Characterization of a new extra-axonemal structure in the Giardia intestinalis flagella.

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature - it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

Stimulation of microvesicle secretion in Trichomonas vaginalis.

Trichomonas vaginalis is an extracellular flagellate protozoan and the etiological agent of human trichomoniasis, a sexually transmitted infection (STI) with a high incidence. Several reports have shown that this protozoan releases microvesicles into the culture medium, which show high potential in modulating cell-to-cell communication and the host response to infections. However, the biogenesis of these vesicles has not been analyzed in detail. In the present study, high-resolution ion scanning microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the surface of control cells and cells incubated in the presence of Ca2+ alone or with A 23187 calcium ionophore. Two different strains of T. vaginalis were analyzed. Most control cells displayed relatively smooth surfaces, whereas cells incubated with Ca2+ had many surface projections of variable shape and size (from 40 nm to around 1 µm). Quantitative analyses were performed directly in the scanning electron microscope and showed a significant increase in the number of cells with surface projections after incubation in the presence of calcium. TEM showed that treated cells presented several cytoplasmic multivesicular structures, suggesting membrane fusion and exosomes in the extracellular medium. The amount and size of the released vesicles were quantitatively analyzed using light scattering and TEM on negatively stained samples. The observations show that incubation of both parasite strains in the presence of Ca2+ significantly increased the release of microvesicles into the extracellular medium in a time-dependent process. Sequential incubation in the presence of Ca2+ and the calcium ionophore A23187 increases the presence of vesicles on the parasite surface only at a short incubation time (5 min). Transmission electron microscopy showed that at least part of the vesicles are originated from cytoplasmic multivesicular structures. This information contributes to a better understanding of the biogenesis of extracellular vesicles secreted by T. vaginalis.

Effects of SQ109 on Trichomonas vaginalis.

Trichomonas vaginalis is a protozoan that causes human trichomoniasis, a sexually transmitted infection (STI) that affects approximately 278 million people worldwide. The current treatment for human trichomoniasis is based on 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, known as Metronidazole (MTZ). Although effective in eliminating parasitic infection, MTZ is related to serious adverse effects and is not recommended during pregnancy. In addition, some strains are resistant to 5'-nitroimidazoles, prompting the development of alternative drugs for trichomoniasis. Here we show that SQ109 [N-adamantan-2-yl-N'-((E)-3,7-dimethyl-octa- 2,6-dienyl)-ethane-1,2-diamine], a drug under development (antitubercular drug candidate that completed Phase IIb/III) for the treatment of tuberculosis, and previously tested in Trypanosoma cruzi and Leishmania. SQ109 inhibited T.vaginalis growth with an IC50 of 3.15 µM. We used scanning and transmission electron microscopy to visualize the ultrastructural alterations induced by SQ109. The microscopy analysis showed morphological changes on the protozoan surface, where the cells became rounded with increasing surface projections. In addition, the hydrogenosomes increased their size and area occupied in the cell. Furthermore, the volume and a significant association of glycogen particles with the organelle were seen to be altered. A bioinformatics search was done about the compound to find its possible targets and mechanisms of action. Our observations identify SQ109 as a promising compound against T. vaginalis in vitro, suggesting its potential utility as an alternative chemotherapy for trichomoniasis.

Expansion Microscopy of trichomonads.

Light microscopy has significantly advanced in recent decades, especially concerning the increased resolution obtained in fluorescence images. Here we present the Expansion Microscopy (ExM) technique in two parasites, Trichomonas vaginalis and Tritrichomonas foetus, which significantly improved the localization of distinct proteins closely associated with cytoskeleton by immunofluorescence microscopy. The ExM techniques have been used in various cell types, tissues and other protist parasites. It requires the embedment of the samples in a swellable gel that is highly hydrophilic. As a result, cells are expanded 4.5 times in an isotropic manner, offering a spatial resolution of ∼70 nm. We used this new methodology not only to observe the structural organization of protozoa in more detail but also to increase the resolution by immunofluorescence microscopy of two major proteins such as tubulin, found in structures formed by microtubules, and costain 1, the only protein identified until now in the T. foetus's costa, a unique rod-shaped like structure. The individualized microtubules of the axostyle were seen for the first time in fluorescence microscopy and several other details are presented after this technique.

Nanoarchitecture of the ventral disc of Giardia intestinalis as revealed by high-resolution scanning electron microscopy and helium ion microscopy.

The parasitic protozoan Giardia intestinalis, the causative agent of giardiasis, presents a stable and elaborated cytoskeleton, which shapes and supports several intracellular structures, including the ventral disc, the median body, the funis, and four pairs of flagella. Giardia trophozoite is the motile form that inhabits the host small intestine and attaches to epithelial cells, leading to infection. The ventral disc is considered one important element of adhesion to the intestinal cells. It is adjacent to the plasma membrane in the ventral region of the cell and consists of a spiral layer of microtubules and microribbons. In this work, we studied the organization of the cytoskeleton in the ventral disc of G. intestinalis trophozoites using high-resolution scanning electron microscopy or helium ion microscopy in plasma membrane-extracted cells. Here, we show novel morphological details about the arrangement of cross-bridges in different regions of the ventral disc. Results showed that the disc is a non-uniformly organized structure that presents specific domains, such as the margin and the ventral groove region. High-resolution scanning electron microscopy allowed observation of the labeling pattern for several anti-tubulin antibodies using secondary gold particle-labeled antibodies. Labeling in the region of the emergence of the microtubules and supernumerary microtubules using an anti-acetylated tubulin antibody was observed. Ultrastructural analysis and immunogold labeling for gamma-tubulin suggest that disc microtubules originate from a region bounded by the bands of the banded collar and merge with microtubules formed at the perinuclear region. Actin-like filaments and microtubules of the disc are associated, showing an interconnection between elements of the cytoskeleton of the trophozoite.

Giardia intestinalis and its endomembrane system.

Giardia intestinalis has unique characteristics, even in the absence of certain organelles. For instance, Golgi and mitochondria are not found. On the other hand, there is a network of peripheral vacuoles (PVs) and mitosomes. The endoplasmic reticulum (ER), nuclear membrane, peroxisomes, and lipid bodies are present. The peripheral vacuole system seems to play several simultaneous roles. It is involved in the endocytic activity of the trophozoite but also has characteristics of early and late endosomes and even lysosomes, establishing a connection with the ER. Some of the PVs contain small vesicles, acting as multivesicular bodies, including the release of exosomes. The mitosomes are surrounded by two membranes, divide during mitosis, and are distributed throughout the cell. They do not contain DNA, enzymes involved in the citric acid cycle, respiratory chain, or ATP synthesis. However, they contain the iron-sulfur complex and transporters as TOM and TIM. Some mitosomes are linked to flagellar axonemes through a fibrillar connection. During encystation, two types of larger cytoplasmic vesicles appear. One originating from the ER contains the cyst wall proteins. Another contains carbohydrates. Both migrate to the cell periphery and fuse with plasma membrane secreting their contents to give rise to the cell wall.

Effects of amiodarone, amioder, and dronedarone on Trichomonas vaginalis.

Trichomonas vaginalis is a protozoan that causes human trichomoniasis, the most common non-viral sexually transmitted infection (STI) affecting approximately 278 million people worldwide. The current treatment for trichomoniasis is based on 1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole, known as metronidazole (MTZ). Although effective in clearing the parasite infection, MTZ is related to provoking severe side effects, and it is not recommended during pregnancy. In addition, some strains present resistance to 5'-nitroimidazoles, making urgent the development of alternative drugs for trichomoniasis. Amiodarone, an antiarrhythmic drug, exerts a significant anti-parasite effect, mainly due to its interference with calcium homeostasis and the biosynthesis of sterols. Therefore, we decided to test the effect of amiodarone and two other related compounds (amioder and dronedarone) on T. vaginalis. Our observations show that amiodarone stimulated, rather than inhibited, parasite growth, induced cell aggregation, and glycogen accumulation. Furthermore, the other two compounds displayed anti-parasite activity with IC50 of 3.15 and 11 µM, respectively, and the apoptosis-like process killed the cells. In addition, cells exhibited morphological changes, including an effect on hydrogenosomes structure.

Giardia intestinalis can interact, change its shape and internalize large particles and microorganisms.

Giardia intestinalis is a parasitic protozoan that inhabits its vertebrate hosts' upper small intestine and is the most common cause of waterborne diarrhoea worldwide. Giardia trophozoites present few organelles, and among them, they possess peripheral vesicles (PVs), which are considered an endosomal-lysosomal system. All experimental procedures carried out until now indicate that Giardia ingests macromolecules by fluid-phase and receptor-mediated endocytic pathways. Still, there is no description concerning the interaction and ingestion of large materials. Here, we tested Giardia's capacity to interact with large particles; once, in vivo, it inhabits an environment with a microbiota. We tested protozoan interaction with yeasts, bacteria, latex beads, ferritin and albumin, in different times of interaction and used several microscopy techniques (light microscopy, scanning electron microscopy and transmission electron microscopy) to follow their fate. Giardia interacted with all of the materials we tested. Projections of the plasma membrane similar to pseudopods were seen. As albumin, small markers were found in the PVs while the larger materials were not seen there. Large vacuoles containing large latex beads were detected intracellularly. Thus, we observed that: (1) Giardia interacts with large materials; (2) Giardia can display an amoeboid shape and exhibit membrane projections when in contact with microorganisms and large inorganic materials; (3) the region of the exit of the ventral flagella is very active when in contact with large materials, although all cell surface also present activity in the interactions; (4) intracellular vacuoles, which are not the PVs, present ingested large beads.

Observation of Giardia sp. in the termite gut of Heterotermes tenuis.

Giardia comprises one genus with several morphologically distinct species described in mammals (including humans, marsupials, rodents), birds, and amphibians. This group of protists provokes diarrhoea diseases in humans and animals worldwide. Transmission of the parasite occurs through the faecal-oral route. Regarding the presence of Giardia in invertebrates, some works have shown that flies can transmit Giardia cysts by contact and transport between contaminated faeces and food. In this way, flies would eventually transmit this parasite. To date, Giardia's presence in the gut of other invertebrates has not been described in the literature. Here we show by first time, using scanning electron microscopy, the presence of Giardia-like trophozoites in the gut of termite Heterotermes tenuis. Two groups of Giardia were found based exclusively on the size and the flange shape of the protozoa: one presented eight flagella, a ventral disc, size, and shape very similar to Giardia intestinalis. In contrast, other cells were smaller and showed some differences in the external morphology. We cannot exclude the possibility that they correspond to the same species and that these differences result from protozoan heterogeneity.

The peripheral vesicles gather multivesicular bodies with different behavior during the Giardia intestinalis life cycle.

Giardia intestinalis presents an intriguing endomembrane system, which includes endoplasmic reticulum and peripheral vesicles (PVs). The PVs have previously been considered to be organelles that display early and late endosomal and lysosomal properties. Some of these vesicles accumulate macromolecules ingested by the protozoan and show acid phosphatase activity. It has been previously shown that the parasite releases microvesicles, which contribute to giardiasis pathogenesis; however, the vesicles' origin and the way in which they are released by the parasite still remain unclear. In this study, we induced the parasites to encyst in vitro and analyzed these events using advanced electron microscopy techniques, including focused ion beam and electron microscopy tomography followed by three-dimensional reconstruction, in order to better understand protozoal multivesicular body (MVB) biogenesis. In addition, we performed an ultrastructural analysis of phosphatase activity during differentiation. We demonstrated that some vegetative trophozoites' PVs exhibited morphological characteristics of MVBs with a mean diameter of 50 nm, containing intraluminal vesicles (ILVs).

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.

TfVPS32 Regulates Cell Division in the Parasite Tritrichomonas foetus.

The flagellated protist Tritrichomonas foetus is a parasite that causes bovine trichomonosis, a major sexually transmitted disease in cattle. Cell division has been described as a key player in controlling cell survival in other cells, including parasites but there is no information on the regulation of this process in T. foetus. The regulation of cytokinetic abscission, the final stage of cell division, is mediated by members of the ESCRT (endosomal sorting complex required for transport) machinery. VPS32 is a subunit within the ESCRTIII complex and here, we report that TfVPS32 is localized on cytoplasmic vesicles and a redistribution of the protein to the midbody is observed during the cellular division. In concordance with its localization, deletion of TfVPS32 C-terminal alpha helices (α5 helix and/or α4-5 helix) leads to abnormal T. foetus growth, an increase in the percentage of multinucleated parasites and cell cycle arrest at G2/M phase. Together, these results indicate a role of this protein in controlling normal cell division.

G-CSF-Induced Suppressor IL-10+ Neutrophils Promote Regulatory T Cells That Inhibit Graft-Versus-Host Disease in a Long-Lasting and Specific Way.

Acute graft-versus-host disease (aGVHD) is the main complication of allogeneic hematopoietic stem cell transplantation, and many efforts have been made to overcome this important limitation. We showed previously that G-CSF treatment generates low-density splenic granulocytes that inhibit experimental aGVHD. In this article, we show that aGVHD protection relies on incoming IL-10+ neutrophils from G-CSF-treated donor spleen (G-Neutrophils). These G-Neutrophils have high phagocytic capacity, high peroxide production, low myeloperoxidase activity, and low cytoplasmic granule content, which accounts for their low density. Furthermore, they have low expression of MHC class II, costimulatory molecules, and low arginase1 expression. Also, they have low IFN-γ, IL-17F, IL-2, and IL-12 levels, with increased IL-10 production and NO synthase 2 expression. These features are in accordance with the modulatory capacity of G-Neutrophils on regulatory T cell (Treg) generation. In vivo, CD25+ Treg depletion shortly after transplantation with splenic cells from G-CSF-treated donors blocks suppression of aGVHD, suggesting Treg involvement in the protection induced by the G-Neutrophils. The immunocompetence and specificity of the semiallogeneic T cells, long-term after the bone marrow transplant using G-Neutrophils, were confirmed by third-party skin graft rejection; importantly, a graft-versus-leukemia assay showed that T cell activity was maintained, and all of the leukemic cells were eliminated. We conclude that G-CSF treatment generates a population of activated and suppressive G-Neutrophils that reduces aGVHD in an IL-10- and Treg-dependent manner, while maintaining immunocompetence and the graft versus leukemia effect.

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

BACKGROUND INFORMATION: The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. RESULTS: Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. CONCLUSIONS: The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. SIGNIFICANCE: Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvß3/VEGFR2 Axis.

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvß3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc.

The fungal metabolite gliotoxin inhibits proteasome proteolytic activity and induces an irreversible pseudocystic transformation and cell death in Tritrichomonas foetus.

Proteasomal proteolysis is required for a wide range of cellular processes, including protein quality control, cell cycle progression, cell death and metabolic adaptation to environment changes or stress responses. Proteasome inhibitors are useful compounds for determining the roles of proteasome in eukaryotic cells. Here, we investigated the effects of gliotoxin, a proteasome inhibitor, on the cell growth, replication, ultrastructure, DNA integrity and proteasomal proteolytic activity of the protist parasite Tritrichomonas foetus. The effect of gliotoxin on the transformation of T. foetus to endoflagellar form (EFF), also known as pseudocyst, was investigated. Gliotoxin inhibited the culture growth, arrested cell cycle, and provoked a trichomonacidal effect in a dose-dependent manner. Parasites treated with gliotoxin displayed features typical of cell death, such as membrane blebbing, concentric membrane whorls containing remnants of organelles, intense cytosolic and nuclear vacuolisation, chromatin condensation, DNA fragmentation, cytoplasmic disintegration and plasma membrane disruption. The proteasomal peptidase activity was inhibited by gliotoxin in a dose-dependent manner. Gliotoxin treatment also induced an irreversible EFF transformation in a dose/time-dependent manner. We compared morphological characteristics between gliotoxin- and cold-induced EFF parasites. Our results suggest that gliotoxin could induce EFF transformation by a mechanism distinct from that provoked by cold temperature. This study further contributes to a better understanding of the role of proteasome system in cell cycle, cell death and EFF transformation in T. foetus.

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites.

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.