Prolonged survival of venereal Tritrichomonas foetus parasite in the gastrointestinal tract, bovine fecal extract, and water.

IMPORTANCE: Nowadays, the routine herd diagnosis is usually performed exclusively on bulls, as they remain permanently infected, and prevention and control of Tritrichomonas foetus transmission are based on identifying infected animals and culling practices. The existence of other forms of transmission and the possible role of pseudocysts or cyst-like structures as resistant forms requires rethinking the current management and control of this parasitic disease in the future in some livestock regions of the world.

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.

Extracellular vesicle-localized miR-203 mediates neural crest-placode communication required for trigeminal ganglia formation.

While interactions between neural crest and placode cells are critical for the proper formation of the trigeminal ganglion, the mechanisms underlying this process remain largely uncharacterized. Here, we show that the microRNA-(miR)203, whose epigenetic repression is required for neural crest migration, is reactivated in coalescing and condensing trigeminal ganglion cells. Overexpression of miR-203 induces ectopic coalescence of neural crest cells and increases ganglion size. Reciprocally, loss of miR-203 function in placode, but not neural crest, cells perturbs trigeminal ganglion condensation. Demonstrating intercellular communication, overexpression of miR-203 in the neural crest in vitro or in vivo represses a miR-responsive sensor in placode cells. Moreover, neural crest-secreted extracellular vesicles (EVs), visualized using pHluorin-CD63 vector, become incorporated into the cytoplasm of placode cells. Finally, RT-PCR analysis shows that small EVs isolated from condensing trigeminal ganglia are selectively loaded with miR-203. Together, our findings reveal a critical role in vivo for neural crest-placode communication mediated by sEVs and their selective microRNA cargo for proper trigeminal ganglion formation.

Tritrichomonas foetus Cell Division Involves DNA Endoreplication and Multiple Fissions.

Tritrichomonas foetus and Trichomonas vaginalis are extracellular flagellated parasites that inhabit animals and humans, respectively. Cell division is a crucial process in most living organisms that leads to the formation of 2 daughter cells from a single mother cell. It has been assumed that T. vaginalis and T. foetus modes of reproduction are exclusively by binary fission. However, here, we showed that multinuclearity is a phenomenon regularly observed in different T. foetus and T. vaginalis strains in standard culture conditions. Additionally, we revealed that nutritional depletion or nutritional deprivation led to different dormant phenotypes. Although multinucleated T. foetus are mostly observed during nutritional depletion, numerous cells with 1 larger nucleus have been observed under nutritional deprivation conditions. In both cases, when the standard culture media conditions are restored, the cytoplasm of these multinucleated cells separates, and numerous parasites are generated in a short period of time by the fission multiple. We also revealed that DNA endoreplication occurs both in large and multiple nuclei of parasites under nutritional deprivation and depletion conditions, suggesting an important function in stress nutritional situations. These results provide valuable data about the cell division process of these extracellular parasites. IMPORTANCE Nowadays, it's known that T. foetus and T. vaginalis generate daughter cells by binary fission. Here, we report that both parasites are also capable of dividing by multiple fission under stress conditions. We also demonstrated, for the first time, that T. foetus can increase its DNA content per parasite without concluding the cytokinesis process (endoreplication) under stress conditions, which represents an efficient strategy for subsequent fast multiplication when the context becomes favorable. Additionally, we revealed the existence of novel dormant forms of resistance (multinucleated or mononucleated polyploid parasites), different than the previously described pseudocysts, that are formed under stress conditions. Thus, it is necessary to evaluate the role of these structures in the parasites' transmission in the future.

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.

Ultrastructural and Functional Analysis of a Novel Extra-Axonemal Structure in Parasitic Trichomonads.

Trichomonas vaginalis and Tritrichomonas foetus are extracellular flagellated parasites that inhabit humans and other mammals, respectively. In addition to motility, flagella act in a variety of biological processes in different cell types, and extra-axonemal structures (EASs) have been described as fibrillar structures that provide mechanical support and act as metabolic, homeostatic, and sensory platforms in many organisms. It has been assumed that T. vaginalis and T. foetus do not have EASs. However, here, we used complementary electron microscopy techniques to reveal the ultrastructure of EASs in both parasites. Such EASs are thin filaments (3-5 nm diameter) running longitudinally along the axonemes and surrounded by the flagellar membrane, forming prominent flagellar swellings. We observed that the formation of EAS increases after parasite adhesion on the host cells, fibronectin, and precationized surfaces. A high number of rosettes, clusters of intramembrane particles that have been proposed as sensorial structures, and microvesicles protruding from the membrane were observed in the EASs. Our observations demonstrate that T. vaginalis and T. foetus can connect to themselves by EASs present in flagella. The protein VPS32, a member of the ESCRT-III complex crucial for diverse membrane remodeling events, the pinching off and release of microvesicles, was found in the surface as well as in microvesicles protruding from EASs. Moreover, we demonstrated that the formation of EAS also increases in parasites overexpressing VPS32 and that T. vaginalis-VPS32 parasites showed greater motility in semisolid agar. These results provide valuable data about the role of the flagellar EASs in the cell-to-cell communication and pathogenesis of these extracellular parasites.

Unveiling the role of EVs in anaerobic parasitic protozoa.

The anaerobic or microaerophilic protozoan parasites such as the enteric human pathogens Entamoeba histolytica, Giardia intestinalis, Cryptosporidium parvum, Blastocystis hominis and urogenital tract parasites Trichomonas vaginalis are able to survival in an environment with oxygen deprivation. Despite living in hostile environments these pathogens adopted different strategies to survive within the hosts. Among them, the release of extracellular vesicles (EVs) has become an active endeavor in the study of pathogenesis for these parasites. EVs are heterogenous, membrane-limited structures that have played important roles in cellular communication, transferring information through cargo and modulating the immune system of the host. In this review, we described several aspects of the recently characterized EVs of the anaerobic protozoa, including their role in adhesion, modulation of the immune response and omics analysis to understand the potential of these EVs in the pathogenesis of these diseases caused by anaerobic parasites.

Toward incorporating epigenetics into regulation of gene expression in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract, causing a highly prevalent sexually transmitted infection. The parasite must change its transcriptional profile in order to establish and maintain infection. However, few core regulatory elements and transcription factors have been identified to date and little is known about other mechanisms that may control these rapid changes in gene expression during parasite infection. In the last years, epigenetic mechanisms involved in the regulation of gene expression have been gaining major attention in this parasite. In this review, we summarize and discuss the major advances of the last few years with regard to epigenetics (DNA methylation, post-translational histone modifications, and histone variants) in the parasite T. vaginalis. These studies can shed light into our current understanding of this parasite's biology with far-reaching implications for the prognosis and treatment of trichomoniasis.

Extracellular vesicles released by anaerobic protozoan parasites: Current situation.

Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism for transferring information between cells and organisms across all three kingdoms of life. Parasitic unicellular eukaryotes use EVs as vehicles for intercellular communication and host manipulation. Pathogenic protozoans are able to modulate the immune system of the host and establish infection by transferring a wide range of molecules contained in different types of EVs. In addition to effects on the host, EVs are able to transfer virulence factors, drug-resistance genes and differentiation factors between parasites. In this review we cover the current knowledge on EVs from anaerobic or microaerophilic extracellular protozoan parasites, including Trichomonas vaginalis, Tritrichomonas foetus, Giardia intestinalis and Entamoeba histolytica, with a focus on their potential role in the process of infection. The role of EVs in host: parasite communication adds a new level of complexity to our understanding of parasite biology, and may be a key to understand the complexity behind their mechanism of pathogenesis.

Adenine DNA methylation, 3D genome organization, and gene expression in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract causing infections that range from asymptomatic to highly inflammatory. Recent works have highlighted the importance of histone modifications in the regulation of transcription and parasite pathogenesis. However, the nature of DNA methylation in the parasite remains unexplored. Using a combination of immunological techniques and ultrahigh-performance liquid chromatography (UHPLC), we analyzed the abundance of DNA methylation in strains with differential pathogenicity demonstrating that N6-methyladenine (6mA), and not 5-methylcytosine (5mC), is the main DNA methylation mark in T. vaginalis Genome-wide distribution of 6mA reveals that this mark is enriched at intergenic regions, with a preference for certain superfamilies of DNA transposable elements. We show that 6mA in T. vaginalis is associated with silencing when present on genes. Interestingly, bioinformatics analysis revealed the presence of transcriptionally active or repressive intervals flanked by 6mA-enriched regions, and results from chromatin conformation capture (3C) experiments suggest these 6mA flanked regions are in close spatial proximity. These associations were disrupted when parasites were treated with the demethylation activator ascorbic acid. This finding revealed a role for 6mA in modulating three-dimensional (3D) chromatin structure and gene expression in this divergent member of the Excavata.

Protein Palmitoylation Plays an Important Role in Trichomonas vaginalis Adherence.

The flagellated protozoan parasite Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. As an obligate extracellular pathogen, adherence to epithelial cells is critical for parasite survival within the human host and a better understanding of this process is a prerequisite for the development of therapies to combat infection. In this sense, recent work has shown S-acylation as a key modification that regulates pathogenesis in different protozoan parasites. However, there are no reports indicating whether this post-translational modification is a mechanism operating in T. vaginalis In order to study the extent and function of S-acylation in T. vaginalis biology, we undertook a proteomic study to profile the full scope of S-acylated proteins in this parasite and reported the identification of 363 proteins involved in a variety of biological processes such as protein transport, pathogenesis related and signaling, among others. Importantly, treatment of parasites with the palmitoylation inhibitor 2-bromopalmitate causes a significant decrease in parasite: parasite aggregation as well as adherence to host cells suggesting that palmitoylation could be modifying proteins that are key regulators of Trichomonas vaginalis pathogenesis.

TfVPS32 Regulates Cell Division in the Parasite Tritrichomonas foetus.

The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division.

Membrane-shed vesicles from the parasite Trichomonas vaginalis: characterization and their association with cell interaction.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Despite the serious consequences associated with trichomoniasis disease, little is known about parasite or host factors involved in attachment of the parasite-to-host epithelial cells. Here, we report the identification of microvesicle-like structures (MVs) released by T. vaginalis. MVs are considered universal transport vehicles for intercellular communication as they can incorporate peptides, proteins, lipids, miRNA, and mRNA, all of which can be transferred to target cells through receptor-ligand interactions, fusion with the cell membrane, and delivery of a functional cargo to the cytoplasm of the target cell. In the present study, we demonstrated that T. vaginalis release MVs from the plasma and the flagellar membranes of the parasite. We performed proteomic profiling of these structures demonstrating that they possess physical characteristics similar to mammalian extracellular vesicles and might be selectively charged with specific protein content. In addition, we demonstrated that viable T. vaginalis parasites release large vesicles (LVs), membrane structures larger than 1 µm that are able to interact with other parasites and with the host cell. Finally, we show that both populations of vesicles present on the surface of T vaginalis are induced in the presence of host cells, consistent with a role in modulating cell interactions.

Epigenetics regulates transcription and pathogenesis in the parasite Trichomonas vaginalis.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Different T. vaginalis strains vary greatly in their adherence and cytolytic capacities. These phenotypic differences might be attributed to differentially expressed genes as a consequence of extra-genetic variation, such as epigenetic modifications. In this study, we explored the role of histone acetylation in regulating gene transcription and pathogenesis in T. vaginalis. Here, we show that histone 3 lysine acetylation (H3KAc) is enriched in nucleosomes positioned around the transcription start site of active genes (BAP1 and BAP2) in a highly adherent parasite strain; compared with the low acetylation abundance in contrast to that observed in a less-adherent strain that expresses these genes at low levels. Additionally, exposition of less-adherent strain with a specific histone deacetylases inhibitor, trichostatin A, upregulated the transcription of BAP1 and BAP2 genes in concomitance with an increase in H3KAc abundance and chromatin accessibility around their transcription start sites. Moreover, we demonstrated that the binding of initiator binding protein, the transcription factor responsible for the initiation of transcription of ~75% of known T. vaginalis genes, depends on the histone acetylation state around the metazoan-like initiator to which initiator binding protein binds. Finally, we found that trichostatin A treatment increased parasite aggregation and adherence to host cells. Our data demonstrated for the first time that H3KAc is a permissive histone modification that functions to mediate both transcription and pathogenesis of the parasite T. vaginalis.

Trichomonas vaginalis homolog of macrophage migration inhibitory factor induces prostate cell growth, invasiveness, and inflammatory responses.

The human-infective parasite Trichomonas vaginalis causes the most prevalent nonviral sexually transmitted infection worldwide. Infections in men may result in colonization of the prostate and are correlated with increased risk of aggressive prostate cancer. We have found that T. vaginalis secretes a protein, T. vaginalis macrophage migration inhibitory factor (TvMIF), that is 47% similar to human macrophage migration inhibitory factor (HuMIF), a proinflammatory cytokine. Because HuMIF is reported to be elevated in prostate cancer and inflammation plays an important role in the initiation and progression of cancers, we have explored a role for TvMIF in prostate cancer. Here, we show that TvMIF has tautomerase activity, inhibits macrophage migration, and is proinflammatory. We also demonstrate that TvMIF binds the human CD74 MIF receptor with high affinity, comparable to that of HuMIF, which triggers activation of ERK, Akt, and Bcl-2-associated death promoter phosphorylation at a physiologically relevant concentration (1 ng/mL, 80 pM). TvMIF increases the in vitro growth and invasion through Matrigel of benign and prostate cancer cells. Sera from patients infected with T. vaginalis are reactive to TvMIF, especially in males. The presence of anti-TvMIF antibodies indicates that TvMIF is released by the parasite and elicits host immune responses during infection. Together, these data indicate that chronic T. vaginalis infections may result in TvMIF-driven inflammation and cell proliferation, thus triggering pathways that contribute to the promotion and progression of prostate cancer.

Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host∶parasite interactions.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.

Reversible association of tetraspanin with Trichomonas vaginalis flagella upon adherence to host cells.

The parasite Trichomonas vaginalis is the causative agent of trichomoniasis, a prevalent sexually transmitted infection. Here, we report the cellular analyses of T. vaginalis tetraspanin 6 (TvTSP6). This family of membrane proteins has been implicated in cell adhesion, migration and proliferation in vertebrates. We observed that TvTSP6 expression is upregulated upon contact with vaginal ectocervical cells (VECs) and that parasite strains that are highly adherent to VECs express higher levels of TvTSP6 mRNA relative to poorly adherent strains. TvTSP6 is localized predominantly on the flagella of parasites cultured in the absence of host cells; however, adherence of the parasite to VECs initially results in a redistribution of the protein to intracellular vesicles and the plasma membrane of the main body of the cell. We found that a 16-amino-acid C-terminal intracellular tail of TvTSP6 is necessary and sufficient for flagellar localization and protein redistribution when the parasite is in contact with VECs. Additionally, deletion of the C-terminal tail reduced parasite migration through Matrigel, a mimic of the extracellular matrix. Together, our data support roles for TvTSP6 in parasite migration in the host and sensory reception during infection.

Toxoplasma gondii Sis1-like J-domain protein is a cytosolic chaperone associated to HSP90/HSP70 complex.

Toxoplasma gondii is an obligate intracellular protozoan parasite in which 36 predicted Hsp40 family members were identified by searching the T. gondii genome. The predicted protein sequence from the gene ID TGME49_065310 showed an amino acid sequence and domain structure similar to Saccharomyces cerevisiae Sis1. TgSis1 did not show differences in its expression profile during alkaline stress by microarray analysis. Furthermore, TgSis1 showed to be a cytosolic Hsp40 which co-immunoprecipitated with T. gondii Hsp70 and Hsp90. Structural modeling of the TgSis1 peptide binding fragment revealed structural and electrostatic properties different from the experimental model of human Sis1-like protein (Hdj1). Based on these differences; we propose that TgSis1 may be a potentially attractive drug target for developing a novel anti-T. gondii therapy.

Trichomonas vaginalis pathobiology new insights from the genome sequence.

The draft genome of the common sexually transmitted pathogen Trichomonas vaginalis encodes one of the largest known proteome with 60,000 candidate proteins. This provides parasitologists and molecular cell biologists alike with exciting, yet challenging, opportunities to unravel the molecular features of the parasite's cellular systems and potentially the molecular basis of its pathobiology. Here, recent investigations addressing selected aspects of the parasite's molecular cell biology are discussed, including surface and secreted virulent factors, membrane trafficking, cell signalling, the degradome, and the potential role of RNA interference in the regulation of gene expression.