The aim of this paper is to describe the prevalence of Mycoplasma genitalium and Trichomonas vaginalis in patients who visited general practitioners in the Netherlands. Additionally, we describe the prevalence of M. genitalium resistance to azithromycin and moxifloxacin. We used data from 7,411 consecutive female patients who were screened for Chlamydia trachomatis, Neisseria gonorrhoeae, M. genitalium, and T. vaginalis and data from 5,732 consecutive male patients screened for C. trachomatis, N. gonorrhoeae, and M. genitalium. The prevalence of M. genitalium and T. vaginalis in female patients was 6.7% (95% CI: 6.2 to 7.4) and 1.9% (95%CI: 1.6 to 2.2%), respectively. M. genitalium prevalence in male patients was 3.7% (3.3 to 4.3). M. genitalium co-occurred with C. trachomatis in 1.4% (0.3 to 0.6%) of female and in 0.7% (0.5 to 0.9) of male patients. Macrolide resistance gene mutations and fluoroquinolone resistance gene mutations were detected in 73.8% and 9.9%, respectively. We concluded that M.genitalium is relatively infrequently found in a large general practitioner population in the Netherlands. It can co-occur with C. trachomatis, and is often resistant to azithromycin. Therefore, when treating sexually transmitted infections, these prevalence and resistance data should be taken into account.

Trichomonas vaginalis is a parasite of the human urogenital tract and the causative agent of trichomoniasis, a sexually transmitted disease of worldwide importance. This parasite is usually found as a motile flagellated trophozoite. However, when subjected to stressful microenvironmental conditions, T. vaginalis trophozoites can differentiate into peculiar cyst-like stages, which exhibit notable physiological resistance to unfavourable conditions. Although well documented in morphological and proteomic terms, patterns of gene expression changes involved in the cellular differentiation into cyst-like stages are mostly unknown. The real-time reverse transcription polymerase chain reaction (RT-qPCR) is recognized as a sensitive and accurate method for quantification of gene expression, providing fluorescence-based data that are proportional to the amount of a target RNA. However, the reliability of relative expression studies depends on the validation of suitable reference genes, which RNAs exhibit a minimum of variation between tested conditions. Here, we attempt to determine suitable reference genes to be used as controls of invariant expression during cold-induced in vitro differentiation of T. vaginalis trophozoites into cyst-like forms. Furthermore, we reveal that the mRNA from the meiotic recombinase Dmc1 is upregulated during this process, indicating that cryptic sexual events may take place in cyst-like stages of T. vaginalis.

Trichomonas vaginalis (Tv) induces host cell damage through cysteine proteinases (CPs) modulated by iron. An immunoproteomic analysis showed that trichomoniasis patient sera recognize various CPs, also some of them are present in vaginal washes (VWs). Thus, the goal of this work was to determine whether TvCP2 is expressed during infection and to assess the effect of iron on TvCP2 expression, localization and contribution to in vitro cellular damage. Western-blotting (WB) assays using TvCP2r and vaginitis patient serum samples showed that 6/9 Tv (+) but none of the Tv (−) patient sera recognized TvCP2r. WB using an anti-TvCP2r antibody and VWs from the same patients showed that in all of the Tv (+) but none of the Tv (−) VWs, the anti-TvCP2r antibody detected a 27 kDa protein band that corresponded to the mature TvCP2, which was confirmed by mass spectrometry analysis. Iron decreased the amount of TvCP2 mRNA and the protein localized on the parasite surface and cytoplasmic vesicles concomitant with the cytotoxic effect of TvCP2 on HeLa cells. Parasites pretreated with the anti-TvCP2r antibody also showed reduced levels of cytotoxicity and apoptosis induction in HeLa cell monolayers. In conclusion, these results show that TvCP2 is expressed during trichomonal infection and plays an important role in the in vitro HeLa cell cytotoxic damage under iron-restricted conditions.

Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract leading to trichomoniasis, the most common sexually-transmitted non-viral disease worldwide. The immune response plays a critical role in the host defense against this parasite. Trichomonas' DNA contains unmethylated CpG motifs (CpGDNA) that in other microorganisms act as modulators of the immune response. However, the molecular mechanisms responsible for CpGDNA immune modulation are still unclear. As macrophages participate in the first line of defense against infection, we investigated the type of immune response of murine macrophages to T. vaginalis DNA (TvDNA). We observed high expression of the proinflammatory cytokines IL-6 and IL-12p40 in macrophages stimulated with TvDNA. In contrast, the anti-inflammatory response, assessed by IL-10 and IL-13 mRNA expression was delayed. This suggests that the immune response induced by TvDNA is modulated through cytokine production, mediated partly by NADPH-oxidase activity, as TvDNA induced reactive species of oxygen production and a rounded morphology in macrophages indicative of an M1 phenotype. Furthermore, infected mice pretreated with TvDNA displayed persistent vulvar inflammation and decreased parasite viability consistent with higher proinflammatory cytokine levels during infection compared to untreated mice. Overall, our findings suggest that TvDNA pretreatment modulates the immune response favouring parasite elimination.

Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

Trichomonas vaginalis is an anaerobic protist, responsible for the most prevalent non-viral sexually transmitted infection in humans. One of the most intriguing aspects of T. vaginalis pathobiology is the complex relationship with intracellular microbial symbionts: a group of dsRNA viruses belonging to family of Totiviridae (T. vaginalis virus), and eubacteria belonging to the Mycoplasma genus, in particular Mycoplasma hominis. Both microorganisms seem to strongly influence the lifestyle of T. vaginalis, suggesting a role of the symbiosis in the high variability of clinical presentation and sequelae during trichomoniasis. In the last few years many aspects of this unique symbiotic relationship have been investigated: M. hominis resides and replicates in the protozoan cell, and T. vaginalis is able to pass the bacterial infection to both mycoplasma-free protozoan isolates and human epithelial cells; M. hominis synergistically upregulates the proinflammatory response of human monocytes to T. vaginalis. Furthermore, the influence of M. hominis over T. vaginalis metabolism and physiology has been characterized. The identification of a novel species belonging to the class of Mollicutes (Candidatus Mycoplasma girerdii) exclusively associated to T. vaginalis opens new perspectives in the research of the complex series of events taking place in the multifaceted world of the vaginal microbiota, both under normal and pathological conditions.

Trichomonas vaginalis is responsible for the most common non-viral, sexually transmitted infection, human trichomoniasis, and is associated with an increased susceptibility to HIV. An escalation in resistance (2.5–10%) to the clinical drug, metronidazole (MTZ), has been detected and this compound also has adverse side-effects. Therefore, new treatment options are urgently required. Herein, we investigate the possible anti-T. vaginalis activity of 1,10-phenanthroline-5,6-dione (phendione) and its metal complexes, [Ag(phendione)2]ClO4 and [Cu(phendione)3](ClO4)2·4H2O. Minimum inhibitory concentration (MIC) against T. vaginalis ATCC 30236 and three fresh clinical isolates and mammalian cells were performed using serial dilution generating IC50 and CC50 values. Drugs combinations with MTZ were evaluated by chequerboard assay. A strong anti-T. vaginalis activity was found for all test compounds. IC50 values obtained for [Cu(phendione)3](ClO4)2·4H2O were similar or lower than those obtained for MTZ. In vitro assays with normal cells showed low cytotoxicity and [Cu(phendione)3](ClO4)2·4H2O presented a high selectivity index (SI) for fibroblasts (SI = 11.39) and erythrocytes (SI > 57.47). Chequerboard assay demonstrated that the combination of [Cu(phendione)3](ClO4)2·4H2O with MTZ leads to synergistic interaction, which suggests distinct mechanisms of action of the copper–phendione complex and avoiding the MTZ resistance pathways. Our results highlight the importance of phendione-based drugs as potential molecules of pharmaceutical interest.

Prior studies have demonstrated that both bacterial vaginosis (BV) and sexually transmitted infections (STIs) are strong independent risk factors for subsequent STI. In observational studies of this biological enhancement (BE) hypothesis, it is important to adjust for the risk of STI exposure so that the independent effect of BE can be assessed. We sought to model if two markers of local sexual network (partner concurrency and cumulative number of STIs) represented residual confounding in the models of risk for subsequent infection in a study that screened 3620 women for STIs every 3 months for a year. Mixed-effects logistic regression was used to calculate the odds ratios for an incident diagnosis of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and BV following a diagnosis of any of these four at the prior visit, controlling for the cumulative number of STIs and partner concurrency variables. We found that partner concurrency and cumulative number of STIs were each associated with incident infection, and in general, controlling for these variables reduced the strength of the association between prior and incident infections. We conclude that the frequently found association between prior and incident STIs is associated with both BE and sexual network structure.

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

A selection of 1,2-disubstituted 5-nitroindazolin-3-ones (1–19) and 3-alkoxy-5-nitroindazoles substituted at positions 1 (20–24) or 2 (25–39) from our in-house compound library were screened in vitro against the most common curable sexually transmitted pathogen, Trichomonas vaginalis. A total of 41% of the studied molecules (16/39) achieved a significant activity of more than 85% growth inhibition at the highest concentration assayed (100 µg mL−1). Among these compounds, 3-alkoxy-5-nitroindazole derivatives 23, 24, 25 and 27 inhibited parasite growth by more than 50% at 10 µg mL−1. In addition, the first two compounds (23, 24) still showed remarkable activity at the lowest dose tested (1 µg mL−1), inhibiting parasite growth by nearly 40%. Their specific activity towards the parasite was corroborated by the determination of their non-specific cytotoxicity against mammalian cells. The four mentioned compounds exhibited non-cytotoxic profiles at all of the concentrations assayed, showing a fair antiparasitic selectivity index (SI > 7·5). In silico studies were performed to predict pharmacokinetic properties, toxicity and drug-score using Molinspiration and OSIRIS computational tools. The current in vitro results supported by the virtual screening suggest 2-substituted and, especially, 1-substituted 3-alkoxy-5-nitroindazoles as promising starting scaffolds for further development of novel chemical compounds with the main aim of promoting highly selective trichomonacidal lead-like drugs with adequate pharmacokinetic and toxicological profiles.

Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (~82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

Enzymes moonlight in a non-enzymatic capacity in a diverse variety of cellular processes. The discovery of these non-enzymatic functions is generally unexpected, and moonlighting enzymes are known in both prokaryotes and eukaryotes. Importantly, this unexpected multi-functionality indicates that caution might be needed on some occasions in interpreting phenotypes that result from the deletion or gene-silencing of some enzymes, including some of the best known enzymes from classic intermediary metabolism. Here, we provide an overview of enzyme moonlighting in parasitic protists. Unequivocal and putative examples of moonlighting are discussed, together with the possibility that the unusual biological characteristics of some parasites either limit opportunities for moonlighting to arise or perhaps contribute to the evolution of novel proteins with clear metabolic ancestry.

Parasites that often grow anaerobically in their hosts have adopted a fermentative strategy relying on the production of partially oxidized end products, including lactate, glycerol, ethanol, succinate and acetate. This review focuses on recent progress in understanding acetate production in protist parasites, such as amoebae, diplomonads, trichomonads, trypanosomatids and in the metazoan parasites helminths, as well as the succinate production pathway(s) present in some of them. We also describe the unconventional organisation of the tricarboxylic acid cycle associated with the fermentative strategy adopted by the procyclic trypanosomes, which may resemble the probable structure of the primordial TCA cycle in prokaryotes.

This study reports that the cytopathic effect of Trichomonas vaginalis, an important human parasite of the urogenital tract, occurs due to mechanical stress and subsequent phagocytosis of the necrotic cells. The investigation was done using a primary culture of bovine oviduct epithelial cells (BOECs), grown either in monolayers or as floating cells. Trophozoites displaying different virulence levels were co-incubated with BOECs for times varying between 1 min and 48 h. Analyses were performed using videomicroscopy, scanning and transmission electron microscopy, colourimetric assays and cytochemistry. Injury was observed as early as 1 h after incubation, while after 12 h the host cells were severely damaged when a fresh trichomonad isolate was used. Trichomonads attack the host cells by clustering around them. Mechanical stress on the microvilli of the host cells was observed and appeared to induce plasma membrane damage and cell death. After membrane injury and lysis, fragments of the necrotic cells were ingested by trichomonads. Phagocytosis occurred by trichomonads avidly eating large portions of epithelial cells containing the nucleus and other organelles, but living or intact cells were not ingested. Necrotic fragments were rapidly digested in lysosomes, as shown by acid phosphatase and ruthenium red assays where only the BOECs were labelled. The lytic capacity of the trichomonads was more pronounced in host cell suspensions.

The ability of Trichomonas vaginalis to adhere to plastic surfaces in the presence of various agents and under different growth conditions was examined in wells of microtitre plates containing unsupplemented TYI medium or the same, with various supplements. Following incubation, the wells were thoroughly washed and adhesion was determined by microscopic counting of the adherent organisms. There was no detectable adhesion in the absence of both serum and carbohydrate. Optimal adhesion (about 10–20% of the total number of parasites) was obtained throughout the growth curve in culture media supplemented with either serum or serum Cohn fractions IV–I (rich in α-globulin) or IV-4 (rich in α + β-globulin) and 25 mM glucose, maltose or fructose, but not in plates pre-coated with the Cohn fractions. Cohn fraction II + III (rich in β + γ-globulin) moderately enhanced adhesion while Cohn fractions II (rich in γ-globulin) or V (albumin), fibronectin, Tamm-Horsfall glycoproteins and polylysine were without effect. Non-metabolizable sugars (methyl derivatives of glucose, mannose or fucose) did not support growth, but, surprisingly, enhanced adhesion. At 4 °C, the trichomonads were not able to adhere and pre-adherent organisms detached from the plastic surface. Optimal adhesion was obtained at a pH range of 6·5–7·5 but was already detectable at pH 5·5. Cytochalasin E markedly suppressed adhesion. The data taken together suggest that the firm, serum-dependent adhesion of T. vaginalis to plastic surfaces is apparently not influenced by the rate of multiplication, requires energy and the binding by the organisms of certain serum proteins, which possibly enhances their hydrophobic interaction with the plastic substratum.

Aerobic resistance of Trichomonas vaginalis to metronidazole was induced in vitro by anaerobic cultivation of drug-susceptible trichomonads with low concentrations of the drug (2–3 μg/ml) for 50 days. Minimal lethal concentrations (MLC) for metronidazole of the resistant derivatives were high in aerobic susceptibility assays (MLC = 216–261.5 μg/ml) but low in anaerobic assays (MLC = 4.2–6.3 μg/ml), surpassing MLC values of their parent strain approximately 50-fold and 3-fold under aerobiosis and anaerobiosis, respectively. Sensitivity to metronidazole under anaerobic conditions and activity of the hydrogenosomal enzyme pyruvate: ferredoxin oxidoreductase indicated that the resistance was of the aerobic type. Dependence of the resistance manifestation on O2 was further confirmed by susceptibility assays in vitro performed in defined gas mixtures of different oxygen content (1–20%). Five percent concentration of O2 proved to be the threshold required for resistance demonstration and the MLC values further increased with increasing O2 concentrations. The in vitro-induced resistance was also demonstrated in vivo by subcutaneous mouse assay. The dose of metronidazole needed to cure 50% of infected mice (DC50) was 223 mg/kg × 3 for resistant derivative MR-3a but 6.6 mg/kg × 3 only for its drug-susceptible parent strain. The metronidazole – resistant strains developed in this study correspond by their properties to drug-resistant T. vaginalis strains isolated from patients refractory to treatment, and promise to be a useful tool in the study of 5-nitroimidazole aerobic resistance.

Immunoprecipitation combined with electrophoresis in gelatin-polyacrylamide gels was successfully used for detection of antibodies against numerous proteinases of Trichomonas vaginalis in infected patients. The method proved to be highly specific as anti-proteinase antibodies were absent in women with negative cultivation of T. vaginalis and no history of trichomoniasis. Sera of 71% and vaginal washes of 86% patients with trichomoniasis were positive for these antibodies. In vaginal washes, but not in sera, antibodies were partly complexed with proteinases, possibly of trichomonad origin. It was also shown that serum antibodies as well as local anti-proteinase antibodies persisted for weeks after patients had been cured.