The long slender bloodstream form Trypanosoma brucei maintains its essential mitochondrial membrane potential (ΔΨm) through the proton-pumping activity of the FoF1-ATP synthase operating in the reverse mode. The ATP that drives this hydrolytic reaction has long been thought to be generated by glycolysis and imported from the cytosol via an ATP/ADP carrier (AAC). Indeed, we demonstrate that AAC is the only carrier that can import ATP into the mitochondrial matrix to power the hydrolytic activity of the FoF1-ATP synthase. However, contrary to expectations, the deletion of AAC has no effect on parasite growth, virulence or levels of ΔΨm. This suggests that ATP is produced by substrate-level phosphorylation pathways in the mitochondrion. Therefore, we knocked out the succinyl-CoA synthetase (SCS) gene, a key mitochondrial enzyme that produces ATP through substrate-level phosphorylation in this parasite. Its absence resulted in changes to the metabolic landscape of the parasite, lowered virulence, and reduced mitochondrial ATP content. Strikingly, these SCS mutant parasites become more dependent on AAC as demonstrated by a 25-fold increase in their sensitivity to the AAC inhibitor, carboxyatractyloside. Since the parasites were able to adapt to the loss of SCS in culture, we also analyzed the more immediate phenotypes that manifest when SCS expression is rapidly suppressed by RNAi. Importantly, when performed under nutrient-limited conditions mimicking various host environments, SCS depletion strongly affected parasite growth and levels of ΔΨm. In totality, the data establish that the long slender bloodstream form mitochondrion is capable of generating ATP via substrate-level phosphorylation pathways.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• fosforylace MeSH
• mitochondrie metabolismus   MeSH
• Trypanosoma brucei brucei * metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH

Trypanosoma brucei, a protist responsible for human African trypanosomiasis (sleeping sickness), is transmitted by the tsetse fly where the procyclic forms of the parasite develop in the proline-rich (1-2 mM) and glucose-depleted digestive tract. Proline is essential for the midgut colonization of the parasite in the insect vector, however other carbon sources could be available and used to feed its central metabolism. Here we show that procyclic trypanosomes can consume and metabolize metabolic intermediates, including those excreted from glucose catabolism (succinate, alanine and pyruvate), with the exception of acetate, which is the ultimate end-product excreted by the parasite. Among the tested metabolites, tricarboxylic acid (TCA) cycle intermediates (succinate, malate and α-ketoglutarate) stimulated growth of the parasite in the presence of 2 mM proline. The pathways used for their metabolism were mapped by proton-NMR metabolic profiling and phenotypic analyses of thirteen RNAi and/or null mutants affecting central carbon metabolism. We showed that (i) malate is converted to succinate by both the reducing and oxidative branches of the TCA cycle, which demonstrates that procyclic trypanosomes can use the full TCA cycle, (ii) the enormous rate of α-ketoglutarate consumption (15-times higher than glucose) is possible thanks to the balanced production and consumption of NADH at the substrate level and (iii) α-ketoglutarate is toxic for trypanosomes if not appropriately metabolized as observed for an α-ketoglutarate dehydrogenase null mutant. In addition, epimastigotes produced from procyclics upon overexpression of RBP6 showed a growth defect in the presence of 2 mM proline, which is rescued by α-ketoglutarate, suggesting that physiological amounts of proline are not sufficient per se for the development of trypanosomes in the fly. In conclusion, these data show that trypanosomes can metabolize multiple metabolites, in addition to proline, which allows them to confront challenging environments in the fly.

• MeSH
• citrátový cyklus účinky léků   MeSH
• glukosa metabolismus   MeSH
• hmyz - vektory parazitologie   MeSH
• moucha tse-tse účinky léků   parazitologie   MeSH
• oxidace-redukce účinky léků   MeSH
• prolin metabolismus   farmakologie   MeSH
• RNA interference fyziologie   MeSH
• Trypanosoma brucei brucei účinky léků   metabolismus   MeSH
• Trypanosoma účinky léků   metabolismus   MeSH
• trypanozomóza africká farmakoterapie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glukosa MeSH
• prolin MeSH

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite Trypanosoma brucei, occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism (i.e. polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.

• Klíčová slova
• Trypanosoma brucei, differentiation, drug action, drug mechanisms, energy homeostasis, glycosomes, metabolomics, parasite metabolism, polypharmacology, proteomics, sleeping sickness, suramin, trypanosome,
• MeSH
• adenosintrifosfát metabolismus   MeSH
• energetický metabolismus účinky léků   MeSH
• flagella účinky léků   metabolismus   ultrastruktura   MeSH
• glykolýza účinky léků   MeSH
• kyselina pyrohroznová metabolismus   MeSH
• membránový potenciál mitochondrií účinky léků   MeSH
• metabolom účinky léků   MeSH
• mikrotělíska účinky léků   metabolismus   ultrastruktura   MeSH
• mitochondrie účinky léků   metabolismus   ultrastruktura   MeSH
• molekulární modely MeSH
• prolin metabolismus   MeSH
• proteom metabolismus   MeSH
• protonové ATPasy metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• suramin farmakologie   MeSH
• Trypanosoma brucei brucei metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenosintrifosfát MeSH
• kyselina pyrohroznová MeSH
• prolin MeSH
• proteom MeSH
• protonové ATPasy MeSH
• protozoální proteiny MeSH
• suramin MeSH

• MeSH
• lidé MeSH
• mitochondriální proteiny metabolismus   MeSH
• proteom analýza   metabolismus   MeSH
• protozoální proteiny metabolismus   MeSH
• stadia vývoje fyziologie   MeSH
• Trypanosoma brucei brucei růst a vývoj   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• proteom MeSH
• protozoální proteiny MeSH

In yeast (Saccharomyces cerevisiae) and animals, the sulfhydryl oxidase Erv1 functions with Mia40 in the import and oxidative folding of numerous cysteine-rich proteins in the mitochondrial intermembrane space (IMS). Erv1 is also required for Fe-S cluster assembly in the cytosol, which uses at least one mitochondrially derived precursor. Here, we characterize an essential Erv1 orthologue from the protist Trypanosoma brucei (TbERV1), which naturally lacks a Mia40 homolog. We report kinetic parameters for physiologically relevant oxidants cytochrome c and O(2), unexpectedly find O(2) and cytochrome c are reduced simultaneously, and demonstrate that efficient reduction of O(2) by TbERV1 is not dependent upon a simple O(2) channel defined by conserved histidine and tyrosine residues. Massive mitochondrial swelling following TbERV1 RNA interference (RNAi) provides evidence that trypanosome Erv1 functions in IMS protein import despite the natural absence of the key player in the yeast and animal import pathways, Mia40. This suggests significant evolutionary divergence from a recently established paradigm in mitochondrial cell biology. Phylogenomic profiling of genes also points to a conserved role for TbERV1 in cytosolic Fe-S cluster assembly. Conversely, loss of genes implicated in precursor delivery for cytosolic Fe-S assembly in Entamoeba, Trichomonas, and Giardia suggests fundamental differences in intracellular trafficking pathways for activated iron or sulfur species in anaerobic versus aerobic eukaryotes.

• MeSH
• cytochromy c chemie   MeSH
• fylogeneze MeSH
• genový knockdown MeSH
• kinetika MeSH
• kyslík chemie   MeSH
• mitochondriální proteiny chemie   genetika   MeSH
• mitochondrie enzymologie   ultrastruktura   MeSH
• molekulární evoluce MeSH
• mutageneze cílená MeSH
• oxidace-redukce MeSH
• oxidancia MeSH
• oxidoreduktasy chemie   genetika   MeSH
• protozoální proteiny chemie   genetika   MeSH
• RNA interference MeSH
• sbalování proteinů MeSH
• substituce aminokyselin MeSH
• transport proteinů MeSH
• Trypanosoma brucei brucei cytologie   enzymologie   MeSH
• zduření mitochondrií MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytochromy c MeSH
• kyslík MeSH
• mitochondriální proteiny MeSH
• oxidancia MeSH
• oxidoreduktasy MeSH
• protozoální proteiny MeSH
• sulfhydryl oxidase MeSH Prohlížeč