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Background The phylum Euglenozoa is a group of flagellated protists comprising the diplonemids, euglenids, symbiontids, and kinetoplastids. The diplonemids are highly abundant and speciose, and recent tools have rendered the best studied representative, Diplonema papillatum, genetically tractable. However, despite the high diversity of diplonemids, their lifestyles, ecological functions, and even primary energy source are mostly unknown. Results We designed a metabolic map of D. papillatum cellular bioenergetic pathways based on the alterations of transcriptomic, proteomic, and metabolomic profiles obtained from cells grown under different conditions. Comparative analysis in the nutrient-rich and nutrient-poor media, as well as the absence and presence of oxygen, revealed its capacity for extensive metabolic reprogramming that occurs predominantly on the proteomic rather than the transcriptomic level. D. papillatum is equipped with fundamental metabolic routes such as glycolysis, gluconeogenesis, TCA cycle, pentose phosphate pathway, respiratory complexes, β-oxidation, and synthesis of fatty acids. Gluconeogenesis is uniquely dominant over glycolysis under all surveyed conditions, while the TCA cycle represents an eclectic combination of standard and unusual enzymes. Conclusions The identification of conventional anaerobic enzymes reflects the ability of this protist to survive in low-oxygen environments. Furthermore, its metabolism quickly reacts to restricted carbon availability, suggesting a high metabolic flexibility of diplonemids, which is further reflected in cell morphology and motility, correlating well with their extreme ecological valence.

Background Mitochondria and peroxisomes are the two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform into anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, collectively called mitochondrion-related organelles (MROs), which vary in enzymatic capacity. Anaerobic peroxisomes were identified only recently, and their putatively most conserved function seems to be the metabolism of inositol. The group Archamoebae includes anaerobes bearing both anaerobic peroxisomes and MROs, specifically hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica, while the organelles within the third lineage represented by Pelomyxa remain uncharacterized. Results We generated high-quality genome and transcriptome drafts from Pelomyxa schiedti using single-cell omics. These data provided clear evidence for anaerobic derivates of mitochondria and peroxisomes in this species, and corresponding vesicles were tentatively identified in electron micrographs. In silico reconstructed MRO metabolism harbors respiratory complex II, electron-transferring flavoprotein, a partial TCA cycle running presumably in the reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, a glycine cleavage system, a sulfate activation pathway, and an expanded set of NIF enzymes for iron-sulfur cluster assembly. When expressed in the heterologous system of yeast, some of these candidates localized into mitochondria, supporting their involvement in the MRO metabolism. The putative functions of P. schiedti peroxisomes could be pyridoxal 5′-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Unexpectedly, out of 67 predicted peroxisomal enzymes, only four were also reported in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and d-lactate dehydrogenase. Localizations in yeast corroborated peroxisomal functions of the latter two. Conclusions This study revealed the presence and partially annotated the function of anaerobic derivates of mitochondria and peroxisomes in P. schiedti using single-cell genomics, localizations in yeast heterologous systems, and transmission electron microscopy. The MRO metabolism resembles that of M. balamuthi and most likely reflects the state in the common ancestor of Archamoebae. The peroxisomal metabolism is strikingly richer in P. schiedti. The presence of myo-inositol 2-dehydrogenase in the predicted peroxisomal proteome corroborates the situation in other Archamoebae, but future experimental evidence is needed to verify additional functions of this organelle.

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.

Pelomyxa schiedti is a free-living amoeba belonging to the group Archamoebae, which encompasses anaerobes bearing mitochondrion-related organelles (MROs) – hydrogenosomes in free-living Mastigamoeba balamuthi and mitosomes in the human pathogen Entamoeba histolytica. Anaerobic peroxisomes, another adaptation to anaerobic lifestyle, were identified only recently in M. balamuthi. We found evidence for both these organelles in the single-cell-derived genome and transcriptome of P. schiedti, and corresponding vesicles were tentatively revealed in electron micrographs. In silico reconstructed MRO metabolism seems similar to that of M. balamuthi harboring respiratory complex II, electron-transferring flavoprotein, partial TCA cycle running presumably in reductive direction, pyruvate:ferredoxin oxidoreductase, [FeFe]-hydrogenases, glycine cleavage system, and sulfate activation pathway. The cell disposes with an expanded set of NIF enzymes for iron sulfur cluster assembly, but their localization remains unclear. Quite contrary, out of 67 predicted peroxisomal enzymes, only four were reported also in M. balamuthi, namely peroxisomal processing peptidase, nudix hydrolase, inositol 2-dehydrogenase, and D-lactate dehydrogenase. Other putative functions of peroxisomes could be pyridoxal 5I⍰-phosphate biosynthesis, amino acid and carbohydrate metabolism, and hydrolase activities. Future experimental evidence is necessary to define functions of this surprisingly enzyme-rich anaerobic peroxisome.Author summaryA major part of the microbial diversity cannot be cultured in isolation, and so it escapes from traditional ways of investigation. In this paper, we demonstrate the successful approach for generating good-quality genome and transcriptome drafts from a peculiar amoeba Pelomyxa schiedti using single-cell methods. P. schiedti is a member of Archamoebae clade harboring microaerobic protists including a free-living Mastigamoeba balamuthi and a human parasite Entamoeba histolytica. Mitochondria and peroxisomes represent two organelles that are most affected during adaptation to microoxic or anoxic environments. Mitochondria are known to transform to anaerobic mitochondria, hydrogenosomes, mitosomes, and various transition stages in between, all of which encompass different enzymatic capacity. Anaerobic peroxisomes have been first noticed in M. balamuthi, but their function remained unclear for now. Data obtained in this study were used for revealing the presence and for the detailed functional annotations of anaerobic derivates of mitochondria and peroxisomes in P. schiedti, which were corroborated by transmission electron microscopy.

Diplonemids are a group of flagellate protists, that belong to the phylum Euglenozoa alongside euglenids, symbiontids and kinetoplastids. They primarily inhabit marine environments, though are also found in freshwater lakes. Diplonemids have been considered as rare and unimportant eukaryotes for over a century, with only a handful of species described until recently. However, thanks to their unprecedented diversity and abundance in the world oceans, diplonemids now attract increased attention. Recent improvements in isolation and cultivation have enabled characterization of several new genera, warranting a re-examination of all available knowledge gathered so far. Here we summarize available data on diplonemids, focusing on the recent advances in the fields of diversity, ecology, genomics, metabolism, and endosymbionts. We illustrate the life stages of cultivated genera, and summarise all reported interspecies associations, which in turn suggest lifestyles of predation and parasitism. This review also includes the latest classification of diplonemids, with a taxonomic revision of the genus Diplonema. Ongoing efforts to sequence various diplonemids suggest the presence of large and complex genomes, which correlate with the metabolic versatility observed in the model species Paradiplonema papillatum. Finally, we highlight its successful transformation into one of few genetically tractable marine protists.

Diplonemids belong to the most diverse and abundant marine protists, which places them among the key players of the oceanic ecosystem. Under in vitro conditions, their best-known representative Diplonema papillatum accumulates in its cytoplasm a crystalline polymer. When grown under the nutrient-poor conditions, but not nutrient-rich conditions, D. papillatum synthesizes a β-1,3-glucan polymer, also known as paramylon. This phenomenon is unexpected, as it is in striking contrast to the accumulation of paramylon in euglenids, since these related flagellates synthesize this polymer solely under nutrient-rich conditions. The capacity of D. papillatum to store an energy source in the form of polysaccharides when the environment is poor in nutrients is unexpected and may contribute to the wide distribution of these protists in the ocean.

In this work, we studied the biochemical properties and evolutionary histories of catalase (CAT) and ascorbate peroxidase (APX), two central enzymes of reactive oxygen species detoxification, across the highly diverse clade Eugenozoa. This clade encompasses free-living phototrophic and heterotrophic flagellates, as well as obligate parasites of insects, vertebrates, and plants. We present evidence of several independent acquisitions of CAT by horizontal gene transfers and evolutionary novelties associated with the APX presence. We posit that Euglenozoa recruit these detoxifying enzymes for specific molecular tasks, such as photosynthesis in euglenids and membrane-bound peroxidase activity in kinetoplastids and some diplonemids.

Summary Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each rep- resenting a novel yet globally distributed and/or species-rich clade, and compare them with well- studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarento- lae. Differences in structure and activities of res- piratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phyloge- netic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for pur- poses other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypa- nosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.

Kinetoplast maxicircle DNA of trypanosomatids encodes eighteen proteins. RNA editing is required to confer translatability to mRNA for twelve of these. Sequence conservation of the predicted hydrophobic polypeptides indicates that they represent functional components of the respiratory chain. Yet, so far only two of those, cytochrome c oxidase subunit I and apocytochrome b of cytochrome c reductase, have been identified with biochemical methods. Here we report on identification of A6 subunit of F1FO ATPase encoded by a pan-edited mRNA in Trypanosoma brucei. The polypeptide was present among the (35)S-labeled mitochondrial translation products characterized by anomalous migration in denaturing 2D gels. It was identified as an ATPase subunit by co-migration with this complex in Blue Native 2D gels. A partial N-terminal sequence of the corresponding polypeptide present in the gel-purified ATPase complex from Leishmania tarentolae was consistent with the predicted A6 sequence.

Trypanosomatids are a very diverse group composed of monoxenous and dixenous parasites belonging to the excavate class Kinetoplastea. Here we studied the respiration of five monoxenous species (Blechomonas ayalai, Herpetomonas muscarum, H. samuelpessoai, Leptomonas pyrrhocoris and Sergeia podlipaevi) introduced into culture, each representing a novel yet globally distributed and/or species-rich clade, and compare them with well-studied flagellates Trypanosoma brucei, Phytomonas serpens, Crithidia fasciculata and Leishmania tarentolae. Differences in structure and activities of respiratory chain complexes, respiration and other biochemical parameters recorded under laboratory conditions reveal their substantial diversity, likely a reflection of different host environments. Phylogenetic relationships of the analysed trypanosomatids do not correlate with their biochemical parameters, with the differences within clades by far exceeding those among clades. As the S. podlipaevi canonical respiratory chain complexes have very low activities, we believe that its mitochondrion is utilised for purposes other than oxidative phosphorylation. Hence, the single reticulated mitochondrion of diverse trypanosomatids seems to retain multipotency, with the capacity to activate its individual components based on the host environment.