Trichomonas vaginalis extracellular vesicles up-regulate and directly transfer adherence factors promoting host cell colonization.

Trichomonas vaginalis, a common sexually transmitted parasite that colonizes the human urogenital tract, secretes extracellular vesicles (TvEVs) that are taken up by human cells and are speculated to be taken up by parasites as well. While the crosstalk between TvEVs and human cells has led to insight into host:parasite interactions, roles for TvEVs in infection have largely been one-sided, with little known about the effect of TvEV uptake by T. vaginalis. Approximately 11% of infections are found to be coinfections of multiple T. vaginalis strains. Clinical isolates often differ in their adherence to and cytolysis of host cells, underscoring the importance of understanding the effects of TvEV uptake within the parasite population. To address this question, our lab tested the ability of a less adherent strain of T. vaginalis, G3, to take up fluorescently labeled TvEVs derived from both itself (G3-EVs) and TvEVs from a more adherent strain of the parasite (B7RC2-EVs). Here, we showed that TvEVs generated from the more adherent strain are internalized more efficiently compared to the less adherent strain. Additionally, preincubation of G3 parasites with B7RC2-EVs increases parasite aggregation and adherence to host cells. Transcriptomics revealed that TvEVs up-regulate expression of predicted parasite membrane proteins and identified an adherence factor, heteropolysaccharide binding protein (HPB2). Finally, using comparative proteomics and superresolution microscopy, we demonstrated direct transfer of an adherence factor, cadherin-like protein, from TvEVs to the recipient parasite's surface. This work identifies TvEVs as a mediator of parasite:parasite communication that may impact pathogenesis during mixed infections.

Structures of Native Doublet Microtubules from Trichomonas vaginalis Reveal Parasite-Specific Proteins as Potential Drug Targets.

Doublet microtubules (DMTs) are flagellar components required for the protist Trichomonas vaginalis ( Tv ) to swim through the human genitourinary tract to cause trichomoniasis, the most common non-viral sexually transmitted disease. Lack of DMT structures has prevented structure-guided drug design to manage Tv infection. Here, we determined the cryo-EM structure of native Tv- DMTs, identifying 29 unique proteins, including 18 microtubule inner proteins and 9 microtubule outer proteins. While the A-tubule is simplistic compared to DMTs of other organisms, the B-tubule features specialized, parasite-specific proteins, like Tv FAP40 and Tv FAP35 that form filaments near the inner and outer junctions, respectively, to stabilize DMTs and enable Tv locomotion. Notably, a small molecule, assigned as IP6, is coordinated within a pocket of Tv FAP40 and has characteristics of a drug molecule. This first atomic model of the Tv -DMT highlights the diversity of eukaryotic motility machinery and provides a structural framework to inform the rational design of therapeutics.

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.

The Effect of a Single Dose of Prophylactic Vancomycin Prior to Peripherally Inserted Central Catheter Removal on Sepsis.

OBJECTIVE: To evaluate the effect of a single dose of prophylactic vancomycin prior to the removal of a peripherally inserted central catheter (PICC) in decreasing sepsis evaluations, positive cultures, and antibiotic usage in neonates. METHODS: A retrospective review was conducted from December 1, 2015, through November 30, 2019, to evaluate outcomes of sepsis evaluations, positive cultures, and antibiotic usage in neonates not receiving prophylactic vancomycin prior to the discontinuation of a PICC as compared with those receiving prophylaxis vancomycin in a neonatal intensive care unit (NICU). RESULTS: Of the 138 neonates enrolled in the study, 82 did not receive vancomycin prophylaxis (Cohort 1), and 56 did (Cohort 2). Both cohorts were similar in sex distribution, gestational age, and PICC days. The frequency of sepsis evaluations, positive cultures, and the need for antibiotics was not found to be significant (p = 0.404, 0.703, 0.808) (Table 2).CONCLUSIONS The results did not show a statistically significant improvement in the incidence of sepsis in neonates who received prophylactic vancomycin prior to PICC discontinuation. However, there were lower percentages of sepsis evaluations, positive cultures, and antibiotics administered in the Cohort 2 patients. Although the advantage of implementing this antibiotic policy is uncertain based on this study, further research across multiple centers including a larger number of subjects may provide more conclusive results.

Atomic Structure of the Trichomonas vaginalis Double-Stranded RNA Virus 2.

Trichomonas vaginalis, the causative pathogen for the most common nonviral sexually transmitted infection worldwide, is itself frequently infected with one or more of the four types of small double-stranded RNA (dsRNA) Trichomonas vaginalis viruses (TVV1 to 4, genus Trichomonasvirus, family Totiviridae). Each TVV encloses a nonsegmented genome within a single-layered capsid and replicates entirely intracellularly, like many dsRNA viruses, and unlike those in the Reoviridae family. Here, we have determined the structure of TVV2 by cryo-electron microscopy (cryoEM) at 3.6 Å resolution and derived an atomic model of its capsid. TVV2 has an icosahedral, T = 2*, capsid comprised of 60 copies of the icosahedral asymmetric unit (a dimer of the two capsid shell protein [CSP] conformers, CSP-A and CSP-B), typical of icosahedral dsRNA virus capsids. However, unlike the robust CSP-interlocking interactions such as the use of auxiliary "clamping" proteins among Reoviridae, only lateral CSP interactions are observed in TVV2, consistent with an assembly strategy optimized for TVVs' intracellular-only replication cycles within their protozoan host. The atomic model reveals both a mostly negatively charged capsid interior, which is conducive to movement of the loosely packed genome, and channels at the 5-fold vertices, which we suggest as routes of mRNA release during transcription. Structural comparison of TVV2 to the Saccharomyces cerevisiae L-A virus reveals a conserved helix-rich fold within the CSP and putative guanylyltransferase domain along the capsid exterior, suggesting conserved mRNA maintenance strategies among Totiviridae This first atomic structure of a TVV provides a framework to guide future biochemical investigations into the interplay between Trichomonas vaginalis and its viruses.IMPORTANCETrichomonas vaginalis viruses (TVVs) are double-stranded RNA (dsRNA) viruses that cohabitate in Trichomonas vaginalis, the causative pathogen of trichomoniasis, the most common nonviral sexually transmitted disease worldwide. Featuring an unsegmented dsRNA genome encoding a single capsid shell protein (CSP), TVVs contrast with multisegmented dsRNA viruses, such as the diarrhea-causing rotavirus, whose larger genome is split into 10 dsRNA segments encoding 5 unique capsid proteins. To determine how TVVs incorporate the requisite functionalities for viral replication into their limited proteome, we derived the atomic model of TVV2, a first for TVVs. Our results reveal the intersubunit interactions driving CSP association for capsid assembly and the properties that govern organization and maintenance of the viral genome. Structural comparison between TVV2 capsids and those of distantly related dsRNA viruses indicates conserved strategies of nascent RNA release and a putative viral guanylyltransferase domain implicated in the cytoplasmic maintenance of viral messenger and genomic RNA.

A Novel Trichomonas vaginalis Surface Protein Modulates Parasite Attachment via Protein:Host Cell Proteoglycan Interaction.

Trichomonas vaginalis is a highly prevalent, sexually transmitted parasite which adheres to mucosal epithelial cells to colonize the human urogenital tract. Despite adherence being crucial for this extracellular parasite to thrive within the host, relatively little is known about the mechanisms or key molecules involved in this process. Here, we have identified and characterized a T. vaginalis hypothetical protein, TVAG_157210 (TvAD1), as a surface protein that plays an integral role in parasite adherence to the host. Quantitative proteomics revealed TvAD1 to be ∼4-fold more abundant in parasites selected for increased adherence (MA parasites) than the isogenic parental (P) parasite line. De novo modeling suggested that TvAD1 binds N-acetylglucosamine (GlcNAc), a sugar comprising host glycosaminoglycans (GAGs). Adherence assays utilizing GAG-deficient cell lines determined that host GAGs, primarily heparan sulfate (HS), mediate adherence of MA parasites to host cells. TvAD1 knockout (KO) parasites, generated using CRISPR-Cas9, were found to be significantly reduced in host cell adherence, a phenotype that is rescued by overexpression of TvAD1 in KO parasites. In contrast, there was no significant difference in parasite adherence to GAG-deficient lines by KO parasites compared with wild-type, which is contrary to that observed for KO parasites overexpressing TvAD1. Isothermal titration calorimetric (ITC) analysis showed that TvAD1 binds to HS, indicating that TvAD1 mediates host cell adherence via HS interaction. In addition to characterizing the role of TvAD1 in parasite adherence, these studies reveal a role for host GAG molecules in T. vaginalis adherence.IMPORTANCE The ability of the sexually transmitted parasite Trichomonas vaginalis to adhere to its human host is critical for establishing and maintaining an infection. Yet how parasites adhere to host cells is poorly understood. In this study, we employed a novel adherence selection method to identify proteins involved in parasite adherence to the host. This method led to the identification of a protein, with no previously known function, that is more abundant in parasites with increased capacity to bind host cells. Bioinformatic modeling and biochemical analyses revealed that this protein binds a common component on the host cell surface proteoglycans. Subsequent creation of parasites that lack this protein directly demonstrated that the protein mediates parasite adherence via an interaction with host cell proteoglycans. These findings both demonstrate a role for this protein in T. vaginalis adherence to the host and shed light on host cell molecules that participate in parasite colonization.

Extracellular vesicles produced by the protozoan parasite Trichomonas vaginalis contain a preferential cargo of tRNA-derived small RNAs.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis, the most prevalent non-viral sexually transmitted infection worldwide. Trichomonas vaginalis releases extracellular vesicles that play a role in parasite:parasite and parasite:host interactions. The aim of this study was to characterise the RNA cargo of these vesicles. Trichomonas vaginalis extracellular vesicles were found to encapsulate a cargo of RNAs of small size. RNA-seq analysis showed that tRNA-derived small RNAs, mostly 5' tRNA halves, are the main type of small RNA in these vesicles. The tRNA-derived small RNAs in T. vaginalis extracellular vesicles were shown to be derived from the specific processing of tRNAs within cells. The specificity of this RNA cargo in T. vaginalis extracellular vesicles suggests a preference for packaging. The RNA cargo of T. vaginalis was shown to be rapidly internalised by human cells via lipid raft-dependent endocytosis. The potential role of these tsRNAs - an emerging class of small RNAs with regulatory functions - on altering host cellular responses requires further examination, suggesting a new mode of parasite:host communication.

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.

Trichomonas vaginalis extracellular vesicles are internalized by host cells using proteoglycans and caveolin-dependent endocytosis.

Trichomonas vaginalis, a human-infective parasite, causes the most prevalent nonviral sexually transmitted infection worldwide. This pathogen secretes extracellular vesicles (EVs) that mediate its interaction with host cells. Here, we have developed assays to study the interface between parasite EVs and mammalian host cells and to quantify EV internalization by mammalian cells. We show that T. vaginalis EVs interact with glycosaminoglycans on the surface of host cells and specifically bind to heparan sulfate (HS) present on host cell surface proteoglycans. Moreover, competition assays using HS or removal of HS from the host cell surface strongly inhibit EV uptake, directly demonstrating that HS proteoglycans facilitate EV internalization. We identified an abundant protein on the surface of T. vaginalis EVs, 4-α-glucanotransferase (Tv4AGT), and show using isothermal titration calorimetry that this protein binds HS. Tv4AGT also competitively inhibits EV uptake, defining it as an EV ligand critical for EV internalization. Finally, we demonstrate that T. vaginalis EV uptake is dependent on host cell cholesterol and caveolin-1 and that internalization proceeds via clathrin-independent, lipid raft-mediated endocytosis. These studies reveal mechanisms used to drive host:pathogen interactions and further our understanding of how EVs are internalized by target cells to allow cross-talk between different cell types.

A Novel Cadherin-like Protein Mediates Adherence to and Killing of Host Cells by the Parasite Trichomonas vaginalis.

Trichomonas vaginalis, a prevalent sexually transmitted parasite, adheres to and induces cytolysis of human mucosal epithelial cells. We have characterized a hypothetical protein, TVAG_393390, with predicted tertiary structure similar to that of mammalian cadherin proteins involved in cell-cell adherence. TVAG_393390, renamed cadherin-like protein (CLP), contains a calcium-binding site at a position conserved in cadherins. CLP is surface localized, and its mRNA and protein levels are significantly upregulated upon parasite adherence to host cells. To test the roles of CLP and its calcium-binding dependency during host cell adherence, we first demonstrated that wild-type CLP (CLP) binds calcium with a high affinity, whereas the calcium-binding site mutant protein (CLP-mut) does not. CLP and CLP-mut constructs were then used to overexpress these proteins in T. vaginalis Parasites overexpressing CLP have ∼3.5-fold greater adherence to host cells than wild-type parasites, and this increased adherence is ablated by mutating the calcium-binding site. Additionally, competition with recombinant CLP decreased parasite binding to host cells. We also found that overexpression of CLP induced parasite aggregation which was further enhanced in the presence of calcium, whereas CLP-mut overexpression did not affect aggregation. Lastly, parasites overexpressing wild-type CLP induced killing of host cells ∼2.35-fold, whereas parasites overexpressing CLP-mut did not have this effect. These analyses describe the first parasitic CLP and demonstrate a role for this protein in mediating parasite-parasite and host-parasite interactions. T. vaginalis CLP may represent convergent evolution of a parasite protein that is functionally similar to the mammalian cell adhesion protein cadherin, which contributes to parasite pathogenesis.IMPORTANCE The adherence of pathogens to host cells is critical for colonization of the host and establishing infection. Here we identify a protein with no known function that is more abundant on the surface of parasites that are better at binding host cells. To interrogate a predicted function of this protein, we utilized bioinformatic protein prediction programs which allowed us to uncover the first cadherin-like protein (CLP) found in a parasite. Cadherin proteins are conserved metazoan proteins with central roles in cell-cell adhesion, development, and tissue structure maintenance. Functional characterization of this CLP from the unicellular parasite Trichomonas vaginalis demonstrated that the protein mediates both parasite-parasite and parasite-host adherence, which leads to an enhanced killing of host cells by T. vaginalis Our findings demonstrate the presence of CLPs in unicellular pathogens and identify a new host cell binding protein family in a human-infective parasite.

Trichomonas vaginalis: Pathogenesis, Symbiont Interactions, and Host Cell Immune Responses.

The parasite Trichomonas vaginalis (Tv) causes a highly prevalent sexually transmitted infection. As an extracellular pathogen, the parasite mediates adherence to epithelial cells to colonize the human host. In addition, the parasite interfaces with the host immune system and the vaginal microbiota. Modes of Tv pathogenesis include damage to host tissue mediated by parasite killing of host cells, disruption of steady-state vaginal microbial ecology, and eliciting inflammation by activating the host immune response. Recent Tv research has uncovered new players that contribute to multifactorial mechanisms of host-parasite adherence and killing, and has examined the relationship between Tv and vaginal bacteria. Mechanisms that may lead to parasite recognition and killing, or the evasion of host immune cells, have also been revealed.

Trichomonas vaginalis Macrophage Migration Inhibitory Factor Mediates Parasite Survival during Nutrient Stress.

Trichomonas vaginalis is responsible for the most prevalent non-viral sexually transmitted disease worldwide, and yet the mechanisms used by this parasite to establish and maintain infection are poorly understood. We previously identified a T. vaginalis homologue (TvMIF) of a human cytokine, human macrophage migration inhibitory factor (huMIF). TvMIF mimics huMIF's role in increasing cell growth and inhibiting apoptosis in human host cells. To interrogate a role of TvMIF in parasite survival during infection, we asked whether overexpression of TvMIF (TvMIF-OE) confers an advantage to the parasite under nutrient stress conditions by comparing the survival of TvMIF-OE parasites to that of empty vector (EV) parasites. We found that under conditions of serum starvation, overexpression of TvMIF resulted in increased parasite survival. Serum-starved parasites secrete 2.5-fold more intrinsic TvMIF than unstarved parasites, stimulating autocrine and paracrine signaling. Similarly, we observed that addition of recombinant TvMIF increased the survival of the parasites in the absence of serum. Recombinant huMIF likewise increased the parasite survival in the absence of serum, indicating that the parasite may use this host survival factor to resist its own death. Moreover, TvMIF-OE parasites were found to undergo significantly less apoptosis and reactive oxygen species (ROS) generation under conditions of serum starvation, consistent with increased survival being the result of blocking ROS-induced apoptosis. These studies demonstrated that a parasitic MIF enhances survival under adverse conditions and defined TvMIF and huMIF as conserved survival factors that exhibit cross talk in host-pathogen interactions.IMPORTANCE Macrophage migration inhibitory factor (MIF) is a conserved protein found in most eukaryotes which has been well characterized in mammals but poorly studied in other eukaryotes. The limited analyses of MIF proteins found in unicellular eukaryotes have focused exclusively on the effect of parasitic MIF on the mammalian host. This was the first study to assess the function of a parasite MIF in parasite biology. We demonstrate that the Trichomonas vaginalis MIF functions to suppress cell death induced by apoptosis, thereby enhancing parasite survival under adverse conditions. Our research reveals a conserved survival mechanism, shared by a parasite and its host, and indicates a role for a conserved protein in mediating cross talk in host-pathogen interactions.

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.

Neutrophils kill the parasite Trichomonas vaginalis using trogocytosis.

T. vaginalis, a human-infective parasite, causes the most common nonviral sexually transmitted infection (STI) worldwide and contributes to adverse inflammatory disorders. The immune response to T. vaginalis is poorly understood. Neutrophils (polymorphonuclear cells [PMNs]) are the major immune cell present at the T. vaginalis-host interface and are thought to clear T. vaginalis. However, the mechanism of PMN clearance of T. vaginalis has not been characterized. We demonstrate that human PMNs rapidly kill T. vaginalis in a dose-dependent, contact-dependent, and neutrophil extracellular trap (NET)-independent manner. In contrast to phagocytosis, we observed that PMN killing of T. vaginalis involves taking "bites" of T. vaginalis prior to parasite death, using trogocytosis to achieve pathogen killing. Both trogocytosis and parasite killing are dependent on the presence of PMN serine proteases and human serum factors. Our analyses provide the first demonstration, to our knowledge, of a mammalian phagocyte using trogocytosis for pathogen clearance and reveal a novel mechanism used by PMNs to kill a large, highly motile target.

CRISPR/Cas9-mediated gene modification and gene knock out in the human-infective parasite Trichomonas vaginalis.

The sexually-transmitted parasite Trichomonas vaginalis infects ~1/4 billion people worldwide. Despite its prevalence and myriad adverse outcomes of infection, the mechanisms underlying T. vaginalis pathogenesis are poorly understood. Genetic manipulation of this single-celled eukaryote has been hindered by challenges presented by its complex, repetitive genome and inefficient methods for introducing DNA (i.e. transfection) into the parasite. Here, we have developed methods to increase transfection efficiency using nucleofection, with the goal of efficiently introducing multiple DNA elements into a single T. vaginalis cell. We then created DNA constructs required to express several components essential to drive CRISPR/Cas9-mediated DNA modification: guide RNA (gRNA), the Cas9 endonuclease, short oligonucleotides and large, linearized DNA templates. Using these technical advances, we have established CRISPR/Cas9-mediated repair of mutations in genes contained on circular DNA plasmids harbored by the parasite. We also engineered CRISPR/Cas9 directed homologous recombination to delete (i.e. knock out) two non-essential genes within the T. vaginalis genome. This first report of the use of the CRISPR/Cas9 system in T. vaginalis greatly expands the ability to manipulate the genome of this pathogen and sets the stage for testing of the role of specific genes in many biological processes.

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.

Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis.

Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells.

Emerging roles for extracellular vesicles in parasitic infections.

Extracellular vesicles (EVs) are released by cells and contain a complex mixture of proteins, genetic information and lipids. EVs mediate cell:cell communication by transferring their molecular cargo between cells. EVs, initially discovered in mammalian systems, have been demonstrated to play critical role in immunology and cancer biology. More recently, EVs have been identified in a broad range of both unicellular and multicellular parasites. In this review we focus on the emerging roles for EVs in parasitic infections. Parasite-derived EVs can transfer virulence factors and drug-resistance markers, modify host cell gene expression and promote parasite adherence and host cell proliferation. EVs can also suppress or stimulate host immune responses. Thus, EVs are likely important in determining the outcome of parasitic infections.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells.

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis.