The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.
- MeSH
- cystein metabolismus MeSH
- cysteinové proteasy * metabolismus MeSH
- fagozomy metabolismus MeSH
- lidé MeSH
- lyzozomy metabolismus MeSH
- proteasy metabolismus MeSH
- proteomika MeSH
- Trichomonas vaginalis * metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The adaptation of eukaryotic cells to anaerobic conditions is reflected by substantial changes to mitochondrial metabolism and functional reduction. Hydrogenosomes belong among the most modified mitochondrial derivative and generate molecular hydrogen concomitant with ATP synthesis. The reduction of mitochondria is frequently associated with loss of peroxisomes, which compartmentalize pathways that generate reactive oxygen species (ROS) and thus protect against cellular damage. The biogenesis and function of peroxisomes are tightly coupled with mitochondria. These organelles share fission machinery components, oxidative metabolism pathways, ROS scavenging activities, and some metabolites. The loss of peroxisomes in eukaryotes with reduced mitochondria is thus not unexpected. Surprisingly, we identified peroxisomes in the anaerobic, hydrogenosome-bearing protist Mastigamoeba balamuthi We found a conserved set of peroxin (Pex) proteins that are required for protein import, peroxisomal growth, and division. Key membrane-associated Pexs (MbPex3, MbPex11, and MbPex14) were visualized in numerous vesicles distinct from hydrogenosomes, the endoplasmic reticulum (ER), and Golgi complex. Proteomic analysis of cellular fractions and prediction of peroxisomal targeting signals (PTS1/PTS2) identified 51 putative peroxisomal matrix proteins. Expression of selected proteins in Saccharomyces cerevisiae revealed specific targeting to peroxisomes. The matrix proteins identified included components of acyl-CoA and carbohydrate metabolism and pyrimidine and CoA biosynthesis, whereas no components related to either β-oxidation or catalase were present. In conclusion, we identified a subclass of peroxisomes, named "anaerobic" peroxisomes that shift the current paradigm and turn attention to the reductive evolution of peroxisomes in anaerobic organisms.
- MeSH
- anaerobióza MeSH
- Archamoebae genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- oxidace-redukce MeSH
- peroxiny genetika metabolismus MeSH
- peroxizomy genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- reaktivní formy kyslíku metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tail-anchored (TA) proteins are membrane proteins that are found in all domains of life. They consist of an N-terminal domain that performs various functions and a single transmembrane domain (TMD) near the C-terminus. In eukaryotes, TA proteins are targeted to the membranes of mitochondria, the endoplasmic reticulum (ER), peroxisomes and in plants, chloroplasts. The targeting of these proteins to their specific destinations correlates with the properties of the C-terminal domain, mainly the TMD hydrophobicity and the net charge of the flanking regions. Trichomonas vaginalis is a human parasite that has adapted to oxygen-poor environment. This adaptation is reflected by the presence of highly modified mitochondria (hydrogenosomes) and the absence of peroxisomes. The proteome of hydrogenosomes is considerably reduced; however, our bioinformatic analysis predicted 120 putative hydrogenosomal TA proteins. Seven proteins were selected to prove their localization. The elimination of the net positive charge in the C-tail of the hydrogenosomal TA4 protein resulted in its dual localization to hydrogenosomes and the ER, causing changes in ER morphology. Domain mutation and swap experiments with hydrogenosomal (TA4) and ER (TAPDI) proteins indicated that the general principles for specific targeting are conserved across eukaryotic lineages, including T. vaginalis; however, there are also significant lineage-specific differences.
- MeSH
- multienzymové komplexy metabolismus MeSH
- mutační analýza DNA MeSH
- mutantní proteiny genetika metabolismus MeSH
- organely metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- transport proteinů MeSH
- Trichomonas vaginalis enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Mitochondria originated from proteobacterial endosymbionts, and their transition to organelles was tightly linked to establishment of the protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, an unexpected diversity of subunits between eukaryotic lineages has been predicted. However, experimental knowledge is limited to a few organisms, and so far, it remains unsettled whether the triplet-pore or the twin-pore structure is the generic form of TOM complex. Here, we analysed the TOM complex in hydrogenosomes, a metabolically specialised anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. We demonstrate that the highly divergent β-barrel T. vaginalis TOM (TvTom)40-2 forms a translocation channel to conduct hydrogenosomal protein import. TvTom40-2 is present in high molecular weight complexes, and their analysis revealed the presence of four tail-anchored (TA) proteins. Two of them, Tom36 and Tom46, with heat shock protein (Hsp)20 and tetratricopeptide repeat (TPR) domains, can bind hydrogenosomal preproteins and most likely function as receptors. A third subunit, Tom22-like protein, has a short cis domain and a conserved Tom22 transmembrane segment but lacks a trans domain. The fourth protein, hydrogenosomal outer membrane protein 19 (Homp19) has no known homology. Furthermore, our data indicate that TvTOM is associated with sorting and assembly machinery (Sam)50 that is involved in β-barrel assembly. Visualisation of TvTOM by electron microscopy revealed that it forms three pores and has an unconventional skull-like shape. Although TvTOM seems to lack Tom7, our phylogenetic profiling predicted Tom7 in free-living excavates. Collectively, our results suggest that the triplet-pore TOM complex, composed of three conserved subunits, was present in the last common eukaryotic ancestor (LECA), while receptors responsible for substrate binding evolved independently in different eukaryotic lineages.
- MeSH
- fylogeneze MeSH
- membránové proteiny metabolismus MeSH
- membránové transportní proteiny metabolismus MeSH
- mitochondrie metabolismus MeSH
- organely MeSH
- transport proteinů fyziologie MeSH
- transportní proteiny mitochondriální membrány metabolismus MeSH
- transportní proteiny genetika metabolismus fyziologie MeSH
- Trichomonas vaginalis metabolismus patogenita fyziologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The secretion of virulence factors by parasitic protists into the host environment plays a fundamental role in multifactorial host-parasite interactions. Several effector proteins are known to be secreted by Trichomonas vaginalis, a human parasite of the urogenital tract. However, a comprehensive profiling of the T. vaginalis secretome remains elusive, as do the mechanisms of protein secretion. In this study, we used high-resolution label-free quantitative MS to analyze the T. vaginalis secretome, considering that secretion is a time- and temperature-dependent process, to define the cutoff for secreted proteins. In total, we identified 2 072 extracellular proteins, 89 of which displayed significant quantitative increases over time at 37 °C. These 89 bona fide secreted proteins were sorted into 13 functional categories. Approximately half of the secreted proteins were predicted to possess transmembrane helixes. These proteins mainly include putative adhesins and leishmaniolysin-like metallopeptidases. The other half of the soluble proteins include several novel potential virulence factors, such as DNaseII, pore-forming proteins, and β-amylases. Interestingly, current bioinformatic tools predicted the secretory signal in only 18% of the identified T. vaginalis-secreted proteins. Therefore, we used β-amylases as a model to investigate the T. vaginalis secretory pathway. We demonstrated that two β-amylases (BA1 and BA2) are transported via the classical endoplasmic reticulum-to-Golgi pathways, and in the case of BA1, we showed that the protein is glycosylated with multiple N-linked glycans of Hex5HexNAc2 structure. The secretion was inhibited by brefeldin A but not by FLI-06. Another two β-amylases (BA3 and BA4), which are encoded in the T. vaginalis genome but absent from the secretome, were targeted to the lysosomal compartment. Collectively, under defined in vitro conditions, our analysis provides a comprehensive set of constitutively secreted proteins that can serve as a reference for future comparative studies, and it provides the first information about the classical secretory pathway in this parasite.
BACKGROUND: Cysteine peptidases of clan CA, family C1 account for a major part of proteolytic activity in the haematophagous monogenean Eudiplozoon nipponicum. The full spectrum of cysteine cathepsins is, however, unknown and their particular biochemical properties, tissue localisation, and involvement in parasite-host relationships are yet to be explored. METHODS: Sequences of cathepsins L and B (EnCL and EnCB) were mined from E. nipponicum transcriptome and analysed bioinformatically. Genes encoding two EnCLs and one EnCB were cloned and recombinant proteins produced in vitro. The enzymes were purified by chromatography and their activity towards selected substrates was characterised. Antibodies and specific RNA probes were employed for localisation of the enzymes/transcripts in tissues of E. nipponicum adults. RESULTS: Transcriptomic analysis revealed a set of ten distinct transcripts that encode EnCLs. The enzymes are significantly variable in their active sites, specifically the S2 subsites responsible for interaction with substrates. Some of them display unusual structural features that resemble cathepsins B and S. Two recombinant EnCLs had different pH activity profiles against both synthetic and macromolecular substrates, and were able to hydrolyse blood proteins and collagen I. They were localised in the haematin cells of the worm's digestive tract and in gut lumen. The EnCB showed similarity with cathepsin B2 of Schistosoma mansoni. It displays molecular features typical of cathepsins B, including an occluding loop responsible for its exopeptidase activity. Although the EnCB hydrolysed haemoglobin in vitro, it was localised in the vitelline cells of the parasite and not the digestive tract. CONCLUSIONS: To our knowledge, this study represents the first complex bioinformatic and biochemical characterisation of cysteine peptidases in a monogenean. Eudiplozoon nipponicum adults express a variety of CLs, which are the most abundant peptidases in the worms. The properties and localisation of the two heterologously expressed EnCLs indicate a central role in the (partially extracellular?) digestion of host blood proteins. High variability of substrate-binding sites in the set of EnCLs suggests specific adaptation to a range of biological processes that require proteolysis. Surprisingly, a single cathepsin B is expressed by the parasite and it is not involved in digestion, but probably in vitellogenesis.
- MeSH
- gastrointestinální trakt parazitologie MeSH
- hydrolýza MeSH
- interakce hostitele a parazita MeSH
- kapři parazitologie MeSH
- kathepsin B chemie genetika izolace a purifikace metabolismus MeSH
- kathepsin L chemie genetika izolace a purifikace metabolismus MeSH
- proteolýza MeSH
- rekombinantní proteiny analýza genetika izolace a purifikace MeSH
- stanovení celkové genové exprese MeSH
- Trematoda enzymologie genetika MeSH
- zavlečené druhy MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
p-Cresol and indole are volatile biologically active products of the bacterial degradation of tyrosine and tryptophan respectively. They are typically produced by bacteria in animal intestines, soil and various sediments. Here, we demonstrate that the free-living eukaryote Mastigamoeba balamuthi and its pathogenic relative Entamoeba histolytica produce significant amounts of indole via tryptophanase activity. Unexpectedly, M. balamuthi also produces p-cresol in concentrations that are bacteriostatic to non-p-cresol-producing bacteria. The ability of M. balamuthi to produce p-cresol, which has not previously been observed in any eukaryotic microbe, was gained due to the lateral acquisition of a bacterial gene for 4-hydroxyphenylacetate decarboxylase (HPAD). In bacteria, the genes for HPAD and the S-adenosylmethionine-dependent activating enzyme (AE) are present in a common operon. In M. balamuthi, HPAD displays a unique fusion with the AE that suggests the operon-mediated transfer of genes from a bacterial donor. We also clarified that the tyrosine-to-4-hydroxyphenylacetate conversion proceeds via the Ehrlich pathway. The acquisition of the bacterial HPAD gene may provide M. balamuthi a competitive advantage over other microflora in its native habitat.
- MeSH
- Archamoebae genetika MeSH
- Bacteria genetika MeSH
- bakteriální geny * MeSH
- indoly metabolismus MeSH
- karboxylyasy MeSH
- kresoly metabolismus MeSH
- operon MeSH
- přenos genů horizontální * MeSH
- S-adenosylmethionin metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: The Tim17 family of proteins plays a fundamental role in the biogenesis of mitochondria. Three Tim17 family proteins, Tim17, Tim22, and Tim23, are the central components of the widely conserved multi-subunit protein translocases, TIM23 and TIM22, which mediate protein transport across and into the inner mitochondrial membrane, respectively. In addition, several Tim17 family proteins occupy the inner and outer membranes of plastids. RESULTS: We have performed comprehensive sequence analyses on 5631 proteomes from all domains of life deposited in the Uniprot database. The analyses showed that the Tim17 family of proteins is much more diverse than previously thought and involves at least ten functionally and phylogenetically distinct groups of proteins. As previously shown, mitochondrial inner membrane accommodates prototypical Tim17, Tim22 and Tim23 and two Tim17 proteins, TIMMDC1 and NDUFA11, which participate in the assembly of complex I of the respiratory chain. In addition, we have identified Romo1/Mgr2 as Tim17 family member. The protein has been shown to control lateral release of substrates fromTIM23 complex in yeast and to participate in the production of reactive oxygen species in mammalian cells. Two peroxisomal proteins, Pmp24 and Tmem135, of so far unknown function also belong to Tim17 protein family. Additionally, a new group of Tim17 family proteins carrying a C-terminal coiled-coil domain has been identified predominantly in fungi. CONCLUSIONS: We have mapped the distribution of Tim17 family members in the eukaryotic supergroups and found that the mitochondrial Tim17, Tim22 and Tim23 proteins, as well as the peroxisomal Tim17 family proteins, were all likely to be present in the last eukaryotic common ancestor (LECA). Thus, kinetoplastid mitochondria previously identified as carrying a single Tim17protein family homologue are likely to be the outcome of a secondary reduction. The eukaryotic cell has modified mitochondrial Tim17 family proteins to mediate different functions in multiple cellular compartments including mitochondria, plastids and peroxisomes. Concerning the origin of Tim17 protein family, our analyses do not support the affiliation of the protein family and the component of bacterial amino acid permease. Thus, it is likely that Tim17 protein family is exclusive to eukaryotes. REVIEWERS: The article was reviewed by Michael Gray, Martijn Huynen and Kira Makarova.
The presence of mitochondria and related organelles in every studied eukaryote supports the view that mitochondria are essential cellular components. Here, we report the genome sequence of a microbial eukaryote, the oxymonad Monocercomonoides sp., which revealed that this organism lacks all hallmark mitochondrial proteins. Crucially, the mitochondrial iron-sulfur cluster assembly pathway, thought to be conserved in virtually all eukaryotic cells, has been replaced by a cytosolic sulfur mobilization system (SUF) acquired by lateral gene transfer from bacteria. In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily. This is the first example of a eukaryote lacking any form of a mitochondrion, demonstrating that this organelle is not absolutely essential for the viability of a eukaryotic cell.
BACKGROUND: Low iron bioavailability is a common feature of ocean surface water and therefore micro-algae developed original strategies to optimize iron uptake and metabolism. The marine picoeukaryotic green alga Ostreococcus tauri is a very good model for studying physiological and genetic aspects of the adaptation of the green algal lineage to the marine environment: it has a very compact genome, is easy to culture in laboratory conditions, and can be genetically manipulated by efficient homologous recombination. In this study, we aimed at characterizing the mechanisms of iron assimilation in O. tauri by combining genetics and physiological tools. Specifically, we wanted to identify and functionally characterize groups of genes displaying tightly orchestrated temporal expression patterns following the exposure of cells to iron deprivation and day/night cycles, and to highlight unique features of iron metabolism in O. tauri, as compared to the freshwater model alga Chalamydomonas reinhardtii. RESULTS: We used RNA sequencing to investigated the transcriptional responses to iron limitation in O. tauri and found that most of the genes involved in iron uptake and metabolism in O. tauri are regulated by day/night cycles, regardless of iron status. O. tauri lacks the classical components of a reductive iron uptake system, and has no obvious iron regulon. Iron uptake appears to be copper-independent, but is regulated by zinc. Conversely, iron deprivation resulted in the transcriptional activation of numerous genes encoding zinc-containing regulation factors. Iron uptake is likely mediated by a ZIP-family protein (Ot-Irt1) and by a new Fea1-related protein (Ot-Fea1) containing duplicated Fea1 domains. The adaptation of cells to iron limitation involved an iron-sparing response tightly coordinated with diurnal cycles to optimize cell functions and synchronize these functions with the day/night redistribution of iron orchestrated by ferritin, and a stress response based on the induction of thioredoxin-like proteins, of peroxiredoxin and of tesmin-like methallothionein rather than ascorbate. We briefly surveyed the metabolic remodeling resulting from iron deprivation. CONCLUSIONS: The mechanisms of iron uptake and utilization by O. tauri differ fundamentally from those described in C. reinhardtii. We propose this species as a new model for investigation of iron metabolism in marine microalgae.
- MeSH
- biologická adaptace MeSH
- Chlorophyta klasifikace genetika metabolismus MeSH
- Eukaryota genetika metabolismus MeSH
- fotoperioda MeSH
- fylogeneze MeSH
- fytoplankton genetika metabolismus MeSH
- fyziologický stres MeSH
- homeostáza MeSH
- měď metabolismus MeSH
- oxidace-redukce MeSH
- regulace genové exprese účinky záření MeSH
- rostlinné proteiny genetika metabolismus MeSH
- shluková analýza MeSH
- signální transdukce MeSH
- sloučeniny železa metabolismus MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vysoce účinné nukleotidové sekvenování MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
