Zinc-clotrimazole complexes are effective against Trichomonas vaginalis.

Trichomonas vaginalis is a protozoan parasite that causes trichomoniasis in humans, the most prevalent non-viral sexually transmitted disease (STD). Imidazole compounds are used for the treatment of trichomoniasis, and metronidazole is the most commonly prescribed. However, these compounds can lead to parasite resistance and unwanted side effects. Therefore, there is a need for an alternative treatment for this disease. Here, we explored the potential of clotrimazole (CTZ) and zinc compounds, as well as CTZ complexed with zinc salts ([1] acetate [Zn(CTZ)2(Ac)2] and [2] a chloride [Zn(CTZ)2Cl2] complexes) against T. vaginalis. We synthesized the zinc complexed CTZ compounds and determined their concentration values that inhibited parasite growth by 50% (IC50). We used scanning and transmission electron microscopy to visualize the ultrastructural alterations induced by CTZ and their zinc complexes. The incubation of the parasites with [Zn(CTZ)2(Ac)2] complex inhibited their growth, yielding an IC50 of 4.9 µm. Moreover, there were changes in the shape of treated parasites, including the formation of surface projections that subsequently detached from the cell, in addition to changes in the hydrogenosomes, endoplasmic reticulum and Golgi complex. We found [Zn(CTZ)2(Ac)2] to be a highly effective compound against T. vaginalis in vitro, suggesting its potential utility as an alternative chemotherapy for trichomoniasis.

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.

Trichomonicidal and parasite membrane damaging activity of bidesmosic saponins from Manilkara rufula.

The infection caused by Trichomonas vaginalis is the most common but overlooked non-viral sexually transmitted disease worldwide. Treatment relies on one class of drugs, the 5-nitroimidazoles, but resistance is widespread. New drugs are urgently needed. We reported the effect of crude and purified saponin fractions of Manilkara rufula against Trichomonas vaginalis. The compound responsible for antitrichomonal activity was isolated and identified as an uncommon bidesmosic saponin, Mi-saponin C. This saponin eliminated parasite viability without toxicity against the human vaginal epithelial line (HMVII). In addition, the isolated saponin fraction improved the metronidazole effect against a metronidazole-resistant isolate and dramatically reduced the cytoadherence of T. vaginalis to human cells. Investigation of the mechanism of death showed that the saponin fraction induced the parasite death due to profound membrane damage, inducing a disturbance of intracellular content without nuclear damage. To the best of our knowledge, this is the first report of antitrichomonal activity in the bidesmosic saponins of Manilkara rufula.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

BACKGROUND: Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES: In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS: We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS: It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS: Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

The effect of 3-(biphenyl-4-yl)-3-hydoxyquinuclidine (BPQ-OH) and metronidazole on Trichomonas vaginalis: a comparative study.

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ), a drug that presents some toxicity, causing undesirable side effects. In addition, an increase in metronidazole-resistant parasites has been reported. Thus, the development of alternative treatment is recommended. To date, the search for antiparasitic drugs has been based on different approaches: identification of active natural products, identification of parasite targets, and the use of available compounds active against other pathogenic microorganisms. Here, we analyzed the in vitro antiproliferative and ultrastructural effects on T. vaginalis of BPQ-OH, a hydroxiquinuclidine derivative that inhibits squalene synthase and is active against several protozoa and fungi. We also compared the effects of BPQ-OH on T. vaginalis and mammalian cells with those of MTZ. We found that BPQ-OH inhibits in vitro proliferation of T. vaginalis, with an IC50 of 46 µM after 24 h. Although this IC50 is 16 times higher than that of MTZ (1.8 µM), BPQ-OH is less toxic for human cell lines than MTZ, with LC50 values of 2,300 and 70 µM, and selective indexes of 50 and 39, respectively. Ultrastructural analyses demonstrated that BPQ-OH induced alterations in T. vaginalis, such as rounded and wrinkled cells, membrane blebbing and intense vacuolization, leading to cell death, whereas MTZ also caused significant changes, including a decrease in hydrogenosomes size and endoflagellar forms. Our observations identify BPQ-OH as a promising leading compound for the development of novel anti-T. vaginalis drugs and highlight the need for further testing this molecule using experimentally infected animals.

Evaluation of the effect of miltefosine on Trichomonas vaginalis.

Trichomonas vaginalis causes trichomoniasis in humans, a sexually transmitted disease commonly treated with metronidazole (MTZ). MTZ is known to cause undesirable side effects, and MTZ-resistant parasites have been reported. Thus, the development of an alternative treatment is desirable. Miltefosine (MLT) is an alkylphosphocholine synthetic lipid analogue that displays antiparasitic activity against Leishmania, Trypanosoma cruzi, Entamoeba histolytica, Acanthamoeba spp., Giardia lamblia, T. vaginalis and some fungi. Moreover, it has been used for oral treatment of visceral leishmaniosis in several countries. Here, we analysed the MLT-induced antiproliferative effect on T. vaginalis as well its effect on the fine structure and viability of the parasite. We observed a dose-dependent effect with an IC50 of 14.5 and 20 µM after 24 and 48 h, respectively. Furthermore, reversibility assays demonstrated that new incubations were necessary in order to maintain the antiproliferative effect. Ultrastructural analyses demonstrated that MLT induced several alterations, including the appearance of wrinkled and rounded cells, membrane blebbing, intense vacuolization and nuclear condensation, all indicative of cell death by apoptosis. In addition, the quantitative analyses of the viability assays using combined markers of live and dead cells demonstrated that treatment with the IC50 concentration of MLT significantly reduced the number of viable parasites compared with untreated cells. Taken together, these observations suggest that MLT is a promising compound for the treatment of trichomoniasis.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus.

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

IL-10 release by bovine epithelial cells cultured with Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus are parasitic protists of the human and bovine urogenital tracts, respectively. Several studies have described the cytotoxic effects of trichomonads on urogenital tract epithelial cells. However, little is known about the host cell response against trichomonads. The aim of this study was to determine whether T. foetus and T. vaginalis stimulated the release of the cytokine interleukin (IL)-10 from cultured bovine epithelial cells. To characterise the inflammatory response induced by these parasites, primary cultures of bovine oviduct epithelial cells were exposed to either T. vaginalis or T. foetus. Within 12 h after parasite challenge, supernatants were collected and cytokine production was analysed. Large amounts of IL-10 were detected in the supernatants of cultures that had been stimulated with T. foetus. Interestingly, T. vaginalis induced only a small increase in the release of IL-10 upon exposure to the same bovine cells. Thus, the inflammatory response of the host cell is species-specific. Only T. foetus and not T. vaginalis induced the release of IL-10 by bovine oviduct epithelial cells.

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection.

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.

Interaction of Trichomonas vaginalis and Tritrichomonas foetus with keratin: an important role in parasite infection

Trichomonas vaginalis and Tritrichomonas foetus are human and bovine parasites, respectively, that provoke the sexually transmitted disease trichomoniasis. These extracellular parasites adhere to the host epithelial cell surface. Although mucinases and proteases have been described as important proteins for parasite adhesion to epithelial cells, no studies have examined the role of the keratin molecules that cornify the vaginal epithelium. Here, we investigated the interaction of T. vaginalis and T. foetus with human keratin in vitro; additionally, adherence assays were performed in cattle with T. foetus to elucidate whether trichomonads were able to interact with keratin in vivo. We demonstrated that both T. vaginalisand T. foetusinteracted directly with keratin. Additionally, the trichomonads ingested and digested keratin, shedding new light on the Trichomonas infection process.

Lycorine induces cell death in the amitochondriate parasite, Trichomonas vaginalis, via an alternative non-apoptotic death pathway.

In this study, the mechanism of action of the pro-apoptotic alkaloid lycorine on an amitochondriate cell, the parasite Trichomonas vaginalis, was investigated. The cytotoxicity of lycorine against T. vaginalis was studied from 2.5 to 1000µM and several important ultrastructural alterations were observed by electron microscopy. Lycorine arrested the T. vaginalis cell cycle, although no hallmarks of apoptosis, such as apoptotic bodies, were observed. Consequently, the underlying mechanism of action fails to completely fulfill the criteria for apoptosis. However, some similarities to paraptotic cell death were observed.

Ultrastructural alterations induced by Δ(24(25))-sterol methyltransferase inhibitors on Trichomonas vaginalis.

Trichomonas vaginalis is an important human parasite that causes trichomoniasis, a cosmopolitan sexually transmitted disease. Currently, the treatment of choice for T. vaginalis infections is metronidazole. The increase in metronidazole-resistant parasites and undesirable side effects of this drug make the search for alternative chemotherapeutic approaches a priority for the management of trichomoniasis. Here, the antiproliferative and ultrastructural effects of sterol biosynthesis inhibitors against T. vaginalis were investigated. It was found that 22,26-azasterol (5 µM) and 24(R,S),25-epiminolanosterol (10 µM), known inhibitors of Δ(24(25))-sterol methyltransferase, exhibited antiproliferative effects on T. vaginalis trophozoites cultured in vitro. Morphological analyses showed that azasterols induced changes in the ultrastructure of T. vaginalis. The most significant alterations were (1) membrane blebbing and disruption, (2) wrinkled cells and (3) the formation of cell clusters. In addition, autophagic vacuoles, Golgi duplication arrest, an abnormal Golgi enlargement and damaged hydrogenosomes were also observed. Nonspecific cytotoxicity assays using the cultured mammalian cell lines Madin-Darby canine kidney cells showed no effect of the azasterols on the viability and proliferation of these cells at a concentration that significantly inhibited the proliferation of T. vaginalis, indicating a selective antiparasitic action. Taken together, these results suggest that azasterols could be important compounds in the development of novel chemotherapeutic approaches against T. vaginalis.

Candimine-induced cell death of the amitochondriate parasite Trichomonas vaginalis.

Candimine (1), an alkaloid from the bulbs of Hippeastrum morelianum, was found to be cytotoxic for the amitochondriate parasite Trichomonas vaginalis. Candimine (1) induced cell death with an unprecedented group of effects that failed to fulfill the criteria for apoptosis and apoptosis-like death already reported in trichomonads. Arrest of the parasite cell cycle, and morphologic and ultrastructural alterations, including marked cytoplasmic vacuolization, were induced by 1. The present findings suggest some similarities to paraptotic cell death, described for multicellular organisms. This study contributes to both a better understanding of the biological effects of 1 and T. vaginalis cell biology.

Methyl jasmonate induces cell death and loss of hydrogenosomal membrane potential in Trichomonas vaginalis.

Trichomonas vaginalis is an important human parasite of the urogenital tract. Jasmonates are a group of small lipids that are produced in plants and function as stress hormones. Naturally occurring methyl jasmonate (MJ) has been used to treat several types of cancer cells and it is cytotoxic to protistan parasites. It has been suggested that mitochondria are the target organelles of jasmonates. Here, we tested this drug against T. vaginalis. Although metronidazole has been the drug of choice for trichomoniasis, side effects from this treatment are common, and nausea and dizziness have been reported in up to 12% of patients. In addition, there has been increased recognition of resistance to metronidazole. We demonstrate here using flow cytometry, JC-1 and scanning and transmission electron microscopy that MJ induced the cell death of T. vaginalis parasites. Our results are discussed with previous findings published by others.

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis.

The parasite Trichomonas vaginalis causes one of the most common non-viral sexually transmitted infections in humans. The coexistence of different sexually transmitted diseases in the same individual is very common, such as vaginal infections by T. vaginalis in association with Mycoplasma fermentans or Mycoplasma hominis. However, the consequences and behavior of mycoplasma during trichomonad infections are virtually unknown. This study was undertaken to elucidate whether mycoplasmas enter and leave trichomonad cells and if so how. M. hominis was analyzed in different trichomonad isolates and the process of internalization and the pathway within the parasite was studied. Parasites naturally and experimentally infected with mycoplasmas were used and transmission electron microscopy, cytochemistry and PCR analyses were performed. The results show that: (1) M. hominis enters T. vaginalis cells by endocytosis; (2) some mycoplasmas use a terminal polar tip as anchor to the trichomonad plasma membrane; (3) some trichomonad isolates are able to digest mycoplasmas, mainly when the trichomonads are experimentally infected; (4) some fresh virulent isolates are able to maintain mycoplasmas as cohabitants in the cell's interior; (5) some mycoplasmas are able to escape from the vacuole to the trichomonad cytosol, and trichomonad plasma membrane budding suggested that mycoplasmas could leave the parasite cell.

Trichomonas adhere and phagocytose sperm cells: adhesion seems to be a prominent stage during interaction.

Tritrichomonas foetus and Trichomonas vaginalis are extracellular parasites of the urogenital tract of cattle and humans, respectively. They cause infertility and abortion, but there is no documented information on the susceptibility of bovine sperm cells to this cattle parasite. The aim of this present work was to study the effects provoked by T. foetus and T. vaginalis when in interaction with bovine and human sperm cells. The bovine and human spermatozoa were obtained from uninfected bulls and men, respectively, and were exposed to living trichomonads over different periods of time. Light microscopy, video microscopy, scanning, and transmission electron microscopy first revealed a tropism, then a close proximity followed by a tight adhesion between these two different cells. A decrease in the spermatozoa motility was observed as well intense semen agglutination. The adhesion between trichomonads to the sperm cell occurred either by the flagella or sperm head. Motile parasites were observed during the next 12 h, whereas sperm cells in contact with the parasites rapidly became immotile. The parasites were able to maintain the sperm cells attached to their cell surface, followed by phagocytosis. This process began with a tight membrane-membrane adhesion and the incorporation of the sperm cell within an intracellular vacuole. Afterwards, the sperm cell was gradually digested in lysosomes. Many trichomonads were injured and/or died on making contact with the spermatozoa possibly due to necrosis. Results from this study demonstrated that both T. foetus and T. vaginalis interact with sperm cells provoking damage and death of these reproductive cells. Differences in the behavior of both trichomonads were evident, showing that T. vaginalis was much more virulent than T. foetus. The possible role of trichomonads in reproductive failure is discussed.

Phagocytosis by Trichomonas vaginalis: new insights.

BACKGROUND INFORMATION: The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease. The phagocytic activity of this parasite has not been completely elucidated. In order to better understand the mechanisms of trichomonal phagocytosis, we have studied the in vitro capacity of T. vaginalis to phagocytose and degrade Saccharomyces cerevisiae cells. RESULTS AND CONCLUSIONS: To analyse the phagocytic ability and capacity, two isolates of T. vaginalis presenting different virulence grades were used. Complementary techniques, such as fluorescence microscopy, computer-based fluorescence analysis, scanning and transmission electron microscopy and the use of drugs that interfere with the actin microfilaments, were used in order to follow the behaviour of the actin cytoskeleton during phagocytosis of yeast cells by T. vaginalis. It was concluded that: (1) T. vaginalis changes its shape rapidly and engulfs the yeast cells, which are almost as large as the parasite; (2) long-term and fresh cultures are able to phagocytose, although the low-virulence strain JT demonstrated a lower activity when compared with the highly virulent T016 isolate; (3) the T016 strain exhibited an amoeboid morphology during the internalization of yeast cells in contrast with the JT strain; (4) attachment of yeast cells to the parasite occurs via the whole cell surface, including both anterior and recurrent flagella; (5) two forms of phagocytosis were observed: a 'sinking' process without any apparent participation of plasma membrane extensions and the classical phagocytosis where pseudopodia are extended toward the target cell; (6) the internalized S. cerevisiae are digested in lysosomes; (7) competitor sugars D-mannose or L-fucose inhibit the phagocytosis, and inhibition was 1.67 times higher in long-term cultured JT than that of the parasites from fresh isolate T016; (8) a thick layer of actin microfilaments was present underlying the plasma membrane, and especially in the pseudopodia and around the phagocytosed particles; (9) a dramatic change in the distribution pattern of fibrillar actin occurred during phagocytosis; (10) cytochalasin D depressed the phagocytosis; (11) a non-specific recognition and phagocytosis of yeast cells by T. vaginalis is mediated by a mannose receptor present on the parasite surface; (12) the phagocytic process may occur simultaneously during mitosis of the parasite.

HeLa cell nucleus, a source of thymidine for Trichomonas vaginalis growing in vitro.

Trichomonas vaginalis is a parasitic protist incapable of de novo purine and pyrimidine biosynthesis. The lack of these de novo syntheses of nucleotides is supplemented with purine and pyrimidine salvage pathways. Likewise, T. vaginalis is incapable of converting its ribonucleotides to deoxyribonucleotides. Therefore, the parasite must rely on the salvage of exogenous deoxyribonucleosides for DNA synthesis. It has been demonstrated that the parasite can incorporate external adenine and guanine in vitro, but no in vivo nucleotide source has been identified so far. Accordingly, we set out to determine if the parasite could incorporate 3H-thymidine from the nuclei of a cervical-derived cell line into its own DNA. By light and electron microscopy we found that the parasite was able to interact directly, both with mechanically isolated HeLa cell nuclei and with the nuclei released after the disruption of HeLa cell monolayers by the parasite. This study shows that T. vaginalis was capable of incorporating 3H-thymidine from labeled HeLa cells into its own DNA suggesting that the nuclei of this cervical cell line could be an in vivo source of nucleotides for T. vaginalis.

Trichomonads under Microscopy.

Trichomonads are flagellate protists, and among them Trichomonas vaginalis and Tritrichomonas foetus are the most studied because they are parasites of the urogenital tract of humans and cattle, respectively. Microscopy provides new insights into the cell biology and morphology of these parasites, and thus allows better understanding of the main aspects of their physiology. Here, we review the ultrastructure of T. foetus and T. vaginalis, stressing the participation of the axostyle in the process of cell division and showing that the pseudocyst may be a new form in the trichomonad cell cycle and not simply a degenerative form. Other organelles, such as the Golgi and hydrogenosomes, are also reviewed. The virus present in trichomonads is discussed.