Costain 1 (ARM19800.1) - The first identified protein of the costa of the pathogenic protozoan Tritrichomonas foetus.

Protists members of the Trichomonadidae and Tritrichomonadidae families include agents of trichomoniasis that constitute important parasitic diseases in humans and in animals of veterinary interest. One of the characteristic features of these eukaryotic microorganisms is that they contain a fibrous structure known as the costa as an important cytoskeleton structure, that differs in several aspects from other cytoskeleton structures found in eukaryotic cells. Previous proteomic analysis of an enriched costa fraction revealed the presence of several hypothetical proteins. Here we describe the localization of one of the most prevalent protein found in this previously made proteomic assay to confirm its presence in the costa of Tritrichomonas foetus. A peptide sequence of the hypothetical protein ARM19800.1 was selected for the production of specific polyclonal antibodies and its specificity was confirmed by Western Blotting using an enriched costa fraction. Next, the specific localization of the selected protein was evaluated by immunofluorescence and electron microscopy immunocytochemistry. Our observations clearly showed that the ARM 19800.1 protein is indeed localized in the costa and displays an almost periodic labeling pattern. Since this is the first protein identified in the costa, it was designated as costain 1. A better understanding of a structure as peculiar as the costa is of great biological and evolutionary importance due to the fact that it contains unique proteins, it may represent a possible chemotherapy target and it may correspond to antigens of interest in immunodiagnosis and/or vaccine development.

New advances in scanning microscopy and its application to study parasitic protozoa.

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities.

Iron-modulated pseudocyst formation in Tritrichomonas foetus.

Iron is an essential element for the survival of trichomonads during host-parasite interaction. The availability of this metal modulates several metabolic pathways of the parasites and regulates the expression of virulence factors such as adhesins and proteolytic enzymes. In this study, we investigated the effect of iron depletion on the morphology and life cycle of Tritrichomonas foetus. Scanning and transmission electron microscopy analyses revealed that depletion of iron from the culture medium (named TYM-DIP inducer medium) induces morphological transformation of typical pear-shaped trophozoites into spherical and non-motile pseudocysts. Remarkably, inoculation of pseudocysts into an iron-rich medium (standard TYM medium), or addition of FeSO4 to a TYM-DIP inducer medium reverted the morphological transformation process and typical trophozoites were recovered. These results show that pseudocysts are viable forms of the parasite and highlight the role of iron as a modulator of the parasite phenotype. Although iron is required for the survival of T. foetus, iron depletion does not cause a cellular collapse of pseudocysts, but instead induces phenotypic alterations, probably in order to allow the parasite to survive conditions of nutritional stress. Together, these findings support previous studies that suggest pseudocysts are a resistance form in the life cycle of T. foetus and enable new approaches to understanding the multifactorial role of iron in the cell biology of this protozoan parasite.

Host origin determines pH tolerance of Tritrichomonas foetus isolates from the feline gastrointestinal and bovine urogenital tracts.

The ability for protozoan parasites to tolerate pH fluctuations within their niche is critical for the establishment of infection and require the parasite to be capable of adapting to a distinct pH range. We used two host adapted Tritrichomonas foetus isolates, capable of infecting either the digestive tract (pH 5.3-6.6) of feline hosts or the reproductive tract (pH 7.4-7.8) of bovine hosts to address their adaptability to changing pH. Using flow cytometry, we investigated the pH tolerance of the bovine and feline T. foetus isolates over a range of physiologically relevant pH in vitro. Following exposure to mild acid stress (pH 6), the bovine T. foetus isolates showed a significant decrease in cell viability and increased cytoplasmic granularity (p-value < 0.003, p-value < 0.0002) compared to pH 7 and 8 (p-value > 0.7). In contrast, the feline genotype displayed an enhanced capacity to maintain cell morphology and viability (p-value > 0.05). Microscopic assessment revealed that following exposure to a weak acidic stress (pH 6), the bovine T. foetus transformed into rounded parasites with extended cell volumes and displays a decrease in viability. The higher tolerance for acidic extracellular environment of the feline isolate compared to the bovine isolate suggests that pH could be a critical factor in regulating T. foetus infections and host-specificity.

New insights on the Golgi complex of Tritrichomonas foetus.

Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

Tritrichomonas foetus: characterisation of ecto-phosphatase activities in the endoflagelar form and their possible participation on the parasite's transformation and cytotoxicity.

The protist parasite Tritrichomonas foetus displays a pear-shaped (PS) and a pseudocystic or endoflagellar form (EFF). Here, we characterised the ecto-phosphatase activity on the surface of EFF and compare its biochemical properties to that of the PS regarding rate of substrate hydrolysis, pH activation profile and sensitivity to well-known phosphatases inhibitors. Two strains exhibiting low- and high-cytotoxicity were used. The enzyme activities of PS and EFF exhibited similar characteristics of protein tyrosine phosphatases (PTP). However, the ecto-phosphatase activities for both forms presented distinct kinetic parameters and different inhibition patterns by PTP inhibitors, suggesting the presence of distinct ecto-enzyme activities between PS and EFF, as well, between both strains. Ultrastructural cytochemistry confirmed the differential distribution of the ecto-phosphatase activity during the EFF transformation. An increase in the percentage of the EFF resulted in a proportional increase in the ecto-phosphatase activity. During EFF reversion, ecto-phosphatase activity decreased and was restored to the level found in the parasites before EFF induction. PS and EFF from the high-cytotoxic strain exhibited higher ecto-phosphatase activities than PS and EFF from the low-cytotoxic strain, respectively. In both strains, the EFF was more cytotoxic and exhibited higher ecto-phosphatase activity when compared to the PS. A large part of the ecto-phosphatase activities of EFF from both strains and PS from the high-cytotoxic strain was irreversibly inhibited when the parasites were pre-treated with a specific antibody against amoebic PTP (anti-EhPRL). Immunoreaction assays revealed that the anti-EhPRL antibody cross-reacted with a 24-kDa protein differentially expressed on the cell surface of PS and EFF T. foetus. A positive correlation was observed between the surface expression of 24-kDa protein and ecto-phosphatase activity. Irreversible inhibition of a part of the ecto-phosphatase activities partially blocked the EFF induction and the cytotoxic effects exerted by both forms. These results suggest that the ecto-phosphatase activities could play a role on the EFF transformation and cytotoxicity of T. foetus.

High-resolution scanning electron microscopy of the cytoskeleton of Tritrichomonas foetus.

Tritrichomonas foetus is a pathogenic protozoan that causes bovine trichomoniasis. In addition to its importance in veterinary medicine, this parasite is also a good representative of one the earliest eukaryotic cells available for study. T. foetus contains organelles that are common to all eukaryotic cells as well as uncommon cell structures such as hydrogenosomes and a complex and elaborate cytoskeleton that constitutes the mastigont system. The mastigont system is mainly formed by several proteinaceous structures that are associated with basal bodies, the pelta-axostylar complex and the costa. Although the structural organization of trichomonad cytoskeletons has been analyzed using several techniques, observation using a new generation of scanning electron microscopes with a resolution of 0.8nm has allowed more detailed visualization of the three-dimensional organization of the mastigont system. Moreover, this study revealed the presence of new structures, such as the costa accessory filament, and the presence of two groups of microtubules that form the pelta-axostylar system.

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.

The hydrogenosome peripheral vesicle: similarities with the endoplasmic reticulum.

The hydrogenosome, an organelle that produces molecular hydrogen and ATP from the oxidation of pyruvate or malate under anaerobic conditions, presents some characteristics common to mitochondria. The hydrogenosome of Tritrichomonas foetus, a cattle parasite, is a spherical organelle that presents a peripheral vesicle the origin and behavior of which is poorly known. In this article it is reported an ultrastructural and microanalytical study using energy dispersive X-ray analysis, 3D reconstruction and cytochemistry of the hydrogenosome peripheral vesicle and then compare the results with the endoplasmic reticulum and the nuclear envelope of T. foetus. Similarities between the hydrogenosome peripheral vesicle and the ER are presented. This study included: (1) the detection of ER enzymes by cytochemistry, such as glucose-6-phosphatase, IDPase, acid phosphatase and Ca(2+) -ATPase; (2) elemental composition by X-ray microanalysis and the mapping of calcium, phosphorus and oxygen in both ER and hydrogenosome peripheral vesicle; (3) freeze-fracture; (4) TEM of routine and cryofixed cells by high-pressure freezing and freeze-substitution; (5) 3D reconstruction, (6) monoclonal antibody anti-trichomonads ER; and (6) other cytochemical techniques that detects ER, such as the ZIO and lectins. We found a similar composition of the tested enzymes and other elements present in the ER when compared with the hydrogenosome's peripheral vesicle. It was concluded that, like mitochondria, hydrogenosome presents relationships with the ER, especially the peripheral vesicle.

Effects of three benzimidazoles on growth, general morphology and ultrastructure of Tritrichomonas foetus.

Tritrichomonas foetus is a venereal pathogen of cattle, which causes infertility, early embryonic death or abortion. In order to evaluate the potential trichomonicidal activity of benzimidazoles, the effects of thiabendazole, mebendazole and albendazole were analyzed on the multiplication, general morphology and ultrastructure of T. foetus. It was found that mebendazole presented the highest IC(50%) (2.3 microM), when compared with albendazole (IC(50%)=9.4 microM) and thiabendazole (IC(50%)=142.6 microM), and that such effects were irreversible. Concerning microscopic analysis, thiabendazole- and mebendazole-treated cells presented increased volume, internalization of the flagella, disruption or multiplication of the nucleus, multiple organelles and cytoplasmic vacuolization. Albendazole-treated cells exhibited slight alterations, because the parasite became slightly rounded, its flagella were not internalized but the cytoplasm was vacuolated. Mebendazole was indeed highly effective as an in vitro trichomonicidal agent, and this might open up new possibilities for the use of mebendazole in the therapy of bovine trichomoniasis.

Ultrastructural changes in Tritrichomonas foetus after treatments with AlPcS4 and photodynamic therapy.

The Tritrichomonas foetus is an amitochondrial parasitic protist which causes bovine trichomoniasis, a major sexually transmitted disease in cattle. No effective drugs for this disease have been approved to this date. Photodynamic therapy (PDT) is an experimental treatment that shows great potential for treating bacteria, fungi, yeasts, and viruses. However, the cytotoxic effect of PDT on protozoan has been poorly studied. In this study, PDT with aluminum phthalocyanine tetrasulfonated (AlPcS4) photosensitizer was efficient in killing T. foetus. The mode of cell death in T. foetus after PDT was investigated by transmission electron microscopy. Morphological changes, such as membrane projections, nucleus fragmentation with peripheral masses of heterochromatin, endoplasmic reticulum proliferation, intense cytoplasmic vacuolization, fragmented axostyle-pelta complex, and internalized flagella could be observed. This is the first report to demonstrate cell death in T. foetus after PDT, and thus will open up new lines of investigation to develop new treatments for bovine trichomoniasis.

First record of natural Tritrichomonas foetus infection of the feline uterus.

Tritrichomonas foetus was found in the uterus of a cat with pyometra and in the faeces of three other cats in the same household, one of which had chronic diarrhoea. This is the first report of a feline uterine infection with T. foetus and also the first time T. foetus has ever been diagnosed in animals in Norway. The diagnosis was made by microscopic examination and sequencing studies.

Ca(2+) sequestering in the early-branching amitochondriate protozoan Tritrichomonas foetus: an important role of the Golgi complex and its Ca(2+)-ATPase.

Total membrane vesicles isolated from Tritrichomonas foetus showed an ATP-dependent Ca(2+) uptake, which was not sensitive to 10 microM protonophore FCCP but was blocked by orthovanadate, the inhibitor of P-type ATPases (I(50)=130 microM), and by the Ca(2+)/H(+) exchanger, A-23187. The Ca(2+) uptake was prevented also by thapsigargin, an inhibitor of the SERCA Ca(2+)-ATPases. The sensitivity of the Ca(2+) uptake by the protozoan membrane vesicles to thapsigargin was similar to that of Ca(2+)-ATPase from rabbit muscle sarcoplasmic reticulum. Fractionation of the total membrane vesicles in sucrose density gradient revealed a considerable peak of Ca(2+) transport activity that co-migrated with the Golgi marker guanosine diphosphatase (GDPase). Electron microscopy confirmed that membrane fractions of the peak were enriched with the Golgi membranes. The Golgi Ca(2+)-ATPase contributed to the Ca(2+) uptake by all membrane vesicles 80-85%. We conclude that: (i) the Golgi and/or Golgi-like vesicles form the main Ca(2+) store compartment in T. foetus; (ii) Ca(2+) ATPase is responsible for the Ca(2+) sequestering in this protozoan, while Ca(2+)/H(+) antiporter is not involved in the process; (iii) the Golgi pump of this ancient eukaryotic microorganism appears to be similar to the enzymes of the SERCA family by its sensitivity to thapsigargin.

Are Tritrichomonas foetus and Tritrichomonas suis synonyms?

Tritrichomonas suis, a tritrichomonad of pigs, and the related species Tritrichomonas foetus, a tritrichomonad of cattle, are morphologically identical. The taxonomic relationship between these two tritrichomonads has been questioned ever since they were established as distinct species in 1843 and 1928, respectively. Here, we compare the similarities of morphology, ultrastructure, distribution, host specificity, characteristics of in vitro cultivation, immunology, biochemistry and analysis of molecular data from published sources between these two species. All data indicate that these two tritrichomonad species are identical. Thus, we propose that T. foetus and T. suis are synonyms.

Appearance of virus-like particles in Tritrichomonas foetus after drug treatment.

Tritrichomonas foetus is a parasitic protist that infects the urogenital tract of cattle causing bovine trichomonosis. Virus-like particles (VLPs) in protozoa have been reported in several parasites including Trichomonas vaginalis, a human flagellate, but viruses were never described in T. foetus so far. Herein we show for the first time the presence of VLPs in T. foetus after several drug treatments. They were detected by electron microscopy and were confirmed by immunofluorescence microscopy using antibodies anti-virus proteins. These VLPs were always observed in clusters of variable size. Their preferential locations were at the cell periphery, close to the axostyle, and interestingly in some cases, inside the nucleus. Their appearance occurred when the parasites were under drug-treatments, such as cytoskeleton-affecting drugs (colchicine, vinblastine, taxol, nocodazole, and griseofulvin) or drugs inducing cell death, such as lactacystin and H(2)O(2). We propose that cytoskeleton participates in trichomonads of the process of virus release or maturation. These virus particles were not described previously probably because they were either in low amount or in a latent state.

Changes in the structural organization of the cytoskeleton of Tritrichomonas foetus during trophozoite-pseudocyst transformation.

Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. It grows in axenic media as a trophozoite with a pear-shaped body, three anterior flagella, and one recurrent flagellum. However, under some well-controlled experimental conditions in vitro, as well as in vivo in infected bulls, the parasite acquires a spherical or elliptical shape, and the flagella are internalized but the cells do not display a cyst wall. This form, known as the endoflagellar or pseudocystic form, is viable, and can be transformed back to trophozoites with pear-shaped body. We used confocal laser scanning microscopy, and high resolution scanning electron microscopy to examine the changes that take place in the protozoan cytoskeleton during trophozoite-pseudocyst transformation. Results confirmed previous studies and added new structural information to the organization of cytoskeletal structures during the transformation process. We observed that changes take place in the pseudocysts' axostyle and costa, which acquired a curved shape. In addition, the costa of multinucleated/polymastigont pseudocysts took variable conformations while curved. The costa accessory structure, as well as a network of filaments connecting this structure to the region where the recurrent flagellum associates to the protozoan body, was not seen in pseudocysts. In addition, the axostyle was fragmented during trophozoite-pseudocyst transformation.

Isolation and biochemical characterization of the costa of Tritrichomonas foetus.

The costae are cytoskeletal structures found in Trichomonadidae. Both the structural organization and composition of this organelle are still unknown. In the present work we have introduced a new methodology for the costa isolation. Using sucrose density-gradient centrifugation an enriched costa fraction was obtained. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the costa contains several proteins, with major bands corresponding to apparent molecular masses of 122, 115, 112, 93, 87, 82, 59, 52, 44, 41, 32, and 26 kDa. No significant amount of carbohydrates was detected in the costa fraction. The fractionation methodology described here has the advantage of using normal centrifugation methods and is being applied to trichomonas in the size range of Tritrichomonas foetus and Trichomonas vaginalis.

Structure and division of the Golgi complex in Trichomonas vaginalis and Tritrichomonas foetus.

We present observations on the fine structure and the division process of the Golgi complex in the protists Trichomonas vaginalis and Tritrichomonas foetus, parasites of the urogenital tract of humans and cattle, respectively. The Golgi in trichomonads is a prominent structure, associated with striated parabasal filaments to which this organelle seems to be connected. We followed by immunofluorescence and electron microscopy the Golgi in interphasic and mitotic cells. Ultrastructural studies were performed using fast-freezing fixation, immunocytochemistry using antisera to the known adhesins AP65 and AP51, cytochemistry (acid phosphatase, Ca++-ATPase, zinc iodide-osmium tetroxide technique (ZIO), for analysis of distribution of the endoplasmic reticulum and Golgi complex, and Thiéry's techniques), routine and serial thin-sections. Three-dimensional reconstruction, NBD-ceramide, fluorescent lectin (WGA) and nocodazole treatments were also used. We demonstrate that: (1) the Golgi in trichomonads is a single-copy organelle; (2) presents a fenestrated structure; (3) is formed by 8-12 saccules; (4) is connected to the parabasal filaments by thin filamentous bridges; (5) by cytochemistry, presents a positive reaction for the lectin WGA, Ca++-ATPase, acid phosphatase, ZIO and Thiéry's techniques; (6) does not appear to break down at any point of the cell cycle; (7) elongates during the cell cycle by lateral growth; (8) is labeled by anti-glutamylated tubulin antibodies, but it is not fragmented by nocodazole treatment; (9) before mitosis, the already elongated Golgi ribbon undergoes progressive medial fission, cisternae by cisternae, starting at the cisternae adjacent to the cell surface and ending with the cis-most cisternae; (10) the Golgikinesis originates two small Golgi ribbons; (11) the Golgi is intensely labeled with the antisera to the AP65 and AP51 adhesins in T. vaginalis, thus seeming to be a key station in the production of adhesins.

Pseudocysts in trichomonads--new insights.

Tritrichomonas foetus and Trichomonas vaginalis, parasitic protists of the urogenital tract, display a trophozoite and a pseudocyst stage. The ultrastructure of the trophozoite was compared with the pseudocyst form. The latter appears under unfavorable environmental conditions when the flagella are internalized, and a true cell wall is not formed. Although some authors consider this form as a degenerate stage, the cell behaves as a resistant form. Pseudocysts were found in natural culture conditions and also under induction by hydroxyurea or cycles of cooling and warming cultures. They were studied by light and scanning and transmission electron microscopy, using immunofluorescence and videomicroscopy. This report presents evidence that the trichomonad pseudocysts appear under stress conditions and that they are competent to divide. Pseudocysts differ from trophozoites in that: (1) the flagella are located in endocytic vacuoles and remain beating; (2) the axostyle and the costa are not depolymerized but present a curved shape; (3) the axostyle does not exhibit staining with antitubulin antibodies when the mitotic spindle is observed; (4) the mitotic process occurs within pseudocysts but differs from that described for trophozoites; (5) a nuclear canal is formed connecting the two spindle poles; and (6) the process is reversible if the cells are transferred to fresh medium.