Trichomoniasis, a globally distributed sexually transmitted infection, is caused by the urogenital parasite Trichomonas vaginalis Donné, 1836 affecting both women and men. The treatment of choice is metronidazole (MTZ). In the present study, 15 samples of vaginal discharge and urine were analysed by sequencing nitroreductase genes (ntr4 and ntr6). An in silico model was structured to illustrate the location of point mutations (PM) in the protein. The ntr4 gene presented four PMs: G76C (10/10), C213G (9/10), C318A (5/10) and G424A (1/10), while the ntr6 gene had eight PMs; G593A (13/13) the most frequent, G72T and G627C, both in 8/13. The PM C213G and A438T generated a stop codon causing a truncated nitroreductase 4 and 6 protein. Docking analysis demonstrated that some models had a decrease in binding affinity to MTZ (p < 0.0001). A high frequency of mutations was observed in the samples analysed that could be associated with resistance to MTZ in Chile.
- MeSH
- antiprotozoální látky farmakologie MeSH
- bodová mutace * MeSH
- léková rezistence * MeSH
- lidé MeSH
- metronidazol * farmakologie MeSH
- nitroreduktasy * genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- trichomonádová vaginitida parazitologie MeSH
- Trichomonas vaginalis * genetika účinky léků enzymologie MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Chile MeSH
The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.
- MeSH
- inhibitory proteasomu farmakologie chemie MeSH
- katalytická doména * MeSH
- proteasomový endopeptidasový komplex * metabolismus chemie MeSH
- protozoální proteiny chemie metabolismus antagonisté a inhibitory genetika MeSH
- substrátová specifita MeSH
- Trichomonas vaginalis * enzymologie MeSH
- Publikační typ
- časopisecké články MeSH
Roup dětský (Enterobius vermicularis, E. vermicularis) je aktuálně nejčastějším helmintem lidského střeva. Ze statistik Národní referenční laboratoře pro střevní parazitózy je zřejmé, že je v našich podmínkách téměř jediným zástupcem ze skupiny helmintů (1) (Tab. 1). Vyskytuje se, resp. diagnostikujeme tuto nákazu (enterobióza, oxyurióza) zejména u dětí. Vzhledem k rychlému dozrávání larev ve vajíčku (4–6 hod.), jejich nadprodukci (dospělá samička naklade 5 000–12 000 vajíček (2), (Obr. 1a, b, Obr. 2a, b) a vysoké odolnosti vajíček vůči vnějším vlivům předpokládám současný výskyt u většiny členů rodiny, včetně dospělých. Vzhledem k poměrně vysokému výskytu patogenní trichomonády Dientamoeba fragilis (D. fragilis) ve vzorcích stolice u dětských pacientů se přikláním k hypotéze, kterou vyslovil již v roce 1940 Dobell (3), že právě roup může být vektorem jejich přenosu, a kterou podporuje např. Stark a kol. (4). To naznačují i výsledky studie Prevalence a klinický význam parazitárních infekcí v populaci 0–15letých dětí na Ostravsku, kterou realizovalo naše pracoviště spolu s Dětským oddělením Vítkovické nemocnice. Zde byla D. fragilis přítomna u 26,5 % respondentů, v 11 % společně s E. vermicularis (5). Se zavedením molekulárně biologických diagnostických metod (RT PCR) do rutinní praxe některých laboratoří v ČR se výrazně zvýšil počet jejich nálezů. D. fragilis tak ve statistikách NRL aktuálně již několik let vede (11) (Tab. 2, 3). Způsob přenosu je v zájmu parazitologů (4, 5, 6, 7, 8). Stark a kol. uvádí existenci precystických a cystických stadií (9), nicméně je to i nadále předmětem odborných diskuzí.
Enterobius vermicularis (E. vermicularis) is currently the most common helminth of the human intestine. The statistics of the National Reference Laboratory for Intestinal Parasitoses show that it is almost the only representative of the helminth group in our conditions (1) (Table 1). Occurs or diagnoses this disease (enterobiosis, oxyuriasis) especially in children. Due to the rapid maturation of larvae in the egg (4-6 h), their overproduction (an adult female lays 5 000-12 000 eggs (2) (Fig. 1a, b, Fig. 2a, b) and the high resistance of eggs to external influences, I assume the current occurrence in most family members, including adults. Due to the relatively high prevalence of the pathogenic trichomonad Dientamoeba fragilis (D. fragilis) in stool samples of paediatric patients, I am inclined to the hypothesis, expressed already in 1940 by Dobell (3), that the roup may be the vector of their transmission and which is supported by e.g. Stark et al. (4) This is also suggested by the results of the study Prevalence and clinical significance of parasitic infections in the population of 0-15-year-old children in Ostrava region, which was carried out by our department together with the Children's Department of Vítkovice Hospital.Here, D. fragilis was present in 26,5 % of respondents, in 11% together with E. vermicularis (5). With the introduction of molecular biological diagnostic methods (RT PCR) into the routine practice of some laboratories in the Czech Republic, the number of their findings has increased significantly. Thus, D. fragilis has currently been leading in the NRL statistics for several years (11), (Table 2, 3). The mode of transmission is of interest to parasitologists (4, 5, 6, 7, 8). Stark et al. report the existence of precystic and cystic stages (9), but this remains a matter of professional debate.
- MeSH
- benzimidazoly terapeutické užití MeSH
- Dientamoeba parazitologie patogenita MeSH
- dítě MeSH
- enterobióza diagnóza farmakoterapie přenos MeSH
- Enterobius * parazitologie patogenita MeSH
- lidé MeSH
- metronidazol terapeutické užití MeSH
- nemoci střev etiologie parazitologie MeSH
- parazitární nemoci střev diagnóza klasifikace parazitologie přenos MeSH
- Trichomonadida parazitologie patogenita MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- přehledy MeSH
The eukaryotic phylum Parabasalia is composed primarily of anaerobic, endobiotic organisms such as the veterinary parasite Tritrichomonas foetus and the human parasite Trichomonas vaginalis, the latter causing the most prevalent, non-viral, sexually transmitted disease world-wide. Although a parasitic lifestyle is generally associated with a reduction in cell biology, T. vaginalis provides a striking counter-example. The 2007 T. vaginalis genome paper reported a massive and selective expansion of encoded proteins involved in vesicle trafficking, particularly those implicated in the late secretory and endocytic systems. Chief amongst these were the hetero-tetrameric adaptor proteins or 'adaptins', with T. vaginalis encoding ∼3.5 times more such proteins than do humans. The provenance of such a complement, and how it relates to the transition from a free-living or endobiotic state to parasitism, remains unclear. In this study, we performed a comprehensive bioinformatic and molecular evolutionary investigation of the heterotetrameric cargo adaptor-derived coats, comparing the molecular complement and evolution of these proteins between T. vaginalis, T. foetus and the available diversity of endobiotic parabasalids. Notably, with the recent discovery of Anaeramoeba spp. as the free-living sister lineage to all parabasalids, we were able to delve back to time points earlier in the lineage's history than ever before. We found that, although T. vaginalis still encodes the most HTAC subunits amongst parabasalids, the duplications giving rise to the complement took place more deeply and at various stages across the lineage. While some duplications appear to have convergently shaped the parasitic lineages, the largest jump is in the transition from free-living to endobiotic lifestyle with both gains and losses shaping the encoded complement. This work details the evolution of a cellular system across an important lineage of parasites and provides insight into the evolutionary dynamics of an example of expansion of protein machinery, counter to the more common trends observed in many parasitic systems.
- MeSH
- lidé MeSH
- Parabasalidea * MeSH
- paraziti * MeSH
- Trichomonas vaginalis * genetika MeSH
- Tritrichomonas foetus * genetika MeSH
- výpočetní biologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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.
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
Předkládaná kazuistika popisuje nález Trichomonas vaginalis u pacientky (34 let) s předčasným odtokem plodové vody a následnou sekcí v 25. týdnu těhotenství, kde přítomnost T. vaginalis nebyla jediným rizikovým faktorem předčasného porodu. I když se jedná o vzácný nález u gravidní ženy, je nutné na přítomnost tohoto mikroorganismu při obdobných situacích pomýšlet.
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- MeSH
- antibakteriální látky terapeutické užití MeSH
- dospělí MeSH
- hypertenze MeSH
- lidé MeSH
- methamfetamin škodlivé účinky MeSH
- perinatální smrt etiologie MeSH
- předčasný odtok plodové vody etiologie MeSH
- předčasný porod etiologie MeSH
- rizikové faktory MeSH
- růstová retardace plodu etiologie farmakoterapie MeSH
- těhotenství MeSH
- Trichomonas vaginalis * patogenita MeSH
- trichomoniáza diagnóza farmakoterapie komplikace MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- těhotenství MeSH
- ženské pohlaví MeSH
- Publikační typ
- kazuistiky 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.
- 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
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.
- 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
