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.

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.

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.

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.

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.

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.

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 flavin reductase 1 and its role in metronidazole resistance.

The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defence in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis.

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.

Mitochondrion-related organelles in eukaryotic protists.

The discovery of mitochondrion-type genes in organisms thought to lack mitochondria led to the demonstration that hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as hydrogenosomes, mitosomes, or mitochondrion-like organelles, are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite their diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. This review examines the metabolism and biogenesis of these organelles in divergent unicellular microbes, with a focus on parasitic protists.

A dynamin-related protein contributes to Trichomonas vaginalis hydrogenosomal fission.

Trichomonas vaginalis is a highly divergent, unicellular eukaryote of the phylum Metamonada, class Parabasalia, and the source of a common sexually transmitted infection. This parasite lacks mitochondria, but harbors an evolutionarily related organelle, the hydrogenosome. We explored the role of dynamin-related proteins (DRPs) in the division of the hydrogenosome. Eight DRP homologues [T. vaginalis DRPs (TvDRPs)], which can be grouped into 3 subclasses, are present in T. vaginalis. We examined 5 TvDRPs that are representative of each subclass, by introducing dominant negative mutations analogous to those known to interfere with mitochondrial division in yeast, worms, and mammals. Microscopic and cell fractionation analyses of parasites expressing one of the mutated TvDRPs (TVAG_350040) demonstrated that this protein localizes to hydrogenosomes. Moreover, these organelles were found to be increased in size and reduced in number in cells expressing this dominant negative protein, relative to parasites expressing the corresponding wild-type TvDRP, the other 4 mutant TvDRPs, or an empty vector control. Our data indicate a role for a TvDRP in the fission of T. vaginalis hydrogenosomes, similar to that described for peroxisomes and mitochondria. These findings reveal a conservation of core components involved in the division of diverse eukaryotic organelles across broad phylogenetic distances.

Hydrocortisone for Treatment of Hypotension in the Newborn.

Newborns, and especially premature newborns, are at significant risk for developing hypotension in the first week or two after birth. The etiology of hypotension in the newborn may vary, but the very low birth weight and extremely low birth weight preterm infants are less likely to respond to conventional cardiovascular support when they develop hypotension. This article reviews the least conventional treatment using hydrocortisone for hypotension that is refractory to conventional volume replacement and/or vasopressor medications with the underlying assumption that sick and premature newborns have a relative or measured adrenal insufficiency. The addition of hydrocortisone in the treatment of hypotension in the newborn is becoming more common but is not universally advocated. However, the supportive evidence is growing, and, as reviewed, use of hydrocortisone requires judicious and cautious regard.

Box H/ACA snoRNAs are preferred substrates for the trimethylguanosine synthase in the divergent unicellular eukaryote Trichomonas vaginalis.

The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.

Review of micronutrients in parenteral nutrition for the NICU population.

A recent review of macronutrients in parenteral nutrition (PN) in the NICU population summarized the carbohydrate, protein, and fat nutrients in PN.1 The purpose of this article is to review the other nutrients in PN that are important for growth and homeostasis including minerals or electrolytes, vitamins, and trace elements. Although the macronutrients contribute more dramatically to adequate nutrition and growth, the other components in PN have significant impact on use of macronutrients as well as targeted organ and cellular function.

Review of macronutrients in parenteral nutrition for neonatal intensive care population.

Parenteral nutrition (PN) has become essential in the management of sick and growing newborn populations in the NICU. In the past few decades, PN has become fundamental in the nutritional management of the very low birth weight infant (<1,500 g).1 Although the components in PN are commonly determined and ordered by the physician or neonatal nurse practitioner provider, the NICU nurse is responsible for confirming the components in the daily PN prior to infusion and is responsible for maintaining the infusion of PN. Nurses should understand the nutritional components of PN as well as the indications, side effects, and infusion limitations of each component. The purpose of this article is to review the macronutrients in PN, including carbohydrates, protein, and fat. A subsequent article will review the micronutrients in PN, including electrolytes, minerals, and vitamins.

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 contact-dependent cytolysis of epithelial cells.

Trichomonas vaginalis is an extracellular protozoan parasite that binds to the epithelium of the human urogenital tract during infection. In this study, we examined the propensities of 26 T. vaginalis strains to bind to and lyse prostate (BPH-1) and ectocervical (Ect1) epithelium and to lyse red blood cells (RBCs). We found that only three of the strains had a statistically significant preference for either BPH-1 (MSA1103) or Ect1 (LA1 and MSA1123). Overall, we observed that levels of adherence are highly variable among strains, with a 12-fold range of adherence on Ect1 cells and a 45-fold range on BPH-1 cells. Cytolysis levels displayed even greater variability, from no detectable cytolysis to 80% or 90% cytolysis of Ect1 and BPH-1, respectively. Levels of adherence and cytolysis correlate for weakly adherent/cytolytic strains, and a threshold of attachment was found to be necessary to trigger cytolysis; however, this threshold can be reached without inducing cytolysis. Furthermore, cytolysis was completely blocked when we prevented attachment of the parasites to host cells while allowing soluble factors complete access. We demonstrate that hemolysis was a rare trait, with only 4 of the 26 strains capable of lysing >20% RBCs with a 1:30 parasite/RBC ratio. Hemolysis also did not correlate with adherence to or cytolysis of either male (BPH-1)- or female (Ect1)-derived epithelial cell lines. Our results reveal that despite a broad range of pathogenic properties among different T. vaginalis strains, all strains show strict contact-dependent cytolysis.

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.