The endobiotic flagellate Monocercomonoides exilis is the only known eukaryote to have lost mitochondria and all its associated proteins in its evolutionary past. This final stage of the mitochondrial evolutionary pathway may serve as a model to explain events at their very beginning such as the initiation of protein import. We have assessed the capability of proteins from this eukaryote to enter emerging mitochondria using a specifically designed in vitro assay. Hydrogenosomes (reduced mitochondria) of Trichomonas vaginalis were incubated with a soluble protein pool derived from a cytosolic fraction of M. exilis, and proteins entering hydrogenosomes were subsequently detected by mass spectrometry. The assay detected 19 specifically and reproducibly imported proteins, and in 14 cases the import was confirmed by the overexpression of their tagged version in T. vaginalis. In most cases, only a small portion of the signal reached the hydrogenosomes, suggesting specific but inefficient transport. Most of these proteins represent enzymes of carbon metabolism, and none exhibited clear signatures of proteins targeted to hydrogenosomes or mitochondria, which is consistent with their inefficient import. The observed phenomenon may resemble a primaeval type of protein import which might play a role in the establishment of the organelle and shaping of its proteome in the initial stages of endosymbiosis.

• Klíčová slova
• evolution of protein targeting, hydrogenosome, mitochondrion-free eukaryote, protein import,
• MeSH
• Eukaryota * metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• organely chemie   metabolismus   MeSH
• protozoální proteiny * metabolismus   MeSH
• transport proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• protozoální proteiny * MeSH

The notion that mitochondria cannot be lost was shattered with the report of an oxymonad Monocercomonoides exilis, the first eukaryote arguably without any mitochondrion. Yet, questions remain about whether this extends beyond the single species and how this transition took place. The Oxymonadida is a group of gut endobionts taxonomically housed in the Preaxostyla which also contains free-living flagellates of the genera Trimastix and Paratrimastix. The latter two taxa harbour conspicuous mitochondrion-related organelles (MROs). Here we report high-quality genome and transcriptome assemblies of two Preaxostyla representatives, the free-living Paratrimastix pyriformis and the oxymonad Blattamonas nauphoetae. We performed thorough comparisons among all available genomic and transcriptomic data of Preaxostyla to further decipher the evolutionary changes towards amitochondriality, endobiosis, and unstacked Golgi. Our results provide insights into the metabolic and endomembrane evolution, but most strikingly the data confirm the complete loss of mitochondria for all three oxymonad species investigated (M. exilis, B. nauphoetae, and Streblomastix strix), suggesting the amitochondriate status is common to a large part if not the whole group of Oxymonadida. This observation moves this unique loss to 100 MYA when oxymonad lineage diversified.

• MeSH
• Eukaryota * genetika   MeSH
• fylogeneze MeSH
• genomika MeSH
• mitochondrie genetika   MeSH
• Oxymonadida * genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

All eukaryotic organisms contain mitochondria or organelles that evolved from the same endosymbiotic event like classical mitochondria. Organisms inhabiting low oxygen environments often contain mitochondrial derivates known as hydrogenosomes, mitosomes or neutrally as mitochondrion-like organelles. The detailed investigation has shown unexpected evolutionary plasticity in the biochemistry and protein composition of these organelles in various protists. We investigated the mitochondrion-like organelle in Trimastix pyriformis, a free-living member of one of the three lineages of anaerobic group Metamonada. Using 454 sequencing we have obtained 7 037 contigs from its transcriptome and on the basis of sequence homology and presence of N-terminal extensions we have selected contigs coding for proteins that putatively function in the organelle. Together with the results of a previous transcriptome survey, the list now consists of 23 proteins - mostly enzymes involved in amino acid metabolism, transporters and maturases of proteins and transporters of metabolites. We have no evidence of the production of ATP in the mitochondrion-like organelle of Trimastix but we have obtained experimental evidence for the presence of enzymes of the glycine cleavage system (GCS), which is part of amino acid metabolism. Using homologous antibody we have shown that H-protein of GCS localizes into vesicles in the cell of Trimastix. When overexpressed in yeast, H- and P-protein of GCS and cpn60 were transported into mitochondrion. In case of H-protein we have demonstrated that the first 16 amino acids are necessary for this transport. Glycine cleavage system is at the moment the only experimentally localized pathway in the mitochondrial derivate of Trimastix pyriformis.

• MeSH
• Eukaryota genetika   metabolismus   MeSH
• exprese genu MeSH
• H-protein systému glycinového štěpení genetika   metabolismus   MeSH
• mitochondriální proteiny genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• multienzymové komplexy genetika   metabolismus   MeSH
• organely metabolismus   MeSH
• oxidoreduktasy aminokyselin genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• transferasy genetika   metabolismus   MeSH
• transportní proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glycine cleavage system MeSH Prohlížeč
• H-protein systému glycinového štěpení MeSH
• mitochondriální proteiny MeSH
• multienzymové komplexy MeSH
• oxidoreduktasy aminokyselin MeSH
• transferasy MeSH
• transportní proteiny MeSH

Archamoebae comprises free-living or endobiotic amoebiform protists that inhabit anaerobic or microaerophilic environments and possess mitochondrion-related organelles (MROs) adapted to function anaerobically. We compared in silico reconstructed MRO proteomes of eight species (six genera) and found that the common ancestor of Archamoebae possessed very few typical components of the protein translocation machinery, electron transport chain and tricarboxylic acid cycle. On the other hand, it contained a sulphate activation pathway and bacterial iron-sulphur (Fe-S) assembly system of MIS-type. The metabolic capacity of the MROs, however, varies markedly within this clade. The glycine cleavage system is widely conserved among Archamoebae, except in Entamoeba, probably owing to its role in catabolic function or one-carbon metabolism. MRO-based pyruvate metabolism was dispensed within subgroups Entamoebidae and Rhizomastixidae, whereas sulphate activation could have been lost in isolated cases of Rhizomastix libera, Mastigamoeba abducta and Endolimax sp. The MIS (Fe-S) assembly system was duplicated in the common ancestor of Mastigamoebidae and Pelomyxidae, and one of the copies took over Fe-S assembly in their MRO. In Entamoebidae and Rhizomastixidae, we hypothesize that Fe-S cluster assembly in both compartments may be facilitated by dual localization of the single system. We could not find evidence for changes in metabolic functions of the MRO in response to changes in habitat; it appears that such environmental drivers do not strongly affect MRO reduction in this group of eukaryotes.

• Klíčová slova
• anaerobiosis, comparative genomics, mitochondrion-related organelles, reductive evolution,
• MeSH
• anaerobióza MeSH
• Eukaryota * MeSH
• mitochondrie * genetika   MeSH
• sírany MeSH
• železo MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• sírany MeSH
• železo MeSH

Parasites are widespread and diverse in oceanic plankton and many of them infect single-celled algae for survival. How these parasites develop and scavenge energy within the host and how the cellular organization and metabolism of the host is altered remain open questions. Combining quantitative structural and chemical imaging with time-resolved transcriptomics, we unveil dramatic morphological and metabolic changes of the marine parasite Amoebophrya (Syndiniales) during intracellular infection, particularly following engulfment and digestion of nutrient-rich host chromosomes. Changes include a sequential acristate and cristate mitochondrion with a 200-fold increase in volume, a 13-fold increase in nucleus volume, development of Golgi apparatus and a metabolic switch from glycolysis (within the host) to TCA (free-living dinospore). Similar changes are seen in apicomplexan parasites, thus underlining convergent traits driven by metabolic constraints and the infection cycle. In the algal host, energy-producing organelles (plastid, mitochondria) remain relatively intact during most of the infection. We also observed that sugar reserves diminish while lipid droplets increase. Rapid infection of the host nucleus could be a "zombifying" strategy, allowing the parasite to digest nutrient-rich chromosomes and escape cytoplasmic defense, whilst benefiting from maintained carbon-energy production of the host cell.

• MeSH
• cukry MeSH
• Dinoflagellata * MeSH
• mikrořasy * MeSH
• paraziti * MeSH
• uhlík MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cukry MeSH
• uhlík MeSH

Trimastigids are free-living, anaerobic protists that are closely related to the symbiotic oxymonads, forming together the taxon Preaxostyla (Excavata: Metamonada). We isolated fourteen new strains morphologically corresponding to two species assigned to Trimastix (until now the only genus of trimastigids), Trimastix marina and Trimastix pyriformis. Unexpectedly, marine strains of Trimastix marina branch separately from freshwater strains of this morphospecies in SSU rRNA gene trees, and instead form the sister group of all other Preaxostyla. This position is confirmed by three-gene phylogenies. Ultrastructural examination of a marine isolate of Trimastix marina demonstrates a combination of trimastigid-like features (e.g. preaxostyle-like I fibre) and ancestral characters (e.g. absence of thickened flagellar vane margins), consistent with inclusion of marine T. marina within Preaxostyla, but also supporting its distinctiveness from 'freshwater T. marina' and its deep-branching position within Preaxostyla. Since these results indicate paraphyly of Trimastix as currently understood, we transfer the other better-studied trimastigids to Paratrimastix n. gen. and Paratrimastigidae n. fam. The freshwater form previously identified as T. marina is described as Paratrimastix eleionoma n. sp., and Trimastix pyriformis becomes Paratrimastix pyriformis n. comb. Because of its phylogenetic position, 'true' Trimastix is potentially important for understanding the evolution of mitochondrion-related organelles in metamonads.

• Klíčová slova
• Anaerobe, cytoskeleton, flagellar apparatus, oxymonads, phylogeny, ultrastructure.,
• MeSH
• druhová specificita MeSH
• Eukaryota klasifikace   genetika   izolace a purifikace   ultrastruktura   MeSH
• flagella ultrastruktura   MeSH
• fylogeneze * MeSH
• organely ultrastruktura   MeSH
• RNA ribozomální 18S genetika   MeSH
• vodní organismy klasifikace   genetika   izolace a purifikace   ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• RNA ribozomální 18S MeSH

We have studied topoisomerase II (topo II) in the cells of Bodo saltans, a free-living bodonid (Kinetoplastida). Phylogenetic analysis based on the sequence of the entire topo II gene, which is a single-copy gene, confirmed that B. saltans is a predecessor of parasitic trypanosomatids. Antibodies generated against either an overexpressed unique C-terminal region of topo II or a synthetic oligopeptide derived from the same region did not cross-react with cell lysates of related trypanosomatids, while they recognized a single specific band in the B. saltans lysate. Immunolocalization experiments using both antibodies showed that topo II is evenly dispersed throughout the kinetoplast. This is in striking difference from the localization of topo II in other flagellates, where it occurs in two antipodal centers flanking the kinetoplast disk. Moreover, the same topo II has a distinct localization in multiple loci at the periphery of the nucleus of B. saltans. With a minicircle probe derived from the conserved region we have shown that all relaxed non-catenated minicircles are confined to the globular kinetoplast DNA bundle. Therefore, in the mitochondrion of this primitive eukaryote topo II does not catenate relaxed DNA circles into a network in vivo, while a decatenating activity is present in partially purified cell lysates.

• MeSH
• buněčné jádro enzymologie   MeSH
• DNA primery MeSH
• DNA-topoisomerasy typu II genetika   metabolismus   MeSH
• hybridizace in situ MeSH
• imunohistochemie MeSH
• Kinetoplastida enzymologie   MeSH
• lidé MeSH
• mitochondrie enzymologie   MeSH
• molekulární sekvence - údaje MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• Southernův blotting MeSH
• subcelulární frakce enzymologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• DNA-topoisomerasy typu II MeSH

Naegleria gruberi is a free-living amoeba, closely related to the human pathogen Naegleria fowleri, the causative agent of the deadly human disease primary amoebic meningoencephalitis. Herein, we investigated the effect of iron limitation on different aspects of N. gruberi metabolism. Iron metabolism is among the most conserved pathways found in all eukaryotes. It includes the delivery, storage and utilisation of iron in many cell processes. Nevertheless, most of the iron metabolism pathways of N. gruberi are still not characterised, even though iron balance within the cell is crucial. We found a single homolog of ferritin in the N. gruberi genome and showed its localisation in the mitochondrion. Using comparative mass spectrometry, we identified 229 upregulated and 184 down-regulated proteins under iron-limited conditions. The most down-regulated protein under iron-limited conditions was hemerythrin, and a similar effect on the expression of hemerythrin was found in N. fowleri. Among the other down-regulated proteins were [FeFe]-hydrogenase and its maturase HydG and several heme-containing proteins. The activities of [FeFe]-hydrogenase, as well as alcohol dehydrogenase, were also decreased by iron deficiency. Our results indicate that N. gruberi is able to rearrange its metabolism according to iron availability, prioritising mitochondrial pathways. We hypothesise that the mitochondrion is the center for iron homeostasis in N. gruberi, with mitochondrially localised ferritin as a potential key component of this process.

• Klíčová slova
• Ferritin, Hemerythrin, Iron, Metabolism, Naegleria,
• MeSH
• anaerobióza MeSH
• biologický transport MeSH
• chromatografie kapalinová MeSH
• hemerythrin metabolismus   MeSH
• hmotnostní spektrometrie MeSH
• Naegleria metabolismus   MeSH
• protozoální proteiny genetika   MeSH
• regulace genové exprese enzymů účinky léků   MeSH
• spotřeba kyslíku MeSH
• železo metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hemerythrin MeSH
• protozoální proteiny MeSH
• železo MeSH

The transition of free-living organisms to parasitic organisms is a mysterious process that occurs in all major eukaryotic lineages. Parasites display seemingly unique features associated with their pathogenicity; however, it is important to distinguish ancestral preconditions to parasitism from truly new parasite-specific functions. Here, we sequenced the genome and transcriptome of anaerobic free-living Mastigamoeba balamuthi and performed phylogenomic analysis of four related members of the Archamoebae, including Entamoeba histolytica, an important intestinal pathogen of humans. We aimed to trace gene histories throughout the adaptation of the aerobic ancestor of Archamoebae to anaerobiosis and throughout the transition from a free-living to a parasitic lifestyle. These events were associated with massive gene losses that, in parasitic lineages, resulted in a reduction in structural features, complete losses of some metabolic pathways, and a reduction in metabolic complexity. By reconstructing the features of the common ancestor of Archamoebae, we estimated preconditions for the evolution of parasitism in this lineage. The ancestor could apparently form chitinous cysts, possessed proteolytic enzyme machinery, compartmentalized the sulfate activation pathway in mitochondrion-related organelles, and possessed the components for anaerobic energy metabolism. After the split of Entamoebidae, this lineage gained genes encoding surface membrane proteins that are involved in host-parasite interactions. In contrast, gene gains identified in the M. balamuthi lineage were predominantly associated with polysaccharide catabolic processes. A phylogenetic analysis of acquired genes suggested an essential role of lateral gene transfer in parasite evolution (Entamoeba) and in adaptation to anaerobic aquatic sediments (Mastigamoeba).

• Klíčová slova
• Mastigamoeba, Archamoebae, chitinous cysts, evolution of parasitism, lateral gene transfer, pathway complexity,
• MeSH
• anaerobióza genetika   MeSH
• Archamoebae genetika   metabolismus   MeSH
• biologická adaptace genetika   MeSH
• biologická evoluce * MeSH
• délka genomu MeSH
• Entamoeba histolytica genetika   MeSH
• genom protozoální * MeSH
• paraziti genetika   MeSH
• přenos genů horizontální MeSH
• transkriptom MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.

• Klíčová slova
• Naegleria, hydrogen hypothesis, hydrogenase, maturases, mitochondrial evolution,
• MeSH
• cytosol enzymologie   MeSH
• hydrogenasa genetika   metabolismus   MeSH
• mitochondrie genetika   metabolismus   MeSH
• Naegleria enzymologie   genetika   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• vodík metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hydrogenasa MeSH
• protozoální proteiny MeSH
• vodík MeSH