Trichomonas vaginalis is a parasitic protist that infects the human urogenital tract. During the infection, trichomonads adhere to the host mucosa, acquire nutrients from the vaginal/prostate environment, and release small extracellular vesicles (sEVs) that contribute to the trichomonad adherence and modulate the host-parasite communication. Approximately 40-70% of T. vaginalis strains harbor a double-stranded RNA virus called Trichomonasvirus (TVV). Naked TVV particles have the potential to stimulate a proinflammatory response in human cells, however, the mode of TVV release from trichomonads to the environment is not clear. In this report, we showed for the first time that TVV particles are released from T. vaginalis cells within sEVs. The sEVs loaded with TVV stimulated a higher proinflammatory response of human HaCaT cells in comparison to sEVs from TVV negative parasites. Moreover, a comparison of T. vaginalis isogenic TVV plus and TVV minus clones revealed a significant impact of TVV infection on the sEV proteome and RNA cargo. Small EVs from TVV positive trichomonads contained 12 enriched and 8 unique proteins including membrane-associated BspA adhesine, and about a 2.5-fold increase in the content of small regulatory tsRNA. As T. vaginalis isolates are frequently infected with TVV, the release of TVV via sEVs to the environment represents an important factor with the potential to enhance inflammation-related pathogenesis during trichomoniasis.
- Klíčová slova
- TVV, Trichomonasvirus, exosome, extracellular vesicle, proteomics, tsRNA,
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Trichomonas vaginalis is the causative agent of a sexually transmitted disease in humans. The virulence of the parasite depends on multiple factors, including the presence of endosymbiotic dsRNA viruses. The presence of Trichomonasviruses (TVV) was associated with more severe genital symptoms, increased proinflammatory host reactions, and modulated parasite sensitivity to metronidazole. However, no efficient antiviral drugs are available against TVV to derive isogenic TVV-positive and TVV-negative cell lines that are essential for investigations of the TVV impact on T. vaginalis biology. METHODS: 7-Deaza-2'-C-methyladenosine (7d2CMA) and 2'-C-methylcytidine (2CMC) were used for TVV inhibitory assay. TVV replication was monitored using quantitative reverse transcription PCR (RT qPCR) and western blotting. Modeling of TVV1 RNA-dependent RNA polymerase (RdRp) was performed to visualize the inhibitor-RdRp interaction. Susceptibility to metronidazole was performed under aerobic and anaerobic conditions. RESULTS: We demonstrated that 2CMC but not 7d2CMA is a potent inhibitor of TVV replication. Molecular modeling suggested that the RdRp active site can accommodate 2CMC in the active triphosphate nucleotide form. The effect of 2CMC was shown on strains infected with a single and multiple TVV species. The optimal 2CMC concentration (10 μM) demonstrated strong selectivity for TVVs over trichomonad growth. The presence of TVV has no effect on T. vaginalis metronidazole susceptibility in derived isogenic cell lines. CONCLUSIONS: 2CMC acts against TVVs and represents a new inhibitor against Totiviridae viruses. Our isogenic clones are now available for further studies of various aspects of T. vaginalis biology related to TVV infection.
- Klíčová slova
- Capsid protein, Nucleoside analog, RNA dependent RNA polymerase, TVV, Trichomonasvirus,
- MeSH
- antivirové látky farmakologie MeSH
- cytidin farmakologie MeSH
- lidé MeSH
- metronidazol farmakologie MeSH
- nukleosidy farmakologie MeSH
- paraziti * MeSH
- RNA-dependentní RNA-polymerasa MeSH
- RNA-viry * genetika MeSH
- Totiviridae * genetika MeSH
- Trichomonas vaginalis * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antivirové látky MeSH
- cytidin MeSH
- metronidazol MeSH
- nukleosidy MeSH
- RNA-dependentní RNA-polymerasa MeSH
Accumulated evidence suggests that the endosymbiotic Trichomonasvirus (TVV) may play a role in the pathogenesis and drug susceptibility of Trichomonas vaginalis. Several reports have shown that extracellular vesicles (EVs) released from TVV-positive (TVV+) trichomonads can modulate the immune response in human vaginal epithelial cells and animal models. These results prompted us to examine whether EVs released from TVV+ isolates contained TVV. We isolated small extracellular vesicles (sEVs) from six T. vaginalis isolates that were either TVV free (ATCC 50143), harbored a single (ATCC 30236, ATCC 30238, T1), two (ATCC PRA-98), or three TVV subspecies (ATCC 50148). The presence of TVV subspecies in the six isolates was observed using reverse transcription-polymerase chain reaction (RT-PCR). Transmission electron microscopy (TEM) confirmed the presence of cup-shaped sEVs with a size range from 30-150 nm. Trichomonas vaginalis tetraspanin (TvTSP1; TVAG_019180), the classical exosome marker, was identified in all the sEV preparations. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that all the sEVs isolated from TVV+ isolates contain viral capsid proteins derived from the same TVV subspecies in that isolate as demonstrated by RT-PCR. To provide more comprehensive information on the TVV subspecies population in other T. vaginalis isolates, we investigated the distribution of TVV subspecies in twenty-four isolates by mining the New-Generation Sequencing (NGS) RNAseq datasets. Our results should be beneficial for future studies investigating the role of TVV on the pathogenicity of T. vaginalis and the possible transmission of virus subspecies among different isolates via sEVs.
- Klíčová slova
- New-Generation Sequencing, Trichomonas vaginalis, Trichomonasvirus, extracellular vesicles, proteomics,
- MeSH
- chromatografie kapalinová MeSH
- dvouvláknová RNA MeSH
- extracelulární vezikuly * genetika MeSH
- RNA-viry * genetika MeSH
- tandemová hmotnostní spektrometrie MeSH
- Trichomonas vaginalis * genetika MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- dvouvláknová RNA MeSH
Trichomonads, represented by the highly prevalent sexually transmitted human parasite Trichomonas vaginalis, are anaerobic eukaryotes with hydrogenosomes in the place of the standard mitochondria. Hydrogenosomes form indispensable FeS-clusters, synthesize ATP, and release molecular hydrogen as a waste product. Hydrogen formation is catalyzed by [FeFe] hydrogenase, the hallmark enzyme of all hydrogenosomes found in various eukaryotic anaerobes. Eukaryotic hydrogenases were originally thought to be exclusively localized within organelles, but today few eukaryotic anaerobes are known that possess hydrogenase in their cytosol. We identified a thus-far unknown hydrogenase in T. vaginalis cytosol that cannot use ferredoxin as a redox partner but can use cytochrome b5 as an electron acceptor. Trichomonads overexpressing the cytosolic hydrogenase, while maintaining the carbon flux through hydrogenosomes, show decreased excretion of hydrogen and increased excretion of methylated alcohols, suggesting that the cytosolic hydrogenase uses the hydrogen gas as a source of reducing power for the reactions occurring in the cytoplasm and thus accounts for the overall redox balance. This is the first evidence of hydrogen uptake in a eukaryote, although further work is needed to confirm it. Assembly of the catalytic center of [FeFe] hydrogenases (H-cluster) requires the activity of three dedicated maturases, and these proteins in T. vaginalis are exclusively localized in hydrogenosomes, where they participate in the maturation of organellar hydrogenases. Despite the different subcellular localization of cytosolic hydrogenase and maturases, the H-cluster is present in the cytosolic enzyme, suggesting the existence of an alternative mechanism of H-cluster assembly.
- Klíčová slova
- H-cluster, Trichomonas, cytosolic hydrogenase, hydrogenosome,
- MeSH
- cytosol metabolismus MeSH
- ferredoxiny metabolismus MeSH
- hydrogenasa * metabolismus MeSH
- Trichomonas vaginalis * enzymologie ultrastruktura MeSH
- vodík * metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ferredoxiny MeSH
- hydrogenasa * MeSH
- vodík * MeSH
Entamoeba histolytica is believed to be devoid of peroxisomes, like most anaerobic protists. In this work, we provided the first evidence that peroxisomes are present in E. histolytica, although only seven proteins responsible for peroxisome biogenesis (peroxins) were identified (Pex1, Pex6, Pex5, Pex11, Pex14, Pex16, and Pex19). Targeting matrix proteins to peroxisomes is reduced to the PTS1-dependent pathway mediated via the soluble Pex5 receptor, while the PTS2 receptor Pex7 is absent. Immunofluorescence microscopy showed that peroxisomal markers (Pex5, Pex14, Pex16, Pex19) are present in vesicles distinct from mitosomes, the endoplasmic reticulum, and the endosome/phagosome system, except Pex11, which has dual localization in peroxisomes and mitosomes. Immunoelectron microscopy revealed that Pex14 localized to vesicles of approximately 90-100 nm in diameter. Proteomic analyses of affinity-purified peroxisomes and in silico PTS1 predictions provided datasets of 655 and 56 peroxisomal candidates, respectively; however, only six proteins were shared by both datasets, including myo-inositol dehydrogenase (myo-IDH). Peroxisomal NAD-dependent myo-IDH appeared to be a dimeric enzyme with high affinity to myo-inositol (Km 0.044 mM) and can utilize also scyllo-inositol, D-glucose and D-xylose as substrates. Phylogenetic analyses revealed that orthologs of myo-IDH with PTS1 are present in E. dispar, E. nutalli and E. moshkovskii but not in E. invadens, and form a monophyletic clade of mostly peroxisomal orthologs with free-living Mastigamoeba balamuthi and Pelomyxa schiedti. The presence of peroxisomes in E. histolytica and other archamoebae breaks the paradigm of peroxisome absence in anaerobes and provides a new potential target for the development of antiparasitic drugs.
- MeSH
- anaerobióza MeSH
- Entamoeba histolytica metabolismus MeSH
- fylogeneze MeSH
- inositol metabolismus MeSH
- mutace * MeSH
- peroxiny metabolismus MeSH
- peroxizomální cílové signály * MeSH
- peroxizomy metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- inositol MeSH
- peroxiny MeSH
- peroxizomální cílové signály * MeSH
- protozoální proteiny MeSH
