Trypanosoma brucei:Molecular Cloning and Stage-Regulated Expression of a Malate Dehydrogenase Localized to the Mitochondrion (original) (raw)
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The extraordinary mitochondrion and unusual citric acid cycle in Trypanosoma brucei
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.
International Journal for Parasitology, 2006
Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate. q
Energy generation in insect stages of Trypanosoma brucei: metabolism in flux
Trends in Parasitology, 2005
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 procyclicform T. brucei.
Journal of Bioenergetics and Biomembranes, 2011
Trypanosoma cruzi is a hemoflagellate protozoan that causes Chagas' disease. The life cycle of T. cruzi is complex and involves different evolutive forms that have to encounter different environmental conditions provided by the host. Herein, we performed a functional assessment of mitochondrial metabolism in the following two distinct evolutive forms of T. cruzi: the insect stage epimastigote and the freshly isolated bloodstream trypomastigote. We observed that in comparison to epimastigotes, bloodstream trypomastigotes facilitate the entry of electrons into the electron transport chain by increasing complex II-III activity. Interestingly, cytochrome c oxidase (CCO) activity and the expression of CCO subunit IV were reduced in bloodstream forms, creating an "electron bottleneck" that favored an increase in electron leakage and H 2 O 2 formation. We propose that the oxidative preconditioning provided by this mechanism confers protection to bloodstream trypomastigotes against the host immune system. In this scenario, mitochondrial remodeling during the T. cruzi life cycle may represent a key metabolic adaptation for parasite survival in different hosts.
Molecular and Biochemical Parasitology, 2017
In the slender bloodstream form, Trypanosoma brucei mitochondria are repressed for many functions. Multiple components of mitochondrial complex I, NADH:ubiquinone oxidoreductase, are expressed in this stage, but electron transfer through complex I is not essential. Here we investigate the role of the parasite's second NADH:ubiquinone oxidoreductase, NDH2, which is composed of a single subunit that also localizes to the mitochondrion. While inducible knockdown of NDH2 had a modest growth effect in bloodstream forms, NDH2 null mutants, as well as inducible knockdowns in a complex I deficient background, showed a greater reduction in growth. Altering the NAD + /NADH balance would affect numerous processes directly and indirectly, including acetate production. Indeed, loss of NDH2 led to reduced levels of acetate, which is required for several essential pathways in bloodstream form T. brucei and which may have contributed to the observed growth defect. In conclusion our study shows that NDH2 is important, but not essential, in proliferating bloodstream forms of T. brucei, arguing that the mitochondrial NAD + /NADH balance is important in this stage, even though the mitochondrion itself is not actively engaged in the generation of ATP.
Enigmatic Presence of Mitochondrial Complex I in Trypanosoma brucei Bloodstream Forms
Eukaryotic Cell, 2012
The presence of mitochondrial respiratory complex I in the pathogenic bloodstream stages of Trypanosoma brucei has been vigorously debated: increased expression of mitochondrially encoded functional complex I mRNAs is countered by low levels of enzymatic activity that show marginal inhibition by the specific inhibitor rotenone. We now show that epitope-tagged versions of multiple complex I subunits assemble into ␣ and  subcomplexes in the bloodstream stage and that these subcomplexes require the mitochondrial genome for their assembly. Despite the presence of these large (740-and 855-kDa) multisubunit complexes, the electron transport activity of complex I is not essential under experimental conditions since null mutants of two core genes (NUBM and NUKM) showed no growth defect in vitro or in mouse infection. Furthermore, the null mutants showed no decrease in NADH:ubiquinone oxidoreductase activity, suggesting that the observed activity is not contributed by complex I. This work conclusively shows that despite the synthesis and assembly of subunit proteins, the enzymatic function of the largest respiratory complex is neither significant nor important in the bloodstream stage. This situation appears to be in striking contrast to that for the other respiratory complexes in this parasite, where physical presence in a life-cycle stage always indicates functional significance.
Malleable Mitochondrion of Trypanosoma brucei
International review of cell and molecular biology, 2015
The importance of mitochondria for a typical aerobic eukaryotic cell is undeniable, as the list of necessary mitochondrial processes is steadily growing. Here, we summarize the current knowledge of mitochondrial biology of an early-branching parasitic protist, Trypanosoma brucei, a causative agent of serious human and cattle diseases. We present a comprehensive survey of its mitochondrial pathways including kinetoplast DNA replication and maintenance, gene expression, protein and metabolite import, major metabolic pathways, Fe-S cluster synthesis, ion homeostasis, organellar dynamics, and other processes. As we describe in this chapter, the single mitochondrion of T. brucei is everything but simple and as such rivals mitochondria of multicellular organisms.