Blastocrithidia nonstop is a protist with a highly unusual nuclear genetic code, in which all three standard stop codons are reassigned to encode amino acids, with UAA also serving as a sole termination codon. In this study, we demonstrate that this parasitic flagellate is amenable to genetic manipulation, enabling gene ablation and protein tagging. Using preassembled Cas9 ribonucleoprotein complexes, we successfully disrupted and tagged the non-essential gene encoding catalase. These advances establish this single-celled eukaryote as a model organism for investigating the malleability and evolution of the genetic code in eukaryotes.

• Klíčová slova
• CRISPR‐Cas9, codon reassignment, genetic code, model organism, trypanosomatids,
• MeSH
• genetický kód * genetika   MeSH
• katalasa genetika   MeSH
• protozoální proteiny genetika   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina * genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• katalasa MeSH
• protozoální proteiny MeSH
• terminační kodon MeSH

Leishmania is a genus of the family Trypanosomatidae that unites obligatory parasitic flagellates causing a variety of vector-borne diseases collectively called leishmaniasis. The symptoms range from relatively innocuous skin lesions to complete failures of visceral organs. The disease is exacerbated if a parasite harbors Leishmania RNA viruses (LRVs) of the family Pseudototiviridae. Screening a novel isolate of L. braziliensis, we revealed that it possesses not a toti-, but a bunyavirus of the family Leishbuviridae. To the best of our knowledge, this is a very first discovery of a bunyavirus infecting a representative of the Leishmania subgenus Viannia. We suggest that these viruses may serve as potential factors of virulence in American leishmaniasis and encourage researchers to test leishmanial strains for the presence of not only LRVs, but also other RNA viruses.

• MeSH
• Bunyaviridae klasifikace   genetika   izolace a purifikace   MeSH
• fylogeneze MeSH
• Leishmania braziliensis * genetika   izolace a purifikace   MeSH
• lidé MeSH
• Orthobunyavirus genetika   klasifikace   izolace a purifikace   fyziologie   MeSH
• RNA-viry genetika   klasifikace   izolace a purifikace   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Trypanosomatids (Euglenozoa) are a diverse group of unicellular flagellates predominately infecting insects (monoxenous species) or circulating between insects and vertebrates or plants (dixenous species). Monoxenous trypanosomatids harbor a wide range of RNA viruses belonging to the families Narnaviridae, Totiviridae, Qinviridae, Leishbuviridae, and a putative group of tombus-like viruses. Here, we focus on the subfamily Blastocrithidiinae, a previously unexplored divergent group of monoxenous trypanosomatids comprising two related genera: Obscuromonas and Blastocrithidia. Members of the genus Blastocrithidia employ a unique genetic code, in which all three stop codons are repurposed to encode amino acids, with TAA also used to terminate translation. Obscuromonas isolates studied here bear viruses of three families: Narnaviridae, Qinviridae, and Mitoviridae. The latter viral group is documented in trypanosomatid flagellates for the first time. While other known mitoviruses replicate in the mitochondria, those of trypanosomatids appear to reside in the cytoplasm. Although no RNA viruses were detected in Blastocrithidia spp., we identified an endogenous viral element in the genome of B. triatomae indicating its past encounter(s) with tombus-like viruses.

• Klíčová slova
• Blastocrithidia, Mitoviridae, Narnaviridae, Obscuromonas, Qin-like virus, dsRNA viruses,
• Publikační typ
• časopisecké články MeSH

The canonical stop codons of the nuclear genome of the trypanosomatid Blastocrithidia nonstop are recoded. Here, we investigated the effect of this recoding on the mitochondrial genome and gene expression. Trypanosomatids possess a single mitochondrion and protein-coding transcripts of this genome require RNA editing in order to generate open reading frames of many transcripts encoded as 'cryptogenes'. Small RNAs that can number in the hundreds direct editing and produce a mitochondrial transcriptome of unusual complexity. We find B. nonstop to have a typical trypanosomatid mitochondrial genetic code, which presumably requires the mitochondrion to disable utilization of the two nucleus-encoded suppressor tRNAs, which appear to be imported into the organelle. Alterations of the protein factors responsible for mRNA editing were also documented, but they have likely originated from sources other than B. nonstop nuclear genome recoding. The population of guide RNAs directing editing is minimal, yet virtually all genes for the plethora of known editing factors are still present. Most intriguingly, despite lacking complex I cryptogene guide RNAs, these cryptogene transcripts are stochastically edited to high levels.

• MeSH
• buněčné jádro * genetika   metabolismus   MeSH
• editace RNA * MeSH
• genetický kód MeSH
• genom mitochondriální * MeSH
• guide RNA, Kinetoplastida genetika   metabolismus   MeSH
• kodon genetika   MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA transferová * genetika   metabolismus   MeSH
• terminační kodon genetika   MeSH
• Trypanosomatina genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• guide RNA, Kinetoplastida MeSH
• kodon MeSH
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA transferová * MeSH
• terminační kodon 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

BACKGROUND: Protists of the family Trypanosomatidae (phylum Euglenozoa) have gained notoriety as parasites affecting humans, domestic animals, and agricultural plants. However, the true extent of the group's diversity spreads far beyond the medically and veterinary relevant species. We address several knowledge gaps in trypanosomatid research by undertaking sequencing, assembly, and analysis of genomes from previously overlooked representatives of this protistan group. RESULTS: We assembled genomes for twenty-one trypanosomatid species, with a primary focus on insect parasites and Trypanosoma spp. parasitizing non-human hosts. The assemblies exhibit sizes consistent with previously sequenced trypanosomatid genomes, ranging from approximately 18 Mb for Obscuromonas modryi to 35 Mb for Crithidia brevicula and Zelonia costaricensis. Despite being the smallest, the genome of O. modryi has the highest content of repetitive elements, contributing nearly half of its total size. Conversely, the highest proportion of unique DNA is found in the genomes of Wallacemonas spp., with repeats accounting for less than 8% of the assembly length. The majority of examined species exhibit varying degrees of aneuploidy, with trisomy being the most frequently observed condition after disomy. CONCLUSIONS: The genome of Obscuromonas modryi represents a very unusual, if not unique, example of evolution driven by two antidromous forces: i) increasing dependence on the host leading to genomic shrinkage and ii) expansion of repeats causing genome enlargement. The observed variation in somy within and between trypanosomatid genera suggests that these flagellates are largely predisposed to aneuploidy and, apparently, exploit it to gain a fitness advantage. High heterogeneity in the genome size, repeat content, and variation in chromosome copy numbers in the newly-sequenced species highlight the remarkable genome plasticity exhibited by trypanosomatid flagellates. These new genome assemblies are a robust foundation for future research on the genetic basis of life cycle changes and adaptation to different hosts in the family Trypanosomatidae.

• Klíčová slova
• Dixenous, Genome assembly, Monoxenous, Parasite, Protist, Trypanosomatids, Whole-genome sequencing,
• MeSH
• aklimatizace MeSH
• aneuploidie MeSH
• délka genomu MeSH
• Trypanosomatina * genetika   MeSH
• zemědělství MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite's replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

• MeSH
• buněčný cyklus MeSH
• fosfatidátfosfatasa genetika   MeSH
• Leishmania guyanensis * MeSH
• Leishmaniavirus MeSH
• leishmanióza kožní * MeSH
• lipidy MeSH
• myši MeSH
• paraziti * MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fosfatidátfosfatasa MeSH
• lipidy 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

Euglenozoa is a species-rich group of protists, which have extremely diverse lifestyles and a range of features that distinguish them from other eukaryotes. They are composed of free-living and parasitic kinetoplastids, mostly free-living diplonemids, heterotrophic and photosynthetic euglenids, as well as deep-sea symbiontids. Although they form a well-supported monophyletic group, these morphologically rather distinct groups are almost never treated together in a comparative manner, as attempted here. We present an updated taxonomy, complemented by photos of representative species, with notes on diversity, distribution and biology of euglenozoans. For kinetoplastids, we propose a significantly modified taxonomy that reflects the latest findings. Finally, we summarize what is known about viruses infecting euglenozoans, as well as their relationships with ecto- and endosymbiotic bacteria.

• Klíčová slova
• Diplonemida, Euglenida, Kinetoplastida, microbial eukaryotes, phylogeny, systematics,
• MeSH
• ekosystém MeSH
• Euglenozoa klasifikace   genetika   fyziologie   virologie   MeSH
• fylogeneze MeSH
• Mimiviridae patogenita   MeSH
• symbióza MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

In this work, we analyzed viral prevalence in trypanosomatid parasites (Blechomonas spp.) infecting Siphonaptera and discovered nine species of viruses from three different groups (leishbunyaviruses, narnaviruses, and leishmaniaviruses). Most of the flagellate isolates bore two or three viral types (mixed infections). Although no new viral groups were documented in Blechomonas spp., our findings are important for the comprehension of viral evolution. The discovery of bunyaviruses in blechomonads was anticipated, since these viruses have envelopes facilitating their interspecific transmission and have already been found in various trypanosomatids and metatranscriptomes with trypanosomatid signatures. In this work, we also provided evidence that even representatives of the family Narnaviridae are capable of host switching and evidently have accomplished switches multiple times in the course of their evolution. The most unexpected finding was the presence of leishmaniaviruses, a group previously solely confined to the human pathogens Leishmania spp. From phylogenetic inferences and analyses of the life cycles of Leishmania and Blechomonas, we concluded that a common ancestor of leishmaniaviruses most likely infected Leishmania first and was acquired by Blechomonas by horizontal transfer. Our findings demonstrate that evolution of leishmaniaviruses is more complex than previously thought and includes occasional host switching.IMPORTANCE Flagellates belonging to the genus Leishmania are important human parasites. Some strains of different Leishmania species harbor viruses (leishmaniaviruses), which facilitate metastatic spread of the parasites, thus aggravating the disease. Up until now, these viruses were known to be hosted only by Leishmania Here, we analyzed viral distribution in Blechomonas, a related group of flagellates parasitizing fleas, and revealed that they also bear leishmaniaviruses. Our findings shed light on the entangled evolution of these viruses. In addition, we documented that Blechomonas can be also infected by leishbunyaviruses and narnaviruses, viral groups known from other insects' flagellates.

• Klíčová slova
• Blechomonas, Leishbunyaviridae, Leishmaniavirus, Narnaviridae,
• MeSH
• fylogeneze MeSH
• genetická variace MeSH
• genom virový MeSH
• Leishmaniavirus genetika   MeSH
• molekulární evoluce * MeSH
• RNA-viry klasifikace   izolace a purifikace   MeSH
• Siphonaptera parazitologie   MeSH
• Trypanosomatina virologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH