Effect of Trichomonacide 6-Nitro-1H-benzimidazole Derivative Compounds on Expression Level of Metabolic Genes in Trichomonas vaginalis.

The parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease worldwide. This infection often remains asymptomatic and is related to several health complications. The traditional treatment for trichomoniasis is the use of drugs of the 5-nitroimidazole family, such as metronidazole; however, scientific reports indicate an increasing number of drug-resistant strains. Benzimidazole derivatives could offer an alternative in the search for new anti-trichomonas drugs. In this sense, two attractive candidates are the compounds O2N-BZM7 and O2N-BZM9 (1H-benzimidazole derivatives), since, through in vitro tests, they have shown a higher trichomonacide activity. In this study, we determined the effect on the expression level of metabolic genes in T. vaginalis. The results show that genes involved in redox balance (NADHOX, G6PD::6PGL) are overexpressed, as well as the gene that participates in the first reaction of glycolysis (CK); on the other hand, structural genes such as ACT and TUB are decreased in expression in trophozoites treated with the compound O2N-BZM9, which would probably affect its morphology, motility and virulence. These results align with the trichomonacidal activity of the compounds, with benzimidazole O2N-BZM9 being the most potent, with an IC50 value of 4.8 µM. These results are promising for potential future therapeutic applications.

Introduction of protein vaccine candidate based on AP65, AP33, and α-actinin proteins against Trichomonas vaginalis parasite: an immunoinformatics design.

BACKGROUND: Trichomonas vaginalis is the most common nonviral sexually transmitted disease (STI) worldwide. Vaccination is generally considered to be one of the most effective methods of preventing infectious diseases. Using AP65, AP33 and α-actinin proteins, this research aims to develop a protein vaccine against Trichomonas vaginalis. METHODS: Based on the B-cell and T-cell epitope prediction servers, the most antigenic epitopes were selected, and with the necessary evaluations, epitope-rich domains of three proteins, AP65, AP33, and α-actinin, were selected and linked. Subsequently, the ability of the vaccine to interact with toll-like receptors 2 and 4 (TLR2 and TLR4) was assessed. The stability of the interactions was also studied by molecular dynamics for a duration of 100 nanoseconds. RESULTS: The designed protein consists of 780 amino acids with a molecular weight of 85247.31 daltons. The results of the interaction of the vaccine candidate with TLR2 and TLR4 of the immune system also showed that there are strong interactions between the vaccine candidate protein with TLR2 (-890.7 kcal mol-1) and TLR4 (-967.3 kcal mol-1). All parameters studied to evaluate the stability of the protein structure and the protein-TLR2 and protein-TLR4 complexes showed that the structure of the vaccine candidate protein is stable alone and in complex with the immune system receptors. Investigation of the ability of the designed protein to induce an immune response using the C-ImmSim web server also showed that the designed protein is capable of stimulating B- and T-cell lymphocytes to produce the necessary cytokines and antibodies against Trichomonas vaginalis. CONCLUSIONS: Overall, our vaccine may have potential protection against Trichomonas vaginalis. However, for experimental in vivo and in vitro studies, it may be a good vaccine candidate.

The core exosome proteome of Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis is parasitic protozoan that causes human urogenital infections. Accumulated reports indicated that exosomes released by this parasite play a crucial role in transmitting information and substances between cells during host-parasite interactions. Current knowledge on the protein contents in T. vaginalis exosome is mainly generated from three previous studies that used different T. vaginalis isolates as an experimental model. Whether T. vaginalis exosomes comprise a common set of proteins (core exosome proteome) is still unclear. METHODS: To explore the core exosome proteome in T. vaginalis, we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the contents of sucrose ultracentrifugation-enriched exosome and supernatant fractions isolated from six isolates. RESULTS: Transmission electron microscopy (TEM) confirmed the presence of exosomes in the enriched fraction. Proteomic analysis identified a total of 1870 proteins from exosomal extracts. There were 1207 exosomal-specific proteins after excluding 436 'non-core exosomal proteins'. Among these, 72 common exosomal-specific proteins were expressed in all six isolates. Compared with three published T. vaginalis exosome proteome datasets, we identified 16 core exosomal-specific proteins. These core exosomal-specific proteins included tetraspanin (TvTSP1), the classical exosome marker, and proteins mainly involved in catalytic activity and binding such as ribosomal proteins, ras-associated binding (Rab) proteins, and heterotrimeric G proteins. CONCLUSIONS: Our study highlighted the importance of using supernatant fraction from exosomal extract as a control to eliminate 'non-core exosomal proteins'. We compiled a reference core exosome proteome of T. vaginalis, which is essential for developing a fundamental understanding of exosome-mediated cell communication and host-parasite interaction.

Phenolic chalcones as agents against Trichomonas vaginalis.

Trichomonas vaginalis, a flagellated and anaerobic protozoan, is a causative agent of trichomoniasis. This disease is among the world's most common non-viral sexually transmitted infection. A single class drug, nitroimidazoles, is currently available for the trichomoniasis treatment. However, resistant isolates have been identified from unsuccessfully treated patients. Thus, there is a great challenge for a discovery of innovative anti-T. vaginalis agents. As part of our ongoing search for antiprotozoal chalcones, we designed and synthesized a series of 21 phenolic chalcones, which were evaluated against T. vaginalis trophozoites. Structure-activity relationship indicated hydroxyl group plays a role key in antiprotozoal activity. 4'-Hydroxychalcone (4HC) was the most active compound (IC50 = 27.5 µM) and selected for detailed bioassays. In vitro and in vivo evaluations demonstrated 4HC was not toxic against human erythrocytes and Galleria mellonella larvae. Trophozoites of T. vaginalis were treated with 4HC and did not present significant reactive oxygen species (ROS) accumulation. However, compound 4HC was able to increase ROS accumulation in neutrophils coincubated with T. vaginalis. qRT-PCR Experiments indicated that 4HC did not affect the expression of pyruvate:ferredoxin oxidoreductase (PFOR) and ß-tubulin genes. In silico simulations, using purine nucleoside phosphorylase of T. vaginalis (TvPNP), corroborated 4HC as a promising ligand. Compound 4HC was able to establish interactions with residues D21, G20, M180, R28, R87 and T90 through hydrophobic interactions, π-donor hydrogen bond and hydrogen bonds. Altogether, these results open new avenues for phenolic chalcones to combat trichomoniasis, a parasitic neglected infection.

Trichomonas vaginalis adherence phenotypes and extracellular vesicles impact parasite survival in a novel in vivo model of pathogenesis.

Trichomonas vaginalis is a human infective parasite responsible for trichomoniasis-the most common, non-viral, sexually transmitted infection worldwide. T. vaginalis resides exclusively in the urogenital tract of both men and women. In women, T. vaginalis has been found colonizing the cervix and vaginal tract while in men it has been identified in the upper and lower urogenital tract and in secreted fluids such as semen, urethral discharge, urine, and prostatic fluid. Despite the over 270 million cases of trichomoniasis annually worldwide, T. vaginalis continues to be a highly neglected organism and thus poorly studied. Here we have developed a male mouse model for studying T. vaginalis pathogenesis in vivo by delivering parasites into the murine urogenital tract (MUT) via transurethral catheterization. Parasite burden was assessed ex-vivo using a nanoluciferase-based gene expression assay which allowed quantification of parasites pre- and post-inoculation. Using this model and read-out approach, we show that T. vaginalis can be found within MUT tissue up to 72 hrs post-inoculation. Furthermore, we also demonstrate that parasites that exhibit increased parasite adherence in vitro also have higher parasite burden in mice in vivo. These data provide evidence that parasite adherence to host cells aids in parasite persistence in vivo and molecular determinants found to correlate with host cell adherence in vitro are applicable to infection in vivo. Finally, we show that co-inoculation of T. vaginalis extracellular vesicles (TvEVs) and parasites results in higher parasite burden in vivo. These findings confirm our previous in vitro-based predictions that TvEVs assist the parasite in colonizing the host. The establishment of this pathogenesis model for T. vaginalis sets the stage for identifying and examining parasite factors that contribute to and influence infection outcomes.

Metabolomics analysis reveals changes related to pseudocyst formation induced by iron depletion in Trichomonas vaginalis.

BACKGROUND: Iron is an essential element for cellular functions, such as energy metabolism. Trichomonas vaginalis, a human urogenital tract pathogen, is capable of surviving in the environment without sufficient iron supplementation. Pseudocysts (cyst-like structures) are an environmentally tolerated stage of this parasite while encountering undesired conditions, including iron deficiency. We previously demonstrated that iron deficiency induces more active glycolysis but a drastic downregulation of hydrogenosomal energy metabolic enzymes. Therefore, the metabolic direction of the end product of glycolysis is still controversial. METHODS: In the present work, we conducted an LC‒MS-based metabolomics analysis to obtain accurate insights into the enzymatic events of T. vaginalis under iron-depleted (ID) conditions. RESULTS: First, we showed the possible digestion of glycogen, cellulose polymerization, and accumulation of raffinose family oligosaccharides (RFOs). Second, a medium-chain fatty acid (MCFA), capric acid, was elevated, whereas most detected C18 fatty acids were reduced significantly. Third, amino acids were mostly reduced, especially alanine, glutamate, and serine. Thirty-three dipeptides showed significant accumulation in ID cells, which was probably associated with the decrease in amino acids. Our results indicated that glycogen was metabolized as the carbon source, and the structural component cellulose was synthesized at same time. The decrease in C18 fatty acids implied possible incorporation in the membranous compartment for pseudocyst formation. The decrease in amino acids accompanied by an increase in dipeptides implied incomplete proteolysis. These enzymatic reactions (alanine dehydrogenase, glutamate dehydrogenase, and threonine dehydratase) were likely involved in ammonia release. CONCLUSION: These findings highlighted the possible glycogen utilization, cellulose biosynthesis, and fatty acid incorporation in pseudocyst formation as well as NO precursor ammonia production induced by iron-depleted stress.

An Atypical F-Actin Capping Protein Modulates Cytoskeleton Behaviors Crucial for Trichomonas vaginalis Colonization.

Cytoadherence and migration are crucial for pathogens to establish colonization in the host. In contrast to a nonadherent isolate of Trichomonas vaginalis, an adherent one expresses more actin-related machinery proteins with more active flagellate-amoeboid morphogenesis, amoeba migration, and cytoadherence, activities that were abrogated by an actin assembly blocker. By immunoprecipitation coupled with label-free quantitative proteomics, an F-actin capping protein (T. vaginalis F-actin capping protein subunit α [TvFACPα]) was identified from the actin-centric interactome. His-TvFACPα was detected at the barbed end of a growing F-actin filament, which inhibited elongation and possessed atypical activity in binding G-actin in in vitro assays. TvFACPα partially colocalized with F-actin at the parasite pseudopod protrusion and formed a protein complex with α-actin through its C-terminal domain. Meanwhile, TvFACPα overexpression suppressed F-actin polymerization, amoeboid morphogenesis, and cytoadherence in this parasite. Ser2 phosphorylation of TvFACPα enriched in the amoeboid stage of adhered trophozoites was reduced by a casein kinase II (CKII) inhibitor. Site-directed mutagenesis and CKII inhibitor treatment revealed that Ser2 phosphorylation acts as a switching signal to alter TvFACPα actin-binding activity and the consequent actin cytoskeleton behaviors. Through CKII signaling, TvFACPα also controls the conversion of adherent trophozoites from amoeboid migration to the flagellate form with axonemal motility. Together, CKII-dependent Ser2 phosphorylation regulates TvFACPα binding to actin to fine-tune cytoskeleton dynamics and drive crucial behaviors underlying host colonization by T. vaginalis. IMPORTANCE Trichomoniasis is one of the most prevalent nonviral sexually transmitted diseases. T. vaginalis cytoadherence to urogenital epithelium cells is the first step in the colonization of the host. However, studies on the mechanisms of cytoadherence have focused mainly on the role of adhesion molecules, and their effects are limited when analyzed by loss- or gain-of-function assays. This study proposes an extra pathway in which the actin cytoskeleton mediated by a capping protein α-subunit may play roles in parasite morphogenesis, cytoadherence, and motility, which are crucial for colonization. Once the origin of the cytoskeleton dynamics could be manipulated, the consequent activities would be controlled as well. This mechanism may provide new potential therapeutic targets to impair this parasite infection and relieve the increasing impact of drug resistance on clinical and public health.

Role of cytoneme structures and extracellular vesicles in Trichomonas vaginalis parasite-parasite communication.

Trichomonas vaginalis, the etiologic agent of the most common non-viral sexually transmitted infection worldwide. With an estimated annual prevalence of 276 million new cases, mixed infections with different parasite strains are expected. Although it is known that parasites interact with their host to enhance their own survival and transmission, evidence of mixed infections call into question the extent to which unicellular parasites communicate with each other. Here, we demonstrated that different T. vaginalis strains can communicate through the formation of cytoneme-like membranous cell connections. We showed that cytonemes formation of an adherent parasite strain (CDC1132) is affected in the presence of a different strain (G3 or B7RC2). Our findings provide evidence that this effect is contact-independent and that extracellular vesicles (EVs) are responsible, at least in part, of the communication among strains. We found that EVs isolated from G3, B7RC2, and CDC1132 strains contain a highly distinct repertoire of proteins, some of them involved in signaling and communication, among other functions. Finally, we showed that parasite adherence to host cells is affected by communication between strains as binding of adherent T. vaginalis CDC1132 strain to prostate cells is significantly higher in the presence of G3 or B7RC2 strains. We also observed that a poorly adherent parasite strain (G3) adheres more strongly to prostate cells in the presence of an adherent strain. The study of signaling, sensing, and cell communication in parasitic organisms will enhance our understanding of the basic biological characteristics of parasites, which may have important consequences in pathogenesis.

Iron restriction increases the expression of a cytotoxic cysteine proteinase TvCP2 by a novel mechanism of tvcp2 mRNA alternative polyadenylation in Trichomonas vaginalis.

Trichomonas vaginalis TvCP2 (TVAG_057000) is a cytotoxic cysteine proteinase (CP) expressed under iron-limited conditions. This work aimed to identify one of the mechanisms of tvcp2 gene expression regulation by iron at the posttranscriptional level. We checked tvcp2 mRNA stability under both iron-restricted (IR) and high iron (HI) conditions in the presence of actinomycin D. Greater stability of the tvcp2 mRNA under the IR than in HI conditions was observed, as expected. In silico analysis of the 3' regulatory region showed the presence of two putative polyadenylation signals in the tvcp2 transcript. By 3'-RACE assays, we demonstrated the existence of two isoforms of the tvcp2 mRNA with different 3'-UTR that resulted in more TvCP2 protein under IR than in HI conditions detected by WB assays. Additionally, we searched for homologs of the trichomonad polyadenylation machinery by an in silico analysis in the genome database, TrichDB. 16 genes that encode proteins that could be part of the trichomonad polyadenylation machinery were found. qRT-PCR assays showed that most of these genes were positively regulated by iron. Thus, our results show the presence of alternative polyadenylation as a novel iron posttranscriptional regulatory mechanism in T. vaginalis for the tvcp2 gene expression.

Novel therapeutic opportunities for Toxoplasma gondii, Trichomonas vaginalis, and Giardia intestinalis infections.

INTRODUCTION: Toxoplasma gondii, Trichomonas vaginalis, and Giardia intestinalis are the causative agents of toxoplasmosis, trichomoniasis, and giardiasis, three important infections threatening human health and affecting millions of people worldwide. Although drugs and treatment are available to fight these protozoan parasites, side effects and increasing drug resistance require continuous efforts for the development of novel effective drugs. AREAS COVERED: The patents search was carried out in September/October 2022 with four official scientific databases (Espacenet, Scifinder, Reaxys, Google Patents). Treatments for toxoplasmosis, trichomoniasis, and giardiasis (2015-2022) have been grouped according to their chemotypes. In particular, novel chemical entities have been reported and investigated for their structure-activity relationship, when accessible. On the other hand, drug repurposing, extensively exploited to obtain novel antiprotozoal treatment, has been in-depth described. Finally, natural metabolites and extracts have also been reported. EXPERT OPINION: T. gondii, T. vaginalis, and G. intestinalis are protozoan infections usually controlled by immune system in immunocompetent patients; however, they could represent a threatening health for immunocompromised people. The needs of novel effective drugs, endowed with new mechanisms of actions, arises from the increasing drug resistance affecting antibiotic as well as antiprotozoal therapies. In this review different therapeutic approaches to treat protozoan infections have been reported.

[Effect of Trichomonas vaginalis macrophage migration inhibitory factor on THP-1 macrophages].

OBJECTIVE: To investigate the effect of Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) on THP-1 macrophages. METHODS: Recombinant TvMIF protein was prokaryotic expressed and purified, and endotoxin was removed after identification. Following exposure to TvMIF at concentrations of 0, 1, 5, 10, 50 and 100 ng/mL, the cytotoxicity of the recombinant TvMIF protein to THP-1 macrophages was tested using cell counting kit (CCK)-8 assay, and the apoptosis of THP-1 macrophages and reactive oxygen species (ROS) were detected using flow cytometry. The relative expression of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), caspase-1, interleukin-1ß (IL-1ß) and IL-18 genes was quantified using real-time fluorescent quantitative PCR (qPCR) assay, and the expression of caspase-1, NLRP3, gasdermin D (GSDMD), gasdermin D N-terminal (GSDMD-NT) and pro-IL-1ß proteins were determined using Western blotting assay. RESULTS: Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) displayed successful expression and purification of the recombinant TvMIF protein with a molecular weight of 15.5 kDa, and the endotoxin activity assay showed the successful removal of endotoxin in the recombinant TvMIF protein (endotoxin concentration < 0.1 EU/mL), which was feasible for the subsequent studies on protein functions. Flow cytometry revealed that the recombinant TvMIF protein at a concentration of 10 ng/mL and less promoted the apoptosis of THP-1 macrophages, and the highest apoptotic rate of THP-1 macrophages was seen following exposure to the recombinant TvMIF protein at a concentration of 5 ng/mL, while the recombinant TvMIF protein at concentrations of 50 and100 ng/mL inhibited the apoptosis of THP-1 macrophages. Exposure to the recombinant TvMIF protein at a concentration 1 ng/mL resulted in increased ROS levels in THP-1 macrophages. qPCR assay quantified significantly elevated caspase-1, NLRP3, IL-18 and IL-1ß expression in THP-1 macrophages 8 hours post-treatment with the recombinant TvMIF protein at a concentration 1 ng/mL, and Western blotting determined increased caspase-1, NLRP3, pro-IL-1ß, GSDMD and GSDMD-NT protein expression in THP-1 macrophages following exposure to the recombinant TvMIF protein at a concentration 1 ng/mL. Pretreatment with MCC950 significantly reduced GSDMD and GSDMD-NT protein expression. CONCLUSIONS: High-concentration recombinant TvMIF protein inhibits macrophage apoptosis, while low-concentration recombinant TvMIF protein activates NLRP3 inflammasome and promotes macrophage pyroptosis.

Non-vesicular Lipid Transport Machinery in Trichomonas vaginalis: Novel Drug Targets against Trichomoniasis.

Eukaryotic cells have separate membrane-enclosed organelles with distinct biochemical identities and specialized functions. The unique characteristics of each organelle are regulated by asymmetric distribution and intra-cellular trafficking of two important biomolecules, proteins and lipids. Non-vesicular lipid transport facilitated by lipid transfer proteins performs essential roles in intracellular lipid trafficking and homeostasis, while vesicular transport regulates protein trafficking. Comparative analysis of lipid transport machinery in protists could help us to understand the basis of parasitism and insight into eukaryotic evolution. Trichomonas vaginalis, a parasitic protist, greatly depends on receptor-ligand-mediated signaling pathways for cellular differentiation, nutrient uptake, secretion of virulence factors, and pathogenesis. Lipids, despite being key molecules of signaling cascades, have mechanisms of intracellular transport that are largely unexplored in T. vaginalis. We have identified a repertoire of seventeen potential lipid transfer protein (LTP) homologs in T. vaginalis based on a domain-based search on TrichDB (genome database of Trichomonas) coupled with bioinformatics analyses, which indicates the presence of well-organized lipid transport machinery in this parasite. We emphasized here their evolutionary uniqueness and conservation and discussed their potential implications for parasite biology in regard to future therapeutic targets against Trichomoniasis.

Descriptive and functional analyses of four cyclin proteins in Trichomonas vaginalis.

Trichomonas vaginalis is an early divergent protozoan parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection. In metazoans, there is abundant and detailed research on the cell cycle and the components involved in the regulation mechanisms. Regulators such as the cyclin-dependent kinases (CDKs) and cyclins activate the highly regulated processes of cell division. While CDKs have important roles in the phosphorylation of specific substrates, cyclins are important activating-components of CDKs that allow orderly passage through the different stages of the cell cycle. Cell cycle cyclins are characterized by showing drastic changes in their concentration during the cell cycle progression. However, in protists such as T. vaginalis, some biological processes such as cell cycle regulation remain less well studied. In an attempt to gain insight into cell cycle regulation in T. vaginalis, as an initial approach we characterized four proteins with features of cyclins. The genes encoding these putative cyclins were cloned to produce the recombinant proteins TvCYC1, TvCYC2, TvCYC3, and TvCYC4. The functional activity of TvCYC2, TvCYC3, and TvCYC4 was assessed through their complementation of a yeast cln1,2,3Δ mutant strain; TvCYC1 was not able to complement this mutant. Furthermore, our results suggest that TvCYC1, TvCYC2, and TvCYC3, are able to interact with and activate the kinase activity of TvCRK1, a kinase previously characterized by our group. The present study represents the first characterization of cyclins potentially involved in cell cycle regulation in T. vaginalis.

A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis.

Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from H2O-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: H2O-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.

Omics Analyses of Trichomonas vaginalis Actin and Tubulin and Their Participation in Intercellular Interactions and Cytokinesis.

Actin and tubulin proteins from Trichomonas vaginalis are crucial for morphogenesis and mitosis. This parasite has 10 and 11 genes coding bonafide actin and tubulin proteins, respectively. Hence, the goal of this work was to analyze these actin and tubulin genes, their expression at the mRNA and protein levels, and their parasite localization in intercellular interaction and cytokinesis. Representative bonafide actin (tvact1) and tubulin (tvtubα1) genes were cloned into and expressed in Escherichia coli. The recombinant proteins TvACT1r and TvTUBα1r were affinity purified and used as antigens to produce polyclonal antibodies. These antibodies were used in 1DE and 2DE WB and indirect immunofluorescence assays (IFA). By IFA, actin was detected as a ring on the periphery of ameboid, ovoid, and cold-induced cyst-like parasites, on pseudopods of amoeboid parasites, and in cytoplasmic extensions (filopodia) in cell-cell interactions. Tubulin was detected in the axostyle, flagellum, undulating membrane, and paradesmose during mitosis. Paradesmose was observed by IFA mainly during cytokinesis. By scanning electron microscopy, a tubulin-containing nanotubular structure similar to the tunneling nanotubes (TNTs) was also detected in the last stage of cytokinesis. In conclusion, actin and tubulin are multigene families differentially expressed that play important roles in intercellular interactions and cytokinesis.

Influence of Trichomonas vaginalis macrophage migration inhibitory factor on the proliferation activity of prostate epithelial cell line and its preliminary mechanism.

Currently, the pathogenesis of prostate diseases is still under investigation, but it is generally clinically recognized to be related to the imbalance of prostate cell viability. Trichomonas vaginalis macrophage migration inhibitory factor (TvMIF) has been reported to induce the proliferation and invasion of prostate cancer cells, but for normal PECs, the relationship between them has not been reliably confirmed. Therefore, this research aims to determine the influence of macrophage TvMIF on prostate epithelial cells (PECs) and its preliminary mechanism. The activity of RWPE-1 human normal prostate epithelial cells, the inflammatory response state, the expression of miR-451, and the effect of miR-451 on RWPE-1 were detected after TvMIF intervention. We found that TvMIF can enhance RWPE-1 cell proliferation and activate inflammatory factors by suppressing miR-451, thus taking part in the development and proliferation of diseases such as prostatic hyperplasia and prostatitis.

Latent Upregulation of Nlrp3, Nlrc4 and Aim2 Differentiates between Asymptomatic and Symptomatic Trichomonas vaginalis Infection.

Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis. The involvement of NLRP3 inflammasome in trichomoniasis has been discussed in recent studies. The present study aimed to find out the involvement of Nlrp3, Nlrc4, and Aim2 in the BALB/c mouse model infected with symptomatic and asymptomatic isolates of T. vaginalis by quantitative real-time PCR and immunohistochemistry. Our results showed a significantly increased expression of Nlrp3 in the vaginal tissue of the symptomatic group on the 2nd dpi and 14th dpi in the asymptomatic group, respectively. The cervical tissue of asymptomatic groups expressed higher Nlrp3 on 14th dpi than the symptomatic group. The Nlrc4 was expressed on 14th dpi in the vaginal and cervical tissues of mice infected with asymptomatic group as compared to the symptomatic group. Aim2 expression in vaginal tissue was highest at early time points in both the infected groups as compared to controls. However, in cervical tissues, a significant increase of Aim2 expression was observed on 14th dpi in asymptomatic as compared to the symptomatic group. The significantly higher expression of caspase-1 and caspase-4 was observed in cervical tissues of the asymptomatic group on 14th dpi as compared to the symptomatic group, respectively. All NLRs together resulted in higher IL-1ß expression in the vaginal tissues of the symptomatic and asymptomatic groups. We conclude from this study that early expression of Nlrp3, Nlrc4, and Aim2 was seen in the symptomatic group as compared to the late-onset asymptomatic in the vaginal and cervical tissues.

Proteomic Analysis of Trichomonas vaginalis Phagolysosome, Lysosomal Targeting, and Unconventional Secretion of Cysteine Peptidases.

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 ß-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.

VPS32, a member of the ESCRT complex, modulates adherence to host cells in the parasite Trichomonas vaginalis by affecting biogenesis and cargo sorting of released extracellular vesicles.

Trichomonas vaginalis is a common sexually transmitted extracellular parasite that adheres to epithelial cells in the human urogenital tract. Extracellular vesicles (EVs) have been described as important players in the pathogenesis of this parasite as they deliver proteins and RNA into host cells and modulate parasite adherence. EVs are heterogeneous membrane vesicles released from virtually all cell types that collectively represent a new dimension of intercellular communication. The Endosomal Sorting Complex Required for Transport (ESCRT) machinery contributes to several key mechanisms in which it reshapes membranes. Based on this, some components of the ESCRT have been implicated in EVs biogenesis in other cells. Here, we demonstrated that VPS32, a member of ESCRTIII complex, contribute to the biogenesis and cargo sorting of extracellular vesicles in the parasite T. vaginalis. Moreover, we observe that parasites overexpressing VPS32 have a striking increase in adherence to host cells compared to control parasites; demonstrating a key role for this protein in mediating host: parasite interactions. These results provide valuable information on the molecular mechanisms involved in extracellular vesicles biogenesis, cargo-sorting, and parasite pathogenesis.

Comprehensive characterization of purine and pyrimidine transport activities in Trichomonas vaginalis and functional cloning of a trichomonad nucleoside transporter.

Trichomoniasis is a common and widespread sexually-transmitted infection, caused by the protozoan parasite Trichomonas vaginalis. T. vaginalis lacks the biosynthetic pathways for purines and pyrimidines, making nucleoside metabolism a drug target. Here we report the first comprehensive investigation into purine and pyrimidine uptake by T. vaginalis. Multiple carriers were identified and characterized with regard to substrate selectivity and affinity. For nucleobases, a high-affinity adenine transporter, a possible guanine transporter and a low affinity uracil transporter were found. Nucleoside transporters included two high affinity adenosine/guanosine/uridine/cytidine transporters distinguished by different affinities to inosine, a lower affinity adenosine transporter, and a thymidine transporter. Nine Equilibrative Nucleoside Transporter (ENT) genes were identified in the T. vaginalis genome. All were expressed equally in metronidazole-resistant and -sensitive strains. Only TvagENT2 was significantly upregulated in the presence of extracellular purines; expression was not affected by co-culture with human cervical epithelial cells. All TvagENTs were cloned and separately expressed in Trypanosoma brucei. We identified the main broad specificity nucleoside carrier, with high affinity for uridine and cytidine as well as purine nucleosides including inosine, as TvagENT3. The in-depth characterization of purine and pyrimidine transporters provides a critical foundation for the development of new anti-trichomonal nucleoside analogues.