UNLABELLED: Trypanosomatids are among the most extensively studied protists due to their parasitic interactions with insects, vertebrates, and plants. Recently, Blastocrithidia nonstop was found to depart from the canonical genetic code, with all three stop codons reassigned to encode amino acids (UAR for glutamate and UGA for tryptophan), and UAA having dual meaning also as a termination signal (glutamate and stop). To explore features linked to this phenomenon, we analyzed the genomes of four Blastocrithidia and four Obscuromonas species, the latter representing a sister group employing the canonical genetic code. We found that all Blastocrithidia species encode cognate tRNAs for UAR codons, possess a distinct 4 bp anticodon stem tRNATrpCCA decoding UGA, and utilize UAA as the only stop codon. The distribution of in-frame reassigned codons is consistently non-random, suggesting a translational burden avoided in highly expressed genes. Frame-specific enrichment of UAA codons immediately following the genuine UAA stop codon, not observed in Obscuromonas, points to a specific mode of termination. All Blastocrithidia species possess specific mutations in eukaryotic release factor 1 and a unique acidic region following the prion-like N-terminus of eukaryotic release factor 3 that may be associated with stop codon readthrough. We infer that the common ancestor of the genus Blastocrithidia already exhibited a GC-poor genome with the non-canonical genetic code. Our comparative analysis highlights features associated with this extensive stop codon reassignment. This cascade of mutually dependent adaptations, driven by increasing AU-richness in transcripts and frequent emergence of in-frame stops, underscores the dynamic interplay between genome composition and genetic code plasticity to maintain vital functionality. IMPORTANCE: The genetic code, assigning amino acids to codons, is almost universal, yet an increasing number of its alterations keep emerging, mostly in organelles and unicellular eukaryotes. One such case is the trypanosomatid genus Blastocrithidia, where all three stop codons were reassigned to amino acids, with UAA also serving as a sole termination signal. We conducted a comparative analysis of four Blastocrithidia species, all with the same non-canonical genetic code, and their close relatives of the genus Obscuromonas, which retain the canonical code. This across-genome comparison allowed the identification of key traits associated with genetic code reassignment in Blastocrithidia. This work provides insight into the evolutionary steps, facilitating an extensive departure from the canonical genetic code that occurred independently in several eukaryotic lineages.

• Klíčová slova
• AT-rich genomes, eukaryotic release factors, nuclear genetic code, reassigned codon, tRNA structure, termination of translation,
• MeSH
• buněčné jádro * genetika   MeSH
• fylogeneze MeSH
• genetický kód * MeSH
• genom protozoální * MeSH
• genomika MeSH
• molekulární evoluce MeSH
• RNA transferová genetika   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina * genetika   klasifikace   MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• RNA transferová MeSH
• terminační kodon MeSH

The trypanosomatid flagellates possess in their single mitochondrion a highly complex kinetoplast (k)DNA, which is composed of interlocked circular molecules of two types. Dozens of maxicircles represent a classical mitochondrial genome, and thousands of minicircles encode guide (g)RNAs, which direct the processive and essential uridine insertion/deletion messenger RNA (mRNA) editing of maxicircle transcripts. While the details of kDNA structure and this type of RNA editing are well established, our knowledge mostly relies on a narrow foray of intensely studied human parasites of the genera Leishmania and Trypanosoma. Here, we analyzed kDNA, its expression, and RNA editing of two members of the poorly characterized genus Vickermania with very different cultivation histories. In both Vickermania species, the gRNA-containing heterogeneous large (HL)-circles are atypically large with multiple gRNAs each. Examination of Vickermania spadyakhi HL-circle loci revealed a massive redundancy of gRNAs relative to the editing needs. In comparison, the HL-circle repertoire of extensively cultivated Vickermania ingenoplastis is greatly reduced. It correlates with V. ingenoplastis-specific loss of productive editing of transcripts encoding subunits of respiratory chain complex I and corresponding lack of complex I activity. This loss in a parasite already lacking genes for subunits of complexes III and IV suggests an apparent requirement for its mitochondrial adenosine triphosphate (ATP) synthase to work in reverse to maintain membrane potential. In contrast, V. spadyakhi retains a functional complex I that allows ATP synthase to work in its standard direction.

• Klíčová slova
• ATP synthase, RNA editing, Vickermania, kinetoplast DNA, trypanosomatids,
• MeSH
• editace RNA * genetika   MeSH
• genom mitochondriální MeSH
• genom protozoální * MeSH
• kinetoplastová DNA * genetika   MeSH
• molekulární evoluce * MeSH
• Trypanosomatina * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• kinetoplastová DNA * MeSH

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

Instability is an intriguing characteristic of many protist genomes, and trypanosomatids are not an exception in this respect. Some regions of trypanosomatid genomes evolve fast. For instance, the trypanosomatid mitochondrial (kinetoplast) genome consists of fairly conserved maxicircle and minicircle molecules that can, nevertheless, possess high nucleotide substitution rates between closely related strains. Recent experiments have demonstrated that rapid laboratory evolution can result in the non-functionality of multiple genes of kinetoplast genomes due to the accumulation of mutations or loss of critical genomic components. An example of a loss of critical components is the reported loss of entire minicircle classes in Leishmania tarentolae during laboratory cultivation, which results in an inability to generate some correctly encoded genes. In the current work, we estimated the evolutionary rates of mitochondrial and nuclear genome regions of multiple natural Leishmania spp. We analyzed synonymous and non-synonymous substitutions and, rather unexpectedly, found that the coding regions of kinetoplast maxicircles are among the most variable regions of both genomes. In addition, we demonstrate that synonymous substitutions greatly predominate among maxicircle coding regions and that most maxicircle genes show signs of purifying selection. These results imply that maxicircles in natural Leishmania populations remain functional despite their high mutation rate.

• Klíčová slova
• L. infantum, L. major, L. turanica, SNP, genome instability, leishmania donovani,
• Publikační typ
• časopisecké články MeSH

Trypanosomatids are easy to cultivate and they are (in many cases) amenable to genetic manipulation. Genome sequencing has become a standard tool routinely used in the study of these flagellates. In this review, we summarize the current state of the field and our vision of what needs to be done in order to achieve a more comprehensive picture of trypanosomatid evolution. This will also help to illuminate the lineage-specific proteins and pathways, which can be used as potential targets in treating diseases caused by these parasites.

• Klíčová slova
• genomics, next-generation sequencing, trypanosomatids,
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH

Telomeres are the ends of linear eukaryotic chromosomes facilitating the resolution of the ‘end replication and protection’ problems, associated with linearity. At the nucleotide level, telomeres typically represent stretches of tandemly arranged telomeric repeats, which vary in length and sequence among different groups of organisms. Recently, a composition of the telomere-associated protein complex has been scrutinized in Trypanosoma brucei. In this work, we subjected proteins from that list to a more detailed bioinformatic analysis and delineated a core set of 20 conserved proteins putatively associated with telomeres in trypanosomatids. Out of these, two proteins (Ku70 and Ku80) are conspicuously missing in representatives of the genus Blastocrithidia, yet telomeres in these species do not appear to be affected. In this work, based on the analysis of a large set of trypanosomatids widely different in their phylogenetic position and life strategies, we demonstrated that telomeres of trypanosomatids are diverse in length, even within groups of closely related species. Our analysis showed that the expression of two proteins predicted to be associated with telomeres (those encoding telomerase and telomere-associated hypothetical protein orthologous to Tb927.6.4330) may directly affect and account for the differences in telomere length within the species of the Leishmania mexicana complex.

• Klíčová slova
• Genomes, Trypanosomatidae, telomere maintenance,
• MeSH
• Leishmania mexicana genetika   MeSH
• telomery metabolismus   MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

Receptor adenylate cyclases (RACs) on the surface of trypanosomatids are important players in the host-parasite interface. They detect still unidentified environmental signals that affect the parasites' responses to host immune challenge, coordination of social motility, and regulation of cell division. A lesser known class of oxygen-sensing adenylate cyclases (OACs) related to RACs has been lost in trypanosomes and expanded mostly in Leishmania species and related insect-dwelling trypanosomatids. In this work, we have undertaken a large-scale phylogenetic analysis of both classes of adenylate cyclases (ACs) in trypanosomatids and the free-living Bodo saltans. We observe that the expanded RAC repertoire in trypanosomatids with a two-host life cycle is not only associated with an extracellular lifestyle within the vertebrate host, but also with a complex path through the insect vector involving several life cycle stages. In Trypanosoma brucei, RACs are split into two major clades, which significantly differ in their expression profiles in the mammalian host and the insect vector. RACs of the closely related Trypanosoma congolense are intermingled within these two clades, supporting early RAC diversification. Subspecies of T. brucei that have lost the capacity to infect insects exhibit high numbers of pseudogenized RACs, suggesting many of these proteins have become redundant upon the acquisition of a single-host life cycle. OACs appear to be an innovation occurring after the expansion of RACs in trypanosomatids. Endosymbiont-harboring trypanosomatids exhibit a diversification of OACs, whereas these proteins are pseudogenized in Leishmania subgenus Viannia. This analysis sheds light on how ACs have evolved to allow diverse trypanosomatids to occupy multifarious niches and assume various lifestyles.

• Klíčová slova
• Kinetoplastida, adenylate cyclase, oxygen, phylogenetics, receptor, trypanosomatid,
• MeSH
• adenylátcyklasy genetika   MeSH
• duplikace genu MeSH
• fylogeneze * MeSH
• genom protozoální MeSH
• interakce hostitele a patogenu genetika   MeSH
• molekulární evoluce * MeSH
• Trypanosomatina enzymologie   genetika   MeSH
• upregulace MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adenylátcyklasy MeSH

Here we described a new trypanosomatid species, Phytomonas lipae, parasitizing the dock bug Coreus marginatus based on axenic culture and in vivo material. Using light and electron microscopy we characterized the development of this flagellate in the intestine, hemolymph and salivary glands of its insect host. The intestinal promastigotes of Phytomonas lipae do not divide and occur only in the anterior part of the midgut. From there they pass into hemolymph, increasing in size, and then to salivary glands, where they actively proliferate without attachment to the host's epithelium and form infective endomastigotes. We conducted molecular phylogenetic analyses based on 18s rRNA, gGAPDH and HSP83 gene sequences, of which the third marker performed the best in terms of resolving phylogenetic relationships within the genus Phytomonas. Our inference demonstrated rather early origin of the lineage comprising the new species, right after that of P. oxycareni, which represents the earliest known branch within the Phytomonas clade. This allowed us to compare the development of P. lipae and three other Phytomonas spp. in their insect hosts and reconstruct the vectorial part of the life cycle of their common ancestor.

• MeSH
• fylogeneze MeSH
• Heteroptera parazitologie   MeSH
• Kinetoplastida MeSH
• pravděpodobnostní funkce MeSH
• proteiny teplotního šoku genetika   MeSH
• protozoální proteiny genetika   MeSH
• RNA ribozomální 18S genetika   MeSH
• slinné žlázy parazitologie   MeSH
• stadia vývoje * MeSH
• střeva parazitologie   MeSH
• Trypanosomatina klasifikace   genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Hsp83 protein, protozoan MeSH Prohlížeč
• proteiny teplotního šoku MeSH
• protozoální proteiny MeSH
• RNA ribozomální 18S MeSH

Fungi, nematodes and oomycetes belong to the most prominent eukaryotic plant pathogenic organisms. Unicellular organisms from other eukaryotic lineages, commonly addressed as protists, also infect plants. This review provides an introduction to plant pathogenic protists, including algae infecting oomycetes, and their current state of research.

• Klíčová slova
• algae, phytomonas, phytomyxae, plant pathogens, plasmodiophorids, protist, stramenopiles,
• MeSH
• oomycety patogenita   MeSH
• Plasmodiophorida patogenita   MeSH
• rostliny mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH