The divergent ER-mitochondria encounter structures (ERMES) are conserved in parabasalids but lost in several anaerobic lineages with hydrogenosomes.

BACKGROUND: The endoplasmic reticulum (ER)-mitochondria membrane contact sites (MCS) are extensively studied in aerobic eukaryotes; however, little is known about MCS in anaerobes with reduced forms of mitochondria named hydrogenosomes. In several eukaryotic lineages, the direct physical tether between ER and the outer mitochondrial membrane is formed by ER-mitochondria encounter structure (ERMES). The complex consists of four core proteins (Mmm1, Mmm2, Mdm12, and Mdm10) which are involved in phospholipid trafficking. Here we investigated ERMES distribution in organisms bearing hydrogenosomes and employed Trichomonas vaginalis as a model to estimate ERMES cellular localization, structure, and function. RESULTS: Homology searches revealed that Parabasalia-Anaeramoebae, anaerobic jakobids, and anaerobic fungi are lineages with hydrogenosomes that retain ERMES, while ERMES components were gradually lost in Fornicata, and are absent in Preaxostyla and Archamoebae. In T. vaginalis and other parabasalids, three ERMES components were found with the expansion of Mmm1. Immunofluorescence microscopy confirmed that Mmm1 localized in ER, while Mdm12 and Mmm2 were partially localized in hydrogenosomes. Pull-down assays and mass spectrometry of the ERMES components identified a parabasalid-specific Porin2 as a substitute for the Mdm10. ERMES modeling predicted a formation of a continuous hydrophobic tunnel of TvMmm1-TvMdm12-TvMmm2 that is anchored via Porin2 to the hydrogenosomal outer membrane. Phospholipid-ERMES docking and Mdm12-phospholipid dot-blot indicated that ERMES is involved in the transport of phosphatidylinositol phosphates. The absence of enzymes involved in hydrogenosomal phospholipid metabolism implies that ERMES is not involved in the exchange of substrates between ER and hydrogenosomes but in the unidirectional import of phospholipids into hydrogenosomal membranes. CONCLUSIONS: Our investigation demonstrated that ERMES mediates ER-hydrogenosome interactions in parabasalid T. vaginalis, while the complex was lost in several other lineages with hydrogenosomes.

Double-Stranded RNA Viruses Are Released From Trichomonas vaginalis Inside Small Extracellular Vesicles and Modulate the Exosomal Cargo.

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.

Cytidine nucleoside analog is an effective antiviral drug against Trichomonasvirus.

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.