Further studies on the surface charge of various strains of Trichomonas vaginalis and Tritrichomonas foetus

The surface charge of three strains of Trichomonas vaginalis and five strains of Tritrichomonas foetus was determined by direct measurement of the mean cellular electrophoretic mobility (EPM) of cells suspended in solutions of different ionic strength and pH. No differences were observed in the mean EPM among the two species, although significant differences among the strains exist. Strains that are more pathogenic to mouse, as measured using the subcutaneous assay, had a surface more negative. Treatment of the parasites with trypsin or neuraminidase reduced significantly their mean EPM and increased their isoelectric point. Tritrichomonas foetus was more sensitive to the enzyme treatment than T. vaginalis. Enzyme-treated cells recovered their normal EPM if, after enzyme treatment, they were incubated in fresh culture medium. The recovery process of trypsin-treated cells was inhibited 10-20% by addition of inhibitors of either protein synthesis (puromycin) or N-glycosylation of proteins (tunicamycin) to the incubation medium, suggesting that a cytoplasmic pool of sialoglycoproteins may exist. The recovering of the EPM of T. foetus and T. vaginalis previously treated with neuraminidase was inhibited by puromycin or tunicamycin about 40-50% and 17-30%, respectively. These observations suggest that sialoglycolipids exist on the surface of both parasite species, and that they contribute more to the surface charge of T. vaginalis than to that of T. foetus.     

Genetic differentiation and biochemical polymorphism among trichomonads

Isoenzyme electrophoresis was used to study levels of genetic differentiation among strains and clones of Trichomonas gallinae, Trichomonas vaginalis, Tritrichomonas foetus, Tetratrichomonas gallinarum, and Pentatrichomonas hominis. Strain variation was found within T. gallinae, T. vaginalis, and T. foetus, however, levels of enzyme polymorphism were greater in T. gallinae than in T. vaginalis or T. foetus. Isoenzyme genotypes were not a stable property of T. gallinae clones cultivated in vitro. Retrospective studies of T. gallinae SG and JB6 clones revealed that mutation occurred during in vitro cultivation. Heterozygotes of hexokinase-1 and phosphoglucomutase displayed 2 allomorphs in equal dosage, indicating that trichomonads are diploid for these protein loci. Phenetic clustering of the biochemical data suggests that levels of genetic divergence among the species studied are extensive.     

Effect of the antimicrobial peptide, D-hecate, on trichomonads

Tritrichomonas foetus and Trichomonas vaginalis are protozoan parasites that cause sexually transmitted diseases in cattle and humans, respectively. There is a need for new antimicrobial agents to treat or prevent trichomoniasis because there are currently no approved chemotherapeutic agents against T. foetus and resistance of T. vaginalis to metronidazole does occur. Therefore, we evaluated the effect of a novel antimicrobial peptide, D-hecate, on the viability of 6 isolates of T. foetus and T. vaginalis in vitro. Tritrichomonas foetus and T. vaginalis were grown to mid log phase (24 hr) or late log/stationary phase (48 hr). Parasites at 10(6)/ml were mixed with equal volumes of D-hecate to final concentrations of 10 microM, 20 microM. and 40 microM of D-hecate. Controls had minimal essential medium (MEM) alone. The numbers of viable parasites were determined microscopically after 10, 20, and 30 min of incubation at 37 C with D-hecate or MEM. Our results show that D-hecate killed all 6 isolates of T. foetus and T. vaginalis evaluated. The killing effect was dependent on the concentration of the peptide, incubation time, and phase of growth of the parasites. Ultrastructural studies of parasites treated with 10 microM of D-hecate revealed extensive damage to the plasma membrane of most T. foetus and T. vaginalis cells, while a few cells were distorted but remained intact. D-Hecate may be a useful chemotherapeutic agent for the treatment of trichomoniasis.     

Antigenic heterogeneity in the 115,000 Mr major surface antigen of Trichomonas vaginalis

The 115,000-molecular-weight antigen of Trichomonas vaginalis was characterized using monoclonal antibodies developed to three different strains of T. vaginalis and one strain of Tritrichomonas foetus. The antigen was found to be present on all strains or isolates of T. vaginalis examined and was demonstrated to be located on the external surface plasma membrane by agglutination assays and complement-mediated lysis assays. Characteristics of the antigen were assessed with a proteolytic enzyme and periodate oxidation. Periodate treatment of whole T. vaginalis abrogated binding for eight antibodies while use of pronase-treated antigen resulted in loss of antibody binding for two different antibodies. Screening of 19 axenized clinical isolates of T. vaginalis and one strain each of T. foetus and Giardia lamblia with type-specific antibodies delineated three major groups of T. vaginalis based on antigenic specificities (epitope distributions) within the 115,000-molecular-weight antigen. In addition, one epitope of the 115,000-molecular-weight antigen was found only on the immunizing strain. Two epitopes were present on all T. vaginalis isolates as well as T. foetus and G. lamblia. One epitope was common to all T. vaginalis except one. A minimum of six different epitopes of the 115,000-molecular-weight antigen were identified. Antigens purified with type-specific or "common" monoclonal antibodies shared the same partial peptide maps demonstrating relatedness.     

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.     

Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins

Iron is an essential nutrient for Trichomonas vaginalis and is acquired via highly specific receptor-mediated mechanisms from the host. Responses of T. vaginalis to conditions of iron limitation or iron excess were analysed in order to determine whether iron levels in the growth medium regulate certain properties of the parasite. When compared with organisms grown in excess iron, iron limitation resulted in greater than or equal to 80% lower rates of protein synthesis and greater than or equal to 3-fold decreases in cell densities. These parasites also exhibited generation times of approximately 10 hours, 2.5-fold longer than organisms grown in the usual complex medium. Iron-restricted growth also resulted in increased binding of lactoferrin by trichomonads, which paralleled elevated expression of the lactoferrin-binding receptor protein having a relative molecular mass of 136,000 daltons (136 kDa). A Mr 126 kDa protein was concomitantly repressed in low-iron-grown parasites. The greater amounts of lactoferrin bound by iron-depleted T. vaginalis organisms corresponded with both the expression of additional receptors onto trichomonal surfaces and increased affinity of the receptor for the lactoferrin molecule. Finally, immunoblot analysis of parasites grown under high- and low-iron conditions using sera from patients with trichomoniasis further revealed the synthesis by T. vaginalis of at least 19 iron-regulated immunogens, and patients' sera also detected the lactoferrin receptor. These data not only show the overall importance of iron to the biology of this protozoan, but illustrate the in vivo iron modulation of gene expression of the biofunctional lactoferrin receptor and other immunogens.     

Modulation by phorbol 12-myristate 13-acetate of arachidonic acid release from rat basophilic leukemia cells stimulated with A23187

Rat basophilic leukemia (RBL-2H3) cells were cultured in medium containing [3H]arachidonic acid and labelling of the different lipid fractions was followed with time. After up to 4 h of culture, the label was found mostly in phosphatidylcholine. After 8 h, labelling of phosphatidylethanolamine gradually exceeded that of phosphatidylcholine, until at 24 h, approximate equilibrium labelling of the lipid fractions was attained and 45% of the label was found in phosphatidylethanolamine, 35% in phosphatidylcholine, 18% in the phosphatidylserine/inositide fraction and the remainder in the neutral lipid fraction. Stimulation of cells with A23187 after 30 min of labelling caused release of [3H]arachidonic acid which was accountable by a decrease in radioactivity of phosphatidylcholine, whereas stimulation of cells after 24 h of labelling caused the release of radioactive arachidonic acid, which was accompanied by a decrease of label in both phosphatidylcholine and phosphatidylethanolamine. Incubation of the labelled cells with phorbol 12-myristate 13-acetate prior to ionophore addition enhanced both the release of [3H]arachidonic acid and its metabolites and the decrease in label of the same phospholipids as those affected by ionophore alone. Under our conditions, the enhancement effects of phorbol ester were greatest after 2-5 min of preincubation, prior to ionophore addition. The results suggest that in basophilic leukemia cells, arachidonic acid release proceeds from several pools of phospholipids and that the activity of the phospholipase(s) involved is modulated by protein kinase C.     

Observations on the fertilization and development of preimplantation bovine embryos in vitro in the presence of Tritrichomonas foetus

Tritrichomonas foetus, a world-wide distributed parasitic protozoan is a cause of infertility and abortion. There is no documented information on the susceptibility of bovine embryos to the parasite. To determine the effect of T. foetus on fertilization and embryonic development of preimplantation bovine embryos, we added approximately 10(4)/ml or 10(6)/ml T. foetus (Belfast strain) to sperm cells and oocytes prior to in vitro fertilization (IVF) or to presumptive zygotes 24 h post-fertilization. Light and scanning electron microscopy (SEM) revealed that exposure of oocytes or embryos at any stage of development to T. foetus caused rapid adhesion of the trichomonads to the embryonic intact zona pellucida (ZP) and to trophoblastic cells of hatched blastocysts. Treatment of contaminated embryos with 0.25% trypsin for 3 min did not render them free from T. foetus. Motile parasites were not observed after 18 h incubation in IVF medium, or after 72 h in synthetic oviductal fluid (SOF) embryo culture medium. The percentages of cleaved zygotes, blastocysts and hatched embryos resulting from culture of experimental and uninfected control groups of embryos were not different (P > 0.05). Tritrichomonas foetus was not detected in embryonic cells of ZP-intact or hatched embryos when examined by transmission electron microscopy (TEM). In conclusion, T. foetus has no detrimental effect on the fertilization and development of IVF embryos and the potential risk of transmission of trichomonosis is unlikely, due to the limited survival of the parasite in IVF culture conditions.     

The effect of hypoxia on uptake and degradation of low density lipoproteins by cultured human arterial smooth muscle cells.

Atheroma have been produced in experimental animal by systemic hypoxia. This study assessed the effects of hypoxia on binding, uptake and degradation of human low density lipoprotein (LDL) by human arterial smooth muscle cells, the cell involved in atherogenesis. The LDL content of the smooth muscle cell grown in the usual conditions (95% air [20% O2], 5% CO2) increased with the incubation time of LDL in the medium (7.5 mug protein/ml of medium); the trypsin releasable LDL "binding" reached a plateau by 24 h (2.2 +/- 1.3 [x +/- S.D.]) ng/mug LDL protein added per 10(6) cells whereas the LDL in the cell after trypsinization ("net uptake") continued to increase up to 48 h (6.5 +/- 4.6 ng/mug LDL protein added per 10(6) cells at 48 h). LDL protein degradation increases rapidly between 7 and 48 h (10.4 ng/mug LDL protein added per 10(6) cells at 24 h) after an initial delay of approximately 7 h. Smooth muscle cells grown under hypoxic conditions (5%02) had similar LDL "binding " but showed increased "net uptake" (10.7 +/- 4.8 ng/mug LDL protein added per 10(6) cells) and a 36 +/- 13% decrease in degradation (p less than 0.05; n =8). The impaired degradation of lipoprotein by smooth muscle cells may, in part, explain the role of hypoxia in atherogenesis.     

Tritrichomonas foetus: the role played by iron during parasite interaction with epithelial cells

The aim of this work was to investigate the role played by iron during interaction of Tritrichomonas foetus with cultured epithelial cells. We have observed that the growth rate of T. foetus is influenced by the amount of iron available into culture medium. When organisms maintained for 24h in iron-depleted medium were transferred to an iron-rich one, many protozoan cells exhibited a cytokinesis blockage. Parasites maintained in iron-depleted medium exhibited a significant increase in cytoadhesion when compared with both controls and parasites that had been cultured in medium in which iron was replaced. T. foetus collected from iron-depleted medium also exhibited a reduction in its ability to destroy epithelial cell monolayers and a reduction in the activity of several cysteine proteases. Taken together, the results presented here demonstrate that iron may be an extracellular signal, which seems to modulate the ability of T. foetus to interact with host epithelial cells.     

Biosynthesis of Abscisic Acid under Osmotic Stress: Studies Based on a Dual Labelling Technique

Sterile liquid suspension cultures from grape pericarp tissue incorporate label from mevalonic acid into abscisic acid (ABA), some of which is subsequently released into the surrounding medium. The addition of mannitol to the culture medium caused plasmolysis of individual cells and increased ABA generation and release. Levels in the medium increased up to 30 fold over a period of 8 h in the presence of mannitol (720 milliosmol). A dual labelling technique using ¹⁴C and ³H mevalonic acid was developed to distinguish between labelled compounds produced by turgid tissue and those specifically associated with the substantial increase in ABA synthesis following mannitol treatment.     

A linear double-stranded RNA in Trichomonas vaginalis

A "double-stranded" RNA was identified in the anaerobic, parasitic protozoan Trichomonas vaginalis. Electron microscopic evidence indicated linear double-stranded structure 1.5 micron in length, with no apparent hairpins or loops. Boiling in 30% dimethyl sulfoxide denatured it into single strands of 1.5 micron and shorter fragments. It consists of 23.4% G, 23.4% C, 23.0% A, and 30.3% U and melts at a transition temperature of 81.7 degrees C in 75 mM NaCl and 7.5 mM sodium citrate, pH 7.0, with 7-15% hyperchromicity. The 32P-labeled double-stranded RNA hybridized specifically with T. vaginalis DNA fragments in a single DNA band from EcoRI digest and two DNA bands from HindIII digest. Of the 33 different strains or isolates of T. vaginalis examined, all contained this double-stranded RNA. However, the only two metronidazole-resistant T. vaginalis strains examined thus far (IR78 and 85) contained no detectable double-stranded RNA, although the corresponding DNA sequence was present. DNA fragments of Escherichia coli and Giardia lamblia did not hybridize with the double-stranded RNA. But DNA fragments of a metronidazole-sensitive Tritrichomonas foetus hybridized specifically with the double-stranded RNA, even though this organism does not contain the double-stranded RNA itself.     

Agar Techniques for Colonizing and Cloning Trichomonads

SYNOPSIS. Agar media have been used in plates, Burri tubes, and sealed slide preparations to grow trichomonads as clonal colonies. Periodic observation of slide-culture colonies confirmed their origin from single organisms as clones. Axenic cultures of Trichomonas vaginalis (3 strains), T. gallinae (2 strains), T. gallinarum (1 strain), Tritrichomonas augusta (5 strains), Trit. foetus (4 strains), Trit. suis (?) (6 strains), Pentatrichomonas hominis (1 strain), and an undescribed porcine form were used successfully. Urinary and vaginal specimens with T. vaginalis were also plated directly from patients. In all trials, organisms were recovered from colonies for broth culture and re-plating.
Colonies of the two strains of T. gallinae showed constant differences in their growth under the same conditions. Plated T. vaginalis have shown zones of growth inhibition in tests against drug-impregnated discs.     

An ectonucleotide ATP-diphosphohydrolase activity in Trichomonas vaginalis stimulated by galactose and its possible role in virulence

This work describes the ability of living Trichomonas vaginalis to hydrolyze extracellular ATP (164.0 +/- 13.9 nmol Pi/h x 10(7) cells). This ecto-enzyme was stimulated by ZnCl2, CaCl2 and MgCl2, was insensitive to several ATPase and phosphatase inhibitors and was able to hydrolyze several nucleotides besides ATP. The activity was linear with cell density and with time for at least 60 min. The optimum pH for the T. vaginalis ecto-ATPase lies in the alkaline range. D-galactose, known to be involved in adhesion of T. vaginalis to host cells, stimulated this enzyme by more than 90%. A comparison between two strains of T. vaginalis showed that the ecto-ATPase activity of a fresh isolate was twice as much as that of a strain axenically maintained in culture, through daily passages, for several years. The results suggest a possible role for this ecto-ATPase in adhesion of T. vaginalis to host cells and in its pathogenicity.     

A freeze-fracture electron microscope study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)

Two strains of Trichomonas vaginalis, JH162A , with low pathogenicity, and Balt 44, with high pathogenicity, as well as one highly pathogenic strain, KV-1, of Tritrichomonas foetus were studied by freeze-fracture electron microscopy. The protoplasmic faces ( PFs ) of the cell membranes of all three strains of both species had similar numbers of intramembranous particles (IMPs); however, the particles in the external faces (EFs) of these membranes were least abundant in Trichomonas vaginalis strain Balt 44 and most numerous in those of strain JH162A of this species. In Tritrichomonas foetus strain KV-1 the number of IMPs in the EF was close to but somewhat lower than that in the mild strain of the human urogenital trichomonad . In both species, the anterior, but not the recurrent, flagella had rosette-like formations, consisting of approximately 9 to 12 IMPs on both the PFs and EFs. The numbers and distribution of the rosettes appeared to vary among different flagella and in different areas of individual flagella of a single organism belonging to either species. The freeze-fracture electron micrographs provided a more complete understanding of the fine structure of undulating membranes of Trichomonadinae , as represented by Trichomonas vaginalis, and of Tritrichomonadinae (the Tritrichomonas augusta -type), as exemplified by Tritrichomonas foetus, than was gained from previous transmission and scanning electron microscope studies. Typically three longitudinal rows of IMPs on the PF of the recurrent flagellum of Trichomonas vaginalis were noted in the area of attachment of this flagellum to the undulating membrane. The functional aspects of the various structures and differences between certain organelles revealed in the two trichomonad species by the freeze-fracture method are discussed.     

Thyroid thermogenesis in adult rat hepatocytes in primary monolayer culture: direct action of thyroid hormone in vitro

We have studied the effect of 3,5,3'-triiodothyronine (T3) on the respiration of adult rat hepatocytes in primary monolayer culture prepared from hypothyroid rat liver. After addition of T3 to the culture medium at a concentration of 2 x 10(-7) M, oxygen consumption of the cultured cells increased detectably at 24 h and was maximal at 72--96 h, relative to control cultures (38.0 +/- 1.8 vs. 25.0 +/- 1.5 microliter/h.mg protein). The thyroid-responsive enzymes, Na+ + K+-activated adenosine triphosphatase (NaK-ATPase) and alpha-glycerophosphate dehydrogenase (GPD), each exhibited increased activity in response to T3, in parallel with the change in oxygen consumption, whereas the activity of Mg-dependent ATPase was unaffected. These responses to T3 were dose dependent over similar concentration ranges, the half-maximal response for each occurring at ca 8 x 10(-10) M. In thyroid-treated cells, the observed increase in respiration was almost completely (90%) inhibited after addition of ouabain (10(-3) M) to the culture medium. It was found also that a 4-h exposure of the cultured hepatocytes to T3 was sufficient to elicit a significant thermogenic response, measured at a time (48 h later) when T3 was no longer present in the medium. The response to T3 occurred in fully defined culture medium and was independent of the presence or absence of hypothyroid rat serum, corticosterone, or insulin, and cellular ATP was unaffected by T3 in concentrations up to 2 x 10(-7) M. The findings document that adult rat hepatocytes in primary monolayer culture respond directly to thyroid hormone; the increases in respiration and NaK-ATPase activity elicited by T3 were cotemporal and apparently coordinate.     

Purification of cysteine proteinases from trichomonads using bacitracin-sepharose

Abstract Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus . It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteines from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 μg of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10⁹ cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusuall features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.     

Trichomonads, hydrogenosomes and drug resistance

Trichomonas vaginalis and Tritrichomonas foetus are sexually transmitted pathogens of the genito-urinary tract of humans and cattle, respectively. These organisms are amitochondrial anaerobes possessing hydrogenosomes, double membrane-bound organelles involved in catabolic processes extending glycolysis. The oxidative decarboxylation of pyruvate in hydrogenosomes is coupled to ATP synthesis and linked to ferredoxin-mediated electron transport. This pathway is responsible for metabolic activation of 5-nitroimidazole drugs, such as metronidazole, used in chemotherapy of trichomoniasis. Prolonged cultivation of trichomonads under sublethal pressure of metronidazole results in development of drug resistance. In both pathogenic species the resistance develops in a multistep process involving a sequence of stages that differ in drug susceptibility and metabolic activities. Aerobic resistance, similar to that occurring in clinical isolates of T. vaginalis from treatment-refractory patients, appears as the earliest stage. The terminal stage is characterised by stable anaerobic resistance at which the parasites show very high levels of minimal lethal concentration for metronidazole under anaerobic conditions (approximately 1000 microg ml(-1)). The key event in the development of resistance is progressive decrease and eventual loss of the pyruvate:ferredoxin oxidoreductase so that the drug-activating process is averted. In T. vaginalis at least, the development of resistance is also accompanied by decreased expression of ferredoxin. The pyruvate:ferredoxin oxidoreductase deficiency completely precludes metronidazole activation in T. foetus, while T. vaginalis possesses an additional drug-activating system which must be eliminated before the full resistance is acquired. This alternative pathway involves the hydrogenosomal malic enzyme and NAD:ferredoxin oxidoreductase. Metronidazole-resistant trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways. Trichomonas vaginalis enhances lactate fermentation while T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme, to enhance the pyruvate producing bypass and provide NADPH required by alcohol dehydrogenase. Production of succinate by this species is abolished. Metabolic changes accompanying in-vitro development of metronidazole resistance demonstrate the versatility of trichomonad metabolism and provide an interesting example of how unicellular eukaryotes can adjust their metabolism in response to the pressure of an unfavorable environment.     

Renotropic stimulation in rat kidney cell culture

A circulating renotropic factor specific for renal cells has been described in rats. The addition of sera obtained from unilaterally nephrectomized (uni) rats 24h after operation compared to sham-operated (sham) rats augments 3H-thymidine incorporation into the DNA of incubating kidney slices approximately 10%-30%. Attempting to amplify the sensitivity of the assay for this renotropic agent, we replaced slices with primary rat kidney cultures. The assay system was based on one previously used for rabbits. The cultured cells were synchronized in their growth phase by a period of protein-free starvation. Compared to sera from sham rats, sera from uni rats showed significant stimulation of thymidine incorporation into DNA, 35.5% +/- 9.3 (SEM), p less than .0001, at 16 h; 63.3% +/- 10.0 (SEM), p less than .001, at 24 h; and 19.5% +/- 6.5 (SEM), p less than .01, at 48 h post operation. Accordingly, the maximal stimulation at 24 h was greater than that previously found using the kidney slice assay. Measurable renotropic activity occurred earlier and over a shorter duration than in rabbits. Stimulation was similar when a D-valine medium, relatively specific for renal epithelial cells, replaced DME medium. We conclude that growth synchronized, primary rat renal cells in culture verify the presence of a circulating renotropin arising 24 h post uni.