptidase activities. The major lysosomal cysteine proteinase, cruzipain, has been characterized in considerable detail, and might be involved in the host/parasite relationship, in addition to its obvious role in parasite nutrition. Among the enzymes of amino acid catabolism, two glutamate dehydrogenases (one NADP- and the other NAD-linked), alanine aminotransferase, and the major enzymes of aromatic amino acid catabolism (tyrosine aminotransferase and aromatic alpha-hydroxy acid dehydrogenase), have been characterized and proposed to be involved in the reoxidation of glycolytic NADH.
The constituents of the respiratory chain are believed to differ among the trypanosomatids; bloodstream stages of African trypanosomes and Phytomonas promastigotes oxidize ubiquinol by a ubiquinol:oxygen oxidoreductase, also known as alternative oxidase, whereas Leishmania spp. oxidize ubiquinol via a classic cytochrome-containing respiratory chain. The molecular basis for this elementary difference in ubiquinol oxidation by the mitochondrial electron-transport chain in distinct trypanosomatids was investigated. The presence of a gene encoding the plant-like alternative oxidase could be demonstrated in Phytomonas and Trypanosoma brucei, trypanosomatids that are known to contain alternative oxidase activity. Our results further demonstrated that Leishmania spp. lack a gene encoding the plant-like alternative oxidase, and therefore, all stages of Leishmania spp. will lack the alternative oxidase protein. The observed fundamental differences between the respiratory chains of distinct members of the trypanosomatid family are thus caused by the presence or absence of a gene encoding the plant-like alternative oxidase.
The protozoal parasite Trypanosoma brucei depends on a mitochondrial non-cytochrome terminal oxidase known as the trypanosome alternative oxidase (TAO) in its mammalian host. We have recently cloned the cDNA from T. brucei bloodstream form and have characterized a 33 kDa mitochondrial protein as TAO. Here we report that the TAO is a single copy gene in T. brucei and its expression is down regulated at the level of transcript abundance during differentiation from the bloodstream to the procyclic trypanosomes. Like other alternative oxidases (AOXs) cloned from different plants and fungi, TAO possesses the conserved sequences at the centrally located predicted membrane spanning domains and the signature sequence at the C-terminal hydrophilic domain for a pair of putative iron binding motifs (E-X-X-H). Phylogenetic analysis of the deduced protein sequences of eight different alternative oxidases cloned from different plants and fungi revealed that TAO is more closely related to the alternative oxidases of the fungi clade than that of plants. TAO has been functionally expressed in Escherichia coli. In the first of the two putative iron binding motifs, site-directed mutagenesis of E215 to A, L, N and Q resulted in the loss of the ability of the TAO gene to complement the heme deficiency of the E. coli mutants (SASX41B and GE1387) by conferring on them a CN-insensitive pathway of respiration. The conservative substitution of E215 by aspartate and histidine reduced the growth of the E. coli auxotrophs by approximately 80%. The mutations apparently did not have any effect on the stability of the expressed protein as revealed by the immunoblot analysis of the bacterial protein using TAO monoclonal antibody, which we have developed. Together, these points suggest that E215 plays an important role in the function of TAO. The steady state level of TAO mRNA is down-regulated in the procyclic stage presumably accounting for the low levels of TAO protein in these forms.
The effect of various metabolic inhibitors on the rate of oxygen consumption by procyclic culture forms of Trypanosoma congolense utilizing proline as substrate was investigated. Cyanide inhibited the rate of oxygen consumption by 81.0 +/- 6.7%, malonate inhibited the rate by 51.6 +/- 1.6% and Antimycin A by 73.1 +/- 5.9%. A combination of cyanide and malonate inhibited the rate of oxygen consumption by 84.9 +/- 6.7% while a combination of antimycin A and malonate inhibited the rate by 81.6 +/- 7.6%. Rotenone had no effect on the rate of respiration except when the intact cells were first permeabilized by digitonin after which rotenone decreased the rate of respiration by 20-30%. Salicylhydroxamate (SHAM) did not have any effect on the rate of oxygen consumption. Enzymes involved in the catabolism of proline with high activities were: proline dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, fumarase, NADP-linked malic enzyme, alanine aminotransferase and malate dehydrogenase. Activities of 1-pyrroline-5 carboxylate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and NAD-linked malic enzyme were detectable but lower. The end products of proline catabolism were alanine and glutamate. Unlike the case in Trypanosoma brucei brucei aspartate was not detected. Possible pathways of proline catabolism in procyclic culture forms of T. congolense and of electron transfer are proposed.
The energy metabolism of Trypanosomatidae has been the subject of many reviews during the past decade. In recent years, however, new data have led to a more complete picture of trypanosomatid metabolism and a reappraisal of the role of some characteristic organelles in the energy supply of these parasites. For years, the glycosome was thought to be a peroxisome-like organelle that had evolved to allow the parasites to carry out glycolysis at a high rate using a relatively small amount of enzyme. However, the results of recent studies of trypanosomatid glycolysis and the detection of various other pathways and enzymes in the organelle necessitate a modification of this view. Here, Paul Michels, Véronique Hannaert and Frédéric Bringaud review the new data and discuss the possible implications for our view on the role of the glycosome.
Salivarian trypanosomes are the causative agents of several diseases of major social and economic impact. The most infamous parasites of this group are the African subspecies of the Trypanosoma brucei group, which cause sleeping sickness in humans and nagana in cattle. In terms of geographical distribution, however, Trypanosoma equiperdum and Trypanosoma evansi have been far more successful, causing disease in livestock in Africa, Asia, and South America. In these latter forms the mitochondrial DNA network, the kinetoplast, is altered or even completely lost. These natural dyskinetoplastic forms can be mimicked in bloodstream form T. brucei by inducing the loss of kinetoplast DNA (kDNA) with intercalating dyes. Dyskinetoplastic T. brucei are incapable of completing their usual developmental cycle in the insect vector, due to their inability to perform oxidative phosphorylation. Nevertheless, they are usually as virulent for their mammalian hosts as parasites with intact kDNA, thus questioning the therapeutic value of attempts to target mitochondrial gene expression with specific drugs. Recent experiments, however, have challenged this view. This review summarises the data available on dyskinetoplasty in trypanosomes and revisits the roles the mitochondrion and its genome play during the life cycle of T. brucei.
Although parasitic helminths are a very heterogeneous group of organisms, they share many interesting properties in their energy metabolism. In certain stages of their life cycle, they all have a large capacity for anaerobic functioning. In other stages, an aerobic energy metabolism prevails. Parasites have to adapt to different environments in which the availability of oxygen and food varies widely. These variations in their external conditions strongly influence their energy metabolism. Here, Louis Tielens presents an introduction to the current ideas on the bioenergetics of parasitic helminths, focusing on the differences in energy metabolism between various stages (free-living and parasitic), and paying special attention to the mechanisms involved in the transitions between the different methods of energy generation.
Most mitochondrial mRNAs in kinetoplastids require editing, that is, the posttranscriptional insertion and deletion of uridine nucleotides that are specified by guide RNAs and catalyzed by multiprotein complexes. Recent studies have identified many of the proteins in these complexes, in addition to some of their functions and interactions. Although much remains unknown, a picture of highly organized complexes is emerging that shows that the complex that catalyzes the central steps of editing is partitioned into distinct insertion and deletion editing subcomplexes. These subcomplexes coordinate hundreds of ordered catalytic steps that function to produce a single mature mRNA. The dynamic processes, which might entail interactions among multiprotein complexes and changes in their composition and conformation, remain to be elucidated.
The generation of energy in African trypanosomes is a subject of undoubted importance. In bloodstream-form organisms, substrate-level phosphorylation of glucose is sufficient to provide the energy needs of the parasite. The situation in procyclic-form trypanosomes is more complex. For many years, it was accepted that glucose metabolism followed a conventional scheme involving glycolysis, the tricarboxylic acid cycle and ATP-producing oxidative phosphorylation linked to the electron-transport chain. However, progress in sequencing the Trypanosoma brucei genome and the development of gene-knockout and RNA interference technology has provided novel insight. Coupling these new technologies with classical approaches, including NMR and mass spectrometry to analyse glycolytic intermediates and end products, has yielded several surprises. In this article, we summarize how these recent data have helped to change the view of metabolism in procyclic-form T. brucei.
Kinetoplastid protozoa such as trypanosomes and Leishmania are important because they cause human disease. These parasites are named after one of their most unusual features, a mitochondrial DNA known as kinetoplast DNA (kDNA). Unlike all other DNA in nature, kDNA comprises a giant network of interlocked DNA rings with a topology resembling that of medieval chain mail. The replication of the kDNA network is more complex than previously thought, and the discovery of new proteins involved in this process is currently the best approach for illuminating the replication mechanism.
African trypanosomes are parasitic protozoa of the order of Kinetoplastida, which cause sleeping sickness and nagana. Trypanosomes are not only of scientific interest because of their clinical importance, but also because these protozoa contain several very unusual biological features, such as their special energy metabolism. The energy metabolism of Trypanosoma brucei differs significantly from that of its host, not only because it comprises distinct enzymes and metabolic pathways, but also because some of the glycolytic enzymes are localized in organelles called glycosomes. Furthermore, the energy metabolism changes drastically during the complex life cycle of this parasite. This review will focus on the recent advances made in understanding the process of ATP production in T. brucei during its life cycle and the consequences of the special subcellular compartmentation.
Some development stages of the trypanosomatid protozoan parasites are well adapted to in vitro culture. They can be maintained in rich medium containing large excess of glucose and amino acids, which they use as carbon sources for ATP production. Under these growth conditions, carbon sources are converted into partially oxidized end products by so-called aerobic fermentation. Surprisingly, some species, such as the Trypanosoma brucei, Trypanosoma cruzi and Crithidia insect stages, prefer consuming glucose to amino acids, although their natural habitat is L-proline-rich. This review focuses on recent progress in understanding glucose and l-proline metabolism of insect stages, how these metabolic processes are regulated, and the rationale of the aerobic fermentation strategies developed by these parasites.
Although various members of the family Trypanosomatidae generate energy in a similar way, fundamental differences also exist and are not always recognized. In this review, Louis Tielens and Jaap Van Hellemond discuss the known differences in carbohydrate metabolism among trypanosomatids, and especially compare Leishmania with trypanosomatids such as Trypanosoma brucei and Phytomonas spp. Special attention will be paid to differences in end-products of carbohydrate degradation, to differences in anaerobic capacities between the various trypanosomatids and to the components of their respiratory chains, including the presence or absence of a plant-like alternative oxidase. Furthermore, evidence will be discussed which indicates that the succinate produced by trypanosomatids is formed mainly via an oxidative pathway and not via reduction of fumarate, a process known to occur in parasitic helminths.