PLoS Pathog

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

Regulation of Trypanosoma brucei Total and Polysomal mRNA during Development within Its Mammalian Host

This work was supported by a Wellcome Trust Programme grant to KM and by a Wellcome Trust Strategic award to the Centre for Immunity, Infection and Evolution at the University of Edinburgh. SM was supported by a studentship from the Medical Research Council, UK.The gene expression of Trypanosoma brucei has been examined extensively in the blood of mammalian hosts and in forms found in the midgut of its arthropod vector, the tsetse fly. However, trypanosomes also undergo development within the mammalian bloodstream as they progress from morphologically 'slender forms' to transmissible 'stumpy forms' through morphological intermediates. This transition is temporally progressive within the first wave of parasitaemia such that gene expression can be monitored in relatively pure slender and stumpy populations as well as during the progression between these extremes. The development also represents the progression of cells from translationally active forms adapted for proliferation in the host to translationally quiescent forms, adapted for transmission. We have used metabolic labelling to quantitate translational activity in slender forms, stumpy forms and in forms undergoing early differentiation to procyclic forms in vitro. Thereafter we have examined the cohort of total mRNAs that are enriched throughout development in the mammalian bloodstream (slender, intermediate and stumpy forms), irrespective of strain, revealing those that exhibit consistent developmental regulation rather than sample specific changes. Transcripts that cosediment with polysomes in stumpy forms and slender forms have also been enriched to identify transcripts that escape translational repression prior to transmission. Combined, the expression and polysomal association of transcripts as trypanosomes undergo development in the mammalian bloodstream have been defined, providing a resource for trypanosome researchers. This facilitates the identification of those that undergo developmental regulation in the bloodstream and therefore those likely to have a role in the survival and capacity for transmission of stumpy forms.Publisher PDFPeer reviewe

Identification and Analysis of Ingi-Related Retroposons in the Trypanosomatid Genomes

Transposable elements (TE), defined as discrete pieces of DNA that can move from one site to another site in genomes, represent significant components of eukaryotic genomes, including trypanosomatids. Up to 5% of the trypanosomatid genome content is composed of retroposons of the ingi clade, further divided into subclades and subfamilies ranging from short extinct truncated elements (SIDER) to long active elements (ingi). Important differences in ingi-related retroposon content have been reported between trypanosomatid species. For instance, Leishmania spp. have expanded and recycled a whole SIDER family to fulfill an important biological pathway, i.e., regulation of gene expression, while trypanosome genomes are primarily composed of active elements. Here, we present an overview of the computational methods used to identify, annotate, and analyze ingi-related retroposons for providing a comprehensive picture of all these TE families in newly available trypanosomatid genome sequences

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in <i>Trypanosoma brucei</i>

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by ApbE flavin transferases. ApbE-like domains are present in few eukaryotic taxa, e.g. 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 dead ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least 5-fold 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.</jats:p

Isotopic profiling of C-13-labeled biological samples by two-dimensional heteronuclear J-resolved nuclear magnetic resonance spectroscopy

The use of two-dimensional heteronuclear J-resolved (2D H-JRES) nuclear magnetic resonance (NMR) spectroscopy for fast and reliable measurement of isotopic patterns from C-13-enriched compounds resulting from carbon labeling experiments was evaluated. Its use with biological samples of increasing complexity showed that 2D H-JRES spectroscopy is suitable for high-throughput isotopic profiling of any kind of labeled samples. Moreover, the method enabled accurate quantification of C-13 enrichments and, thus, can be used for metabolic flux analysis. The excellent trade-off between reduced experimental time and the number of measurable isotopic data makes 2D H-JRES NMR a promising approach for high-throughput flux analysis of samples of intermediate complexity. (C) 2012 Elsevier Inc. All rights reserved

Functional characterization of TbMCP5, a conserved and essential ADP/ATP carrier present in the mitochondrion of the human pathogen Trypanosoma brucei

Trypanosoma brucei is a kinetoplastid parasite of medical and veterinary importance. Its digenetic life cycle alternates between the bloodstream form in the mammalian host and the procyclic form (PCF) in the bloodsucking insect vector, the tsetse fly. PCF trypanosomes rely in the glucose-depleted environment of the insect vector primarily on the mitochondrial oxidative phosphorylation of proline for their cellular ATP provision. We previously identified two T. brucei mitochondrial carrier family proteins, TbMCP5 and TbMCP15, with significant sequence similarity to functionally characterized ADP/ATP carriers from other eukaryotes. Comprehensive sequence analysis confirmed that TbMCP5 contains canonical ADP/ATP carrier sequence features, whereas they are not conserved in TbMCP15. Heterologous expression in the ANC-deficient yeast strain JL1Δ2Δ3u - revealed that only TbMCP5 was able to restore its growth on the non-fermentable carbon source lactate. Transport studies in yeast mitochondria showed that TbMCP5 has biochemical properties and ADP/ATP exchange kinetics similar to those of Anc2p, the prototypical ADP/ATP carrier of S. cerevisiae. Immunofluorescence microscopy and Western blot analysis confirmed that TbMCP5 is e xclusively mitochondrial and is differentially expressed with 4.5-fold more TbMCP5 in the procyclic form of the parasite. Silencing of TbMCP5 expression in PCF T. brucei revealed that this ADP/ATP carrier is essential for parasite growth, particularly when depending on proline for energy generation. Moreover, ADP/ATP exchange in isolated T. brucei mitochondria was eliminated upon TbMCP5 depletion. These results confirmed that TbMCP5 functions as the main ADP/ATP carrier in the trypanosome mitochondrion. The important role of TbMCP5 in the T. brucei energy metabolism is further discussed. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc

The role of l-serine and l-threonine in the energy metabolism and nutritional stress response of Trypanosoma cruzi

ABSTRACT l-Serine and l-threonine have versatile roles in metabolism. In addition to their use in protein synthesis, these amino acids participate in the biosynthesis pathways of other amino acids and even phospholipids. Furthermore, l-serine and l-threonine can be substrates for a serine/threonine dehydratase (Ser/ThrDH), resulting in pyruvate and 2-oxobutyrate, respectively, thus being amino acids with anaplerotic potential. Trypanosoma cruzi, the etiological agent of Chagas disease, uses amino acids in several biological processes: metacyclogenesis, infection, resistance to nutritional and oxidative stress, osmotic control, etc. This study investigated the import and metabolism of l-serine, l-threonine, and glycine in T. cruzi. Our results demonstrate that these amino acids are transported from the extracellular environment into T. cruzi cells through a saturable transport system that fits the Michaelis-Menten model. Our results show that l-serine and l-threonine can sustain epimastigote cell viability under nutritional stress conditions and stimulate oxygen consumption, maintaining intracellular ATP levels. Additionally, our findings indicate that serine plays a role in establishing the mitochondrial membrane potential in T. cruzi. Serine is also involved in energy metabolism via the serine-pyruvate pathway, which stimulates the production and subsequent excretion of acetate and alanine. Our results demonstrate the importance of l-serine and l-threonine in the energy metabolism of T. cruzi and provide new insights into the metabolic adaptations of this parasite during its life cycle.IMPORTANCETrypanosoma cruzi, the parasite responsible for Chagas disease, impacts 5–6 million individuals in the Americas and is rapidly spreading globally due to significant human migration. This parasitic organism undergoes a complex life cycle involving triatomine insects and mammalian hosts, thriving in diverse environments, such as various regions within the insect’s digestive tract and mammalian cell cytoplasm. Crucially, its transmission hinges on its adaptive capabilities to varying environments. One of the most challenging environments is the insect’s digestive tract, marked by nutrient scarcity between blood meals, redox imbalance, and osmotic stresses induced by the triatomine’s metabolism. To endure these conditions, T. cruzi has developed a remarkably versatile metabolic network enabling it to metabolize sugars, lipids, and amino acids efficiently. However, the full extent of metabolites this parasite can thrive on remains incompletely understood. This study reveals that, beyond conventional carbon and energy sources (glucose, palmitic acids, proline, histidine, glutamine, and alanine), three additional metabolites (serine, threonine, and glycine) play vital roles in the parasite’s survival during starvation. Remarkably, serine and threonine directly contribute to ATP production through a serine/threonine dehydratase enzyme not previously described in T. cruzi. The significance of this metabolic pathway for the parasite’s survival sheds light on how metabolic networks aid in its endurance under extreme conditions and its ability to thrive in diverse metabolic settings

Persistence behavior in African trypanosomes during adipose tissue colonization

Abstract Persistence is an important and ancient evolutionary adaptive mechanism used by several organisms to survive environmental changes. During its life cycle Trypanosoma brucei, the causative agent of sleeping sickness, inhabits several microenvironments, including the adipose tissue. Here we used a mathematical model to investigate how this large parasite reservoir contributes to the global parasite population dynamics. By modeling the total number of parasites and the proportion of transmissible forms in the blood and the adipose tissue during an infection, we estimated that adipose tissue parasites proliferate more slowly. Intravital microscopy of parasites stained with CellTraceTM Violet confirmed that adipose tissue forms divide twice slower than the blood counterparts. Consistent with a reduced growth, proteome analysis revealed that adipose tissue forms undergo a metabolic adaptation and downregulate proteins involved in translation. Quantification of protein synthesis using L-Homopropargylglycine confirmed that this rate is 24% lower in adipose tissue forms. We propose that in adipose tissue, T. brucei acquire a persistence-like behavior, which could contribute to disease chronicity and treatment failure.</jats:p