Lactoferrin-binding proteins of Tritrichomonas foetus.

Tritrichomonas foetus is a common, sexually transmitted, protozoan parasite of cattle. It has an essential requirement for iron, which it obtains from host lactoferrin. However, specific lactoferrin-binding protein receptors have not yet been identified in T. foetus. To differentiate specific and nonspecific binding of lactoferrin, lactoferrin affinity chromatography and Western blotting was used to identify metabolically or surface-labeled T. foetus lactoferrin-binding proteins. Bovine lactoferrin was shown to bind more efficiently than human lactoferrin, and each of these bound much better than bovine transferrin. This is relevant because T. foetus is both species-specific and only infects the mucosal surface of the reproductive tract, which has little transferrin. Whereas the majority of lactoferrin binding was specific, competitive inhibition studies showed that nonspecific, charge-related binding of lactoferrin to T. foetus may also be involved. In the presence of bovine cervical mucus, binding of lactoferrin to T. foetus was diminished, suggesting that mucus has an effect on lactoferrin binding. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surface biotinylated proteins affinity-purified on lactoferrin-Sepharose showed biotinylated bands at Mr values of 22, 49, 55, 72, and 155 kDa. Because lactoferrin-binding proteins may be susceptible to digestion by T. foetus extracellular cysteine proteinases, it is suspected that the 155-kDa protein is the specific lactoferrin-binding protein and that the lower-Mr lactoferrin-binding molecules may be fragmentation products that contain the lactoferrin-binding site; however, other interpretations are clearly feasible. It is possible that there may be multiple proteins or multimers of the same protein. In summary, the data showed that binding of lactoferrin to T. foetus may be regulated by an interplay of specific receptor interactions as well as by hydrophobic and charge-related interactions.     

Tritrichomonas foetus extracellular cysteine proteinase cleavage of bovine IgG2 allotypes

Bovine trichomoniasis is a sexually transmitted disease associated with reproductive failure. Systemic immunization results in protective IgG antibodies in uterine and vaginal secretions. Because bovine IgG2 is a better opsonin than IgG1, it is potentially important in defense. Yet, Tritrichomonas foetus extracellular cysteine proteinase (TFECP) cleaves bovine IgG2, evading protective IgG2 responses. Variations in resistance of the 2 IgG2 allotypes to digestion may explain inherited differences in protection. To address this hypothesis, TFECP was incubated with both IgG2 allotypes at different concentrations and times. The digestion products were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, stained, and quantitated by image analysis. IgG2a was digested faster by TFECP than IgG2b. Differences in the sizes and numbers of digestion products were observed, but the presence of bands the size of Fc and Fd fragments indicated that both allotypes were cleaved at the hinge. Cysteine in the digestion mixture reduced the antibody molecules and increased the rate of digestion, but IgG2a was still more susceptible to cleavage than IgG2b in the absence of cysteine. Thus, not only reduced H chains can be cleaved by cysteine proteinase secreted by T. foetus but also intact functional antibody molecules. Because parasites may evade protective antibody responses by cleaving IgG2, animals with the more resistant IgG2b allotype may be better protected by immunization than animals with the more readily digested IgG2a allotype.     

Tritrichomonas foetus: ultrastructural localization of basic proteins and carbohydrates

The postformalin ammoniacal silver (AS) and the ethanolic phosphotungstic acid (E-PTA) techniques were used to localize basic proteins at the ultrastructural level in the protozoa, Tritrichomonas foetus. The periodic acid-thiosemicarbazide-silver proteinate technique was used to localize carbohydrates. With the AS technique reaction product was seen only in the hydrogenosomes. With the E-PTA technique, reaction product was seen in the microtubules that form the basal bodies, flagella, pelta, and axostyle, in the costa, in the hydrogenosomes, and at the region of the recurrent flagellum's adhesion to the cell body. Previous acetylation of the cells under conditions that block most free amino groups abolished (AS technique) or greatly reduced (E-PTA technique) staining. Carbohydrates were localized on the cell surface; in the membranes of the hydrogenosomes, Golgi complex, and cytoplasmic vesicles; and in the glycogen particles. The specificity of the AS and E-PTA techniques to detect basic proteins is based on observations made with T. foetus as well as with other cell types.     

Cytotoxic effects exerted by Tritrichomonas foetus pseudocysts

The protozoan parasite Tritrichomonas foetus displays a pear-shaped form and a pseudocyst stage. However, little is known about the biology of the pseudocyst. The aim of this work was to assess whether pseudocysts exert cytotoxic effects during their interaction with MDCK cells (an epithelial kidney canine cell line) and compare their behavior to that of the pear-shaped parasites. Pseudocysts and pear-shaped parasites from both cultured and freshly isolated T. foetus were used. Electron microscopy revealed that the epithelial cells exhibited more signs of injury, such as depletion of microvilli, retraction from neighboring cells and swollen mitochondria with loss of electron density in the matrix, when the pseudocysts were used in interaction experiments. In addition, during the co-incubation with MDCK cells, pseudocysts exhibited a more intense amoeboid transformation than that found in pear-shaped parasites. The MTT viability assay demonstrated that the pseudocysts were more cytotoxic when in contact with host cells as compared to the flagellated pear-shaped parasites. The JC-1 viability assay revealed that pseudocysts induced a higher loss of mitochondrial membrane potential compared to pear-shaped parasites. Pseudocysts undergoing a budding process were observed after 2.5h of co-incubation with MDCK cells. Our results suggest that the T. foetus pseudocyst might be a more aggressive form.     

Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis

Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications. Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins. Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion. Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus. Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus. Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T. mobilensis to CHO cells. Specific anti-T. foetus lectin (TFL) and anti-T. mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus. TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions. TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin. Both lectins bound to the surface membrane of CHO cells. Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads. Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces.     

The Surface Charge of Tritrichomonas foetus

The surface charge of Tritrichomonas foetus was evaluated by means of the binding of colloidal iron hydroxide particles at pH 1.8 and cationized ferritin particles at pH 7.2 to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM), of cells suspended in solutions of different ionic strength and pH. At pH 7.2, T. foetus has a negative surface charge with a mean EPM of ?1.03 μmμs^?1μV^?1μcm. At lower pH, there is a decrease in the negative surface charge with an isoelectric point at pH 1.2. At higher pH (> 9.0), there is an increase in the surface charge reaching an EPM of ?2.5 μmμs^?1μV^?1μcm. These results indicate that the surface of T. foetus contains both negatively and positively charged dissociating groups. Binding of colloidal iron hydroxide and cationized ferritin particles throughout the cell surface of the protozoon was observed. Treatment of T. foetus with neuraminidase or trypsin reduced significantly the EPM of the cells. Enzyme-treated cells recovered their normal EPM when incubated for 6 h in fresh culture medium by a process that is inhibited by puromycin.     

Adenosine triphosphatase activity of Tritrichomonas foetus.

Homogenates of Tritrichomonas foetus exhibited a Mg2+-dependent adenosine triphosphatase (ATPase) activity, with a pH optimum in Tris buffers of 8.2 to 8.3. The activity was not sensitive to oxygen. At high concentrations, quercetin and 4-chloro-7-nitrobenzofurazan inhibited ATPase activity in the cytoplasmic extract by 20 and 70%, respectively, whereas oligomycin, venturicidin, triethyltin, leucinostatin, dibutylchloromethyltin chloride, spegazzinine, efrapeptin, citreoviridin and sodium azide had no effect and N,N'-dicyclohexylcarbodi-imide stimulated the activity somewhat. The activity was localized in a population of small cytoplasmic particles which also contained an acid phosphatase. There was no indication of an association of ATPase with hydrogenosomes. The ATPase activity (or activities) in this aerotolerant anaerobe is different from the ATPases characteristic of mitochondria or of anaerobic bacteria.     

Tritrichomonas foetus: ultrastructure and cytochemistry of endocytosis

Tritrichomonas foetus ingests horseradish peroxidase, native ferritin, cationized ferritin, and 0.08 micron latex beads by a process which involves the formation of pinocytic vesicles. These vesicles fuse with each other and with lysosomes forming large vacuoles. Biochemical determinations on the ingestion of horseradish peroxidase and morphometric analysis on the ingestion of cationized ferritin covered latex beads indicated that T. foetus has high endocytic activity. The process of ingestion of the various tracers used was analyzed by transmission electron microscopy of thin sections and freeze fracture replicas.     

Generation of free radicals from metronidazole and other nitroimidazoles by Tritrichomonas foetus

Metronidazole, ronidazole, secnidazole, benznidazole, and misonidazole are reduced by intact Tritrichomonas foetus cells to nitro anion radicals that can be detected by electron spin resonance spectroscopy. This activity appears to be related to the cellular content of reducing substrates, since nitro anion radical formation is stimulated in the presence of glucose and pyruvate. The nitro anion radicals could not be detected under aerobic conditions. Anaerobic homogenates of T. foetus also reduce metronidazole to the nitro anion radical when pyruvate, NADH, or NADPH is added as the ultimate source of reducing equivalents. Free radical formation may be the basic cause of nitroimidazole toxicity in trichomonads.     

First Report of Feline Intestinal Trichomoniasis Caused by Tritrichomonas foetus in Korea

Feline intestinal tritrichomoniasis by Tritrichomonas foetus was first recognized in USA in 1999 and has so far been reported from UK, Norway, Switzerland, and Australia, but not from the Far East Asian countries. In November 2008, 2 female and male littermate Siamese cats, 6-month old, raised in a household in Korea were referred from a local veterinary clinic with a history of chronic persistent diarrhea. A direct smear examination of fecal specimens revealed numerous trichomonad trophozoites which were isolated by the fecal culture in InPouch™ TF-Feline medium. A PCR testing of the isolate based on the amplification of a conserved portion of the T. foetus internal transcribed spacer (ITS) regions (ITS1 and ITS2) and the 5.8S rRNA gene, and the molecular sequencing of the PCR amplicons confirmed infection with T. foetus. This is the first clinical case of feline intestinal trichomoniasis caused by T. foetus in Korea.     

Tritrichomonas foetus: Fine Structure of Freeze-Fractured Membranes1

ABSTRACT. Freeze-fracture techniques reveal differences in fine structure between the anterior three flagella of Tritrichomonas foetus and its recurrent flagellum. The anterior flagella have rosettes of 9–12 intramembranous particles on both the P and E faces. The recurrent flagellum lacks rosettes but has ribbon-like arrays of particles along the length of the flagellum, which may be involved in the flagellum's attachment to the cell body. This flagellum is attached to the membrane of the cell body along a distinct groove that contains few discernible particles. Some large intramembranous particles are visible on the P face of the cell body membrane at the point where the flagellum emerges from the cell body. The randomly distributed particles on the P and E faces of the plasma membrane have a particle density of 919/μm² and 468/μm² respectively, and there are areas on both faces that are devoid of particles. Freeze-fracture techniques also reveal numerous fenestrations in the membrane of the Golgi complex and about 24 pores per μm² in the nuclear. membrane.     

Nucleus behavior during the closed mitosis of Tritrichomonas foetus

We present observations on the fine structure and the division process of the nucleus in the protist Tritrichomonas foetus, parasite of the urogenital tract of cattle. The nucleus was followed by immunofluorescence and electron microscopy during interphase and mitosis. Conventional karyotyping coupled to image processing and bright field Panotic staining were used to follow nucleus modifications, chromosome number and condensation pattern along the whole cell cycle. Confocal laser scanning microscopy (CLSM) using DNA fluorescent probes, followed by image processing in the SURF-Driver program, produced three-dimensional reconstruction data of the mitotic nucleus under each phase of the division process. Immunocytochemistry in thin-sections revealed the chromosome spatial arrangement after bromodeoxyuridine incorporation and immunogold labeling using anti-DNA monoclonal antibodies. Our results indicate that: (1) the nucleus assumes different size and shapes along mitosis: it appears oval in interphase, becoming lobed or concave in prophase, then undergoing torsion and constriction, displaying an 'S' shape (metaphase). Next, it becomes elongated and it is finally separated in two nuclei at the transition of anaphase to telophase; (2) T. foetus nucleus harbors five chromosomes; (3) chromosomes become condensed in a pre-mitotic phase; (4) the nucleolus persists during the mitosis.     

Ultrastructural characterization of the isolated hydrogenosome in Tritrichomonas foetus

In the present study we show new aspects of the hydrogenosome ultrastructure as well alterations induced by the fractionation technique. The morphology of freshly isolated hydrogenosomes as well those found in whole cells of Tritrichomonas foetus were examined in thin-sections, in replicas of fast-freezing, and conventional freeze-fracture, freeze-etching, and by high resolution scanning electron microscopy (field emission in-lens scanning electron microscopy). The true surface as well the concave and convex fracture faces of the inner and outer membranes are shown. We showed that after fractionation procedures the hydrogenosome ultrastructure can be changed, since isolated hydrogenosomes present patchwork-like structures, rosettes and the inner hydrogenosomal membrane is displaced. The peripheral vesicle is seen as a distinct compartment, since its content and morphological appearance is quite different from the rest of the organelle. The peripheral vesicle shows a smooth surface but presenting pores with 20 nm in diameter with a density of 7/micron 2 when observed after freeze-etching. We report the existence of characteristic intramembrane particles distribution and density on hydrogenosome membranes of isolated and whole T. foetus, suggesting that this organelle can have its morphology changed as consequence of technical modifications or as expression of its metabolic state.     

Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus

A low molecular weight iron-sulfur protein has been purified from Tritrichomonas foetus by deoxycholate extraction of whole cells, ion exchange chromatography, and gel filtration. The purified protein was essentially homogeneous as judged by isoelectric focusing, polyacrylamide gel electrophoresis, and gel filtration. A pI of 4.3 was observed. The molecular weight of the protein was estimated to be 12,000. Chemical and spectral analysis showed the protein to have a [2Fe-2S] cluster. The absorbance spectrum of the oxidized protein showed maxima at 280, 340, 458 and shoulders at 410 and 550 nm. The maximum observed A458/A280 ratio was 0.82 and the absorbance of the oxidized protein at 458 nm was 8,000 M-1 X cm-1. The low temperature EPR spectrum of the protein reduced with dithionite revealed axial symmetry with features at g values of g = 1.94 and g = 2.02. The oxidized protein gave no EPR signal in the g = 1.8 to 2.2 range. Cell fractionation studies indicated the localization of this protein in the hydrogenosome. The protein was able to function as an electron transport component in the reduction of metronidazole (a 5-nitroimidazole derivative) by pyruvate:ferredoxin oxidoreductase and hydrogenase from T. foetus and also from Trichomonas vaginalis and Clostridium pasteurianum as well as in the reduction of cytochrome c by plant NADPH:ferredoxin oxidoreductase. This protein has the characteristics of a ferredoxin and is likely to be a physiological electron carrier in hydrogenosomal pyruvate oxidation.     

Glucokinase and fructokinase of Trichomonas vaginalis and Tritrichomonas foetus

Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33-35 microM and 75-83 microM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 microM and 81 microM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1-phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol., 37:183-190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.     

Acidic glycerol lipids of Trichomonas vaginalis and Tritrichomonas foetus

The isolation and characterization of acidic lipids from both Trichomonas vaginalis and Tritrichomonas foetus have been carried out using radiolabeling, a combination of high performance liquid and thin layer chromatographic techniques, and mass spectrometry. Unique among the eukaryotes, these organisms produce phosphatidylglycerols and O-acyl phosphatidylglycerol-like compounds. In this study, the molecular weight distributions of the phosphatidylglycerols and acyl phosphatidylglycerols were determined by negative-ion liquid secondary ionization mass spectrometry (LSIMS) and the fatty acyl groups within each molecular species were assessed by collision-induced decomposition tandem mass spectrometry (CID MS/MS). Both species were found to contain primarily oleic acid in the sn-2 position. The lipids of T. vaginalis had approximately equal amounts of C16 and C18 in the sn-1 position, with varying degrees of unsaturation, especially in the C18 species. The T. foetus lipids had C18 almost exclusively, but also varied in the unsaturation. Other acidic lipids included inositol phosphosphingolipids and inositol diphosphosphingolipids.     

Frozen Storage of Tritrichomonas foetus for 5.6 Years*

SYNOPSIS. Tritrichomonas foetus was cultivated in Diamond's medium without phosphates or agar; 10% glycerol was added to the medium and the protozoa were frozen slowly and stored at ?28 or ?95 C. About half of the trichomonads died within the first day at either temperature. Thereafter, they continued to die off slowly during storage at ?28, and none remained alive at or after 1024 days. New cultures could not be initiated with them after 64 days. At ?95, almost as many trichomonads were alive after 256 days as after 1 day. Thereafter, however, they decreased in numbers, but 11% were still alive after 2048 days (5.6 years). The survivors appeared in good condition, and new cultures were readily initiated with them.     

Glucokinase and Fructokinase of Trichomonas vaginalis and Tritrichomonas foetus

ABSTRACT. Trichomonas vaginalis and Tritrichomonas foetus contain glucokinase and not a hexokinase of broad hexose specificity. Tritrichomonas foetus also contains a specific fructokinase which could be resolved from glucokinase by anion exchange chromatography. Native T. vaginalis glucokinase had a Mr of 76,000, and SDS-PAG electrophoresis showed two equally stained bands corresponding to Mr 40,000 and 38,000. Glucose and ATP were by far the best substrates for both trichomonad glucokinases, with Km values as low as 33–35 μM and 75–83 μM, respectively. Substrate saturation curves for these enzymes were all hyperbolic. Tritrichomonas foetus fructokinase required fructose and ATP, with Km values of 200 μM and 81 μM. None of the activities was affected by a number of potential regulatory metabolites, including glucose-6-phosphate. The only exception was AMP which in supraphysiological concentrations had an inhibitory effect on T. foetus fructokinase. In conclusion, the absence of regulation at the hexose phosphorylation step described here, as well as the presence of an easily reversible PPi: fructose-6-phosphate 1 -phosphotransferase described previously (Mertens, E., Van Schaftingen, E. & Müller, M. 1989. Mol. Biochem. Parasitol. , 37 :183–190), suggest that the rate of the 1st part of glycolysis in trichomonads is controlled only by the intracellular availability of hexoses.