Patricia J. Johnson

The parasite Trichomonas vaginalis colonizes the human vaginal tract and adheres to and lyses epithelial cells causing an inflammatory infection. The vaginal tract is typically acidic, ranging from pH 3.8 to 5.1; however, pathogenesis studies have previously been conducted on parasites grown at pH 5.9 to 6.2. Here we compared the adherence and cytotoxicity of T. vaginalis grown at pH 5.1 and pH 5.9, identifying changes in the surface proteome that contribute to increased pathogenesis of the parasite at pH 5.1. We show that growth of the parasite at pH 5.1 significantly enhances adherence to and lysis of host cells and that this is strongly amplified by the presence of a common bacterial endosymbiont, Mycoplasma hominis . Quantitative proteomics revealed the upregulation of putative surface proteins, three of which were found to be involved in increased parasite adherence and host cell killing. Mechanistic assays demonstrated that a Ricin B-like protein mediates parasite adherence dependent on host glycosaminoglycans via its carbohydrate-recognition domain, while an EF-hand-like protein is shown to promote Ca ²⁺ dependent adherence. Ricin B overexpression was found to reprogram host metabolism, activating ERK1/2 and HIF-1α and driving a Warburg-like glycolytic shift with greatly increased lactate release, which may create a nutrient-rich niche that supports parasite persistence and host cell cytotoxicity. These studies demonstrate a coordinated upregulation of multiple adhesins rather than a single factor at pH 5.1 in the presence of M. hominis and explore the mechanisms underlying the interaction of parasite surface proteins with the host cell.

Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogenital tract, causing infections that range from asymptomatic to highly inflammatory. As an extracellular pathogen, adherence to host epithelial cells is an important step to colonize the human host. Hence, understanding how the process of attachment to host cells is regulated remains an important goal in parasitology research and human health. T. vaginalis -host interaction is regulated by changes in gene expression, but it is still largely unknown how these changes in transcriptional profiles are controlled, as very few transcriptional regulatory elements have been described. Our recent work highlighted the importance of epigenetics in the regulation of transcription, and a specific role for N6-methyladenine (6mA) in modulating three-dimensional chromatin structure has been suggested. Building on these findings, we analyzed here the role of 6mA and chromatin accessibility during the process of host-parasite interaction by integrating MeDIP-seq and assay for transposase-accessible chromatin sequencing data with RNA-seq in free vs host cell-attached parasites. Consistent with our previous results, we identified transcriptionally active and repressive regions flanked by 6mA modifications, observed both in the presence and absence of host cells. Importantly, we detected differentially accessible chromatin regions that influence the gene expression of key pathogenesis-related genes during T. vaginalis host cell interaction. These findings highlight the importance of chromatin architecture in regulating gene expression during parasitic infection. IMPORTANCE: Trichomonas vaginalis , the most common non-viral sexually transmitted parasite, relies on adherence to host epithelial cells to establish infection. Our previous work highlighted the importance of N6-methyladenine (6mA) DNA methylation in the regulation of transcription and three-dimensional chromatin structure. Now, our study integrates RNA-seq, MeDIP-seq, and assay for transposase-accessible chromatin sequencing data to reveal how 6mA and chromatin accessibility modulate gene expression during T. vaginalis interaction with human host cells. We identified over 3,600 differentially expressed genes upon parasite contact with prostate cells, including pathogenesis-related genes. Moreover, we identified transcriptionally active and repressive regions flanked by 6mA that remain largely stable during the process of host interaction. We mapped genome-wide chromatin accessibility and uncovered differentially accessible regions upon host cell contact associated with a subset of genes involved in adhesion. These results suggest that local chromatin accessibility has a major role in modulating gene expression of key virulence genes during host interaction.

The unicellular parasite Trichomonas vaginalis ( Tv ) causes trichomoniasis, the most common non-viral sexually transmitted infection in humans. Trichomoniasis symptoms and sequelae vary substantially, and in vitro analyses have shown that strains differ extensively in their ability to infect and lyse cells of the urogenital tract. Polymorphonuclear leukocytes (PMNs) are known to kill Tv , yet to date, no studies have examined strain differences in PMN killing of this extracellular pathogen. Here, we have examined four strains of Tv for their susceptibility to killing by PMN trogocytosis, a process by which PMNs incrementally acquire fragments of the parasite’s plasma membrane leading to parasite death. Two strains were found to be rapidly and efficiently killed by trogocytosis, whereas the other two strains, although subjected to PMN trogocytosis, were resistant to killing. Strains that resist trogocytic killing were found to have an enhanced plasma membrane resealing capacity. Furthermore, these trogocytosis-resistant strains were susceptible to a later-stage killing mechanism that is significantly reduced in the presence of DNase, consistent with killing by neutrophil extracellular traps (NETosis). Together, our data reveal the use of multiple mechanisms for PMN killing of Tv and demonstrate strain-dependent resistance to rapid killing by trogocytosis, driven by membrane repair. Trogocytosis-resistant strains are subsequently killed by NETosis, likely circumventing the replacement of trogocytosis-sensitive strains by resistant strains over evolutionary time. Together, our data demonstrate that PMNs use different modalities to kill Tv and that Tv strains differ in their PMN-evasive properties. IMPORTANCE: Trichomonas vaginalis ( Tv ) causes the third most prevalent sexually transmitted infection globally and is the most common cause of vaginitis in the United States. Despite its prevalence, little is known about how the human immune system combats and kills Tv . We have previously shown that the abundant white blood cells called polymorphonuclear leukocytes (PMNs), known to kill pathogens using multiple mechanisms, kill Tv using a rapid-killing mechanism called trogocytosis. Here, we examined whether Tv clinical isolates with different pathogenic properties are killed by PMNs using other mechanisms. We demonstrate that clinical isolates that are resistant to trogocytic killing are better at plasma membrane repair and are efficiently killed by a late-stage mechanism called NETosis. Thus, PMN uses multiple, strain-specific mechanisms to kill Tv isolates with varying virulence properties. This study sheds light on host cell killing mechanisms that may impact pathogenic outcomes and sequelae during Tv infection.

Trichomonas vaginalis is a common, extracellular, sexually transmitted parasite which is often found in symbiosis with the intracellular bacterium Mycoplasma hominis (Mh), an opportunistic pathogen of the female reproductive tract. How this symbiosis affects infection outcomes and the host cell innate immune response is poorly understood. Here, we show that infection with T. vaginalis in symbiosis with M. hominis or M. hominis alone triggers a noncanonical type I interferon, interferon-epsilon (IFNε), but infection with T. vaginalis alone does not. We also demonstrate that extracellular vesicles (TvEVs) produced by the parasite downregulate host cell IFNε, counteracting this symbiont-driven response and elevating infection. We further demonstrate that IFNε, a hormonally regulated cytokine produced in the human reproductive system, is protective against T. vaginalis cytoadherence and cytolysis of host cells. These studies provide insight into how a parasite and its bacterial symbiont work in concert to regulate host cell innate immune responses to drive infection.

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 structures of Tv’s DMT (Tv-DMT) has prevented structure-guided drug design to manage Tv infection. Here, we determine the 16 nm, 32 nm, 48 nm and 96 nm-repeat structures of native Tv-DMT at resolution ranging from 3.4 to 4.4 Å by cryogenic electron microscopy (cryoEM) and built an atomic model for the entire Tv-DMT. These structures show that Tv-DMT is composed of 30 different proteins, including the α- and β-tubulin, 19 microtubule inner proteins (MIPs) and 9 microtubule outer proteins. While the A-tubule of Tv-DMT is simplistic compared to DMTs of other organisms, the B-tubule of Tv-DMT features parasite-specific proteins, such as TvFAP40 and TvFAP35. Notably, TvFAP40 and TvFAP35 form filaments near the inner and outer junctions, respectively, and interface with stabilizing MIPs. This atomic model of the Tv-DMT highlights diversity of eukaryotic motility machineries and provides a structural framework to inform rational design of therapeutics against trichomoniasis.

Trichomonas vaginalis is a commonly acquired sexually transmitted infection (STI) often found in symbiosis with the intracellular bacterium Mycoplasma hominis , an opportunistic pathogen of the female reproductive tract associated with bacterial vaginosis. How this symbiosis affects infection outcomes, and the host cell innate immune response is still poorly understood. Here we show that T. vaginalis extracellular vesicles down-regulate a non-canonical type I interferon, interferon-epsilon, and suppress type I interferon responses. Transcriptomic analysis reveals that infection with T. vaginalis in symbiosis with M. hominis or M. hominis alone upregulates genes involved in the type I IFN response, but infection with T. vaginalis alone does not. Finally, we show that interferon-epsilon stimulation is protective against T. vaginalis cytoadherence and cytolysis of host cells and increases the ability of neutrophils to kill the parasite. These studies provide insight into the innate immune response induced by a highly prevalent STI and its bacterial symbiont.

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, such as TvFAP40 and TvFAP35 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 TvFAP40 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 rational design of therapeutics.

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 byT. vaginalis. Approximately 11% of infections are found to be coinfections of multipleT. vaginalisstrains. 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 ofT. 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.

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 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.