Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.

• Klíčová slova
• Krebs cycle, NAD+, NADP+, TCA cycle, cofactor preference, isocitrate dehydrogenase,
• MeSH
• isocitrátdehydrogenasa * genetika   metabolismus   MeSH
• isocitráty metabolismus   MeSH
• NAD * metabolismus   MeSH
• NADP metabolismus   MeSH
• protein - isoformy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isocitrátdehydrogenasa * MeSH
• isocitráty MeSH
• isocitric acid MeSH Prohlížeč
• NAD * MeSH
• NADP MeSH
• protein - isoformy MeSH

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 trypanosomatid 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 metabolism contains all typical features of these diverse and important parasites. We also provided experimental validation 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.

• Klíčová slova
• Blastocrithidia, In silico, Metabolic predictions, Non-canonical genetic code, Trypanosomatid,
• MeSH
• Eukaryota genetika   MeSH
• genetický kód MeSH
• paraziti * genetika   MeSH
• terminační kodon MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosomatina * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• terminační kodon MeSH

Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.

• Klíčová slova
• Chagas disease, RNA editing, electron transport chain, epimastigote, metabolism,
• MeSH
• editace RNA MeSH
• messenger RNA genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• RNA metabolismus   MeSH
• savci genetika   MeSH
• transkriptom MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma cruzi * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH
• RNA MeSH

Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai, Herpetomonas tarakana, Kentomonas sorsogonicus, Leptomonas seymouri, Novymonas esmeraldas, Sergeia podlipaevi and Wallacemonas raviniae. We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens, in which its presence and activity have been previously documented. In addition to P. serpens, the complex I is functionally active in N. esmeraldas and S. podlipaevi. We also identified 24-32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.

• Klíčová slova
• Monoxenous trypanosomatids, NADH dehydrogenase, Phytomonas,
• MeSH
• druhová specificita MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• NADH-dehydrogenasa genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• Trypanosomatina enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• mitochondriální proteiny MeSH
• NADH-dehydrogenasa MeSH
• protozoální proteiny MeSH

A recently redescribed two-flagellar trypanosomatid Vickermania ingenoplastis is insensitive to the classical inhibitors of respiration and thrives under anaerobic conditions. Using genomic and transcriptomic data, we analyzed its genes of the core metabolism and documented that subunits of the mitochondrial respiratory complexes III and IV are ablated, while those of complexes I, II, and V are all present, along with an alternative oxidase. This explains the previously reported conversion of glucose to acetate and succinate by aerobic fermentation. Glycolytic pyruvate is metabolized to acetate and ethanol by pyruvate dismutation, whereby a unique type of alcohol dehydrogenase (shared only with Phytomonas spp.) processes an excess of reducing equivalents formed under anaerobic conditions, leading to the formation of ethanol. Succinate (formed to maintain the glycosomal redox balance) is converted to propionate by a cyclic process involving three enzymes of the mitochondrial methyl-malonyl-CoA pathway, via a cyclic process, which results in the formation of additional ATP. The unusual structure of the V. ingenoplastis genome and its similarity with that of Phytomonas spp. imply their relatedness or convergent evolution. Nevertheless, a critical difference between these two trypanosomatids is that the former has significantly increased its genome size by gene duplications, while the latter streamlined its genome.

• Klíčová slova
• Phytomonas, Vickermania ingenoplastis, genome sequencing, metabolism,
• Publikační typ
• časopisecké články MeSH

In the present work, we investigated molecular mechanisms governing thermal resistance of a monoxenous trypanosomatid Crithidia luciliae thermophila, which we reclassified as a separate species C. thermophila. We analyzed morphology, growth kinetics, and transcriptomic profiles of flagellates cultivated at low (23°C) and elevated (34°C) temperature. When maintained at high temperature, they grew significantly faster, became shorter, with genes involved in sugar metabolism and mitochondrial stress protection significantly upregulated. Comparison with another thermoresistant monoxenous trypanosomatid, Leptomonas seymouri, revealed dramatic differences in transcription profiles of the two species with only few genes showing the same expression pattern. This disparity illustrates differences in the biology of these two parasites and distinct mechanisms of their thermotolerance, a prerequisite for living in warm-blooded vertebrates.

• MeSH
• biochemické jevy genetika   MeSH
• Crithidia genetika   MeSH
• exprese genu genetika   MeSH
• hmyz genetika   MeSH
• teplota MeSH
• transkriptom genetika   MeSH
• upregulace genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

The class Kinetoplastea encompasses both free-living and parasitic species from a wide range of hosts. Several representatives of this group are responsible for severe human diseases and for economic losses in agriculture and livestock. While this group encompasses over 30 genera, most of the available information has been derived from the vertebrate pathogenic genera Leishmaniaand Trypanosoma. Recent studies of the previously neglected groups of Kinetoplastea indicated that the actual diversity is much higher than previously thought. This article discusses the known segment of kinetoplastid diversity and how gene-directed Sanger sequencing and next-generation sequencing methods can help to deepen our knowledge of these interesting protists.

• MeSH
• biodiverzita * MeSH
• biologické markery MeSH
• databáze genetické MeSH
• fylogeneze * MeSH
• Kinetoplastida klasifikace   cytologie   genetika   MeSH
• metagenomika trendy   MeSH
• protozoální DNA genetika   MeSH
• RNA protozoální genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• taxonomické DNA čárové kódování trendy   MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• životní prostředí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• biologické markery MeSH
• protozoální DNA MeSH
• RNA protozoální MeSH
• RNA ribozomální 18S MeSH