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

Kinetoplastid Phylogenomics Reveals the Evolutionary Innovations Associated with the Origins of Parasitism

The evolution of parasitism is a recurrent event in the history of life and a core problem in evolutionary biology. Trypanosomatids are important parasites and include the human pathogens Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp., which in humans cause African trypanosomiasis, Chagas disease, and leishmaniasis, respectively. Genome comparison between trypanosomatids reveals that these parasites have evolved specialized cell-surface protein families, overlaid on a well-conserved cell template. Understanding how these features evolved and which ones are specifically associated with parasitism requires comparison with related non-parasites. We have produced genome sequences for Bodo saltans, the closest known non-parasitic relative of trypanosomatids, and a second bodonid, Trypanoplasma borreli. Here we show how genomic reduction and innovation contributed to the character of trypanosomatid genomes. We show that gene loss has “streamlined” trypanosomatid genomes, particularly with respect to macromolecular degradation and ion transport, but consistent with a widespread loss of functional redundancy, while adaptive radiations of gene families involved in membrane function provide the principal innovations in trypanosomatid evolution. Gene gain and loss continued during trypanosomatid diversification, resulting in the asymmetric assortment of ancestral characters such as peptidases between Trypanosoma and Leishmania, genomic differences that were subsequently amplified by lineage-specific innovations after divergence. Finally, we show how species-specific, cell-surface gene families (DGF-1 and PSA) with no apparent structural similarity are independent derivations of a common ancestral form, which we call “bodonin.” This new evidence defines the parasitic innovations of trypanosomatid genomes, revealing how a free-living phagotroph became adapted to exploiting hostile host environments

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

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

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

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

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

Insect stage trypanosomes use an "acetate shuttle" to transfer mitochondrial acetyl-CoA to the cytosol for the essential fatty acid biosynthesis. The mitochondrial acetate sources are acetate: succinate CoA-transferase (ASCT) and an unknown enzymatic activity. We have identified a gene encoding acetyl-CoA thioesterase (ACH) activity, which is shown to be the second acetate source. First, RNAi-mediated repression of ASCT in the ACH null background abolishes acetate production from glucose, as opposed to both single ASCT and ACH mutants. Second, incorporation of radiolabeled glucose into fatty acids is also abolished in this ACH/ASCT double mutant. ASCT is involved in ATP production, whereas ACH is not, because the ASCT null mutant is similar to 1000 times more sensitive to oligomycin, a specific inhibitor of the mitochondrial F-0/F-1-ATP synthase, than wild-type cells or the ACH null mutant. This was confirmed by RNAi repression of the F-0/F-1-ATP synthase F-1 beta subunit, which is lethal when performed in the ASCT null background but not in the wild-type cells or the ACH null background. We concluded that acetate is produced from both ASCT and ACH; however, only ASCT is responsible, together with the F-0/F-1-ATP synthase, for ATP production in the mitochondrion

Pathways whose components exhibit elevated, or decreased, expression in stumpy forms.

<p>Pathways whose components exhibit elevated, or decreased, expression in stumpy forms.</p