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.     

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.     

Effect of a phorbol ester on basic surface properties of trichomonads

The effect of nanomolar concentrations of 12-O-tetradecanoil-phorbol-13-acetate (TPA) on the cell surface of the urogenital parasitic protozoaTrichomonas vaginalis andTritrichomonas foetus was evaluated by means of measurements of the parasites’ surface tension, electrokinesis, lectin agglutination tests, and adhesion to inert substrates. TPA-treated parasites had their adhesion increased to both plastic and glass substrates. This was accompanied by increases in the parasites’ net negative surface charge and also by changes in their surface tension. The lectin agglutination assays suggest that the increase in surface negativeness may be related in some extent to alterations in the oligosaccharide composition. Successive treatment of the microorganisms with TPA and sphingosine, a well-known competitive inhibitor of the phorbol ester active site, depressed the tendency of trichomonads to exhibit a phenotype of activated cells.     

Antigenic Heterogeneity in the 115,000 Mr Major Surface Antigen of Trichomonas vaginalis1

ABSTRACT 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.     

Heterologous expression in <Emphasis Type="Italic">Tritrichomonas foetus</Emphasis> of functional <Emphasis Type="Italic">Trichomonas vaginalis</Emphasis> AP65 adhesin

Background  

Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.     

Cell death in trichomonads: new insights

Tritrichomonas foetus is an amitochondriate parasite that possesses hydrogenosomes, unusual anerobic energy-producing organelles. In these organisms the “mitochondrial cell death machinery” is supposed to be absent, and the mechanisms that lead to cell demise remain to be elucidated. The presence of a cell death program in trichomonads has already been reported, suggesting the existence of a caspase-like execution pathway in such organisms. Here we demonstrate the alterations provoked by the fungicide griseofulvin and raise the possibility that other cell death pathways may exist in T. foetus. Dramatic changes in trichomonads morphology are presented after griseofulvin treatment, such as intense plasma membrane and nuclear envelope blebbing, nucleus fragmentation, and an abnormal number of oversized vacuoles. One important finding was the exposition of phosphatidylserine (PS) in the outer leaflet of the plasma membrane in cells after drug treatment, and also the presence of a high amount of misshapen flagella and tubulin precipitates as vacuolar contents, suggesting an autophagic process of abnormal cellular elements. Interestingly, immunoreactivity for activated caspase-3 was not detected during griseofulvin treatment, a finding distinct from the observed when this cell was treated with H₂O₂. The possibility of the existence of different pathways to cell death in trichomonads is discussed.     

A Freeze-Fracture Electron Microscope Study of Trichomonas vaginalis Donné and Tritrichomonas foetus (Riedmüller)1

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 ～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.     

Sialoglycoproteins and sialoglycolipids contribute to the negative surface charge of epimastigote and trypomastigote forms of Trypanosoma cruzi

Epimastigote and trypomastigote forms of Trypanosoma cruzi have a net negative surface charge, as determined by direct measurement of the mean cellular electrophoretic mobility. Treatment of the parasites with neuraminidase reduces by 17 and 52% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms, respectively. Neuraminidase-treated cells recover their normal electrophoretic mobility if incubated for 2 h in the presence of fresh culture medium. The recovering process of epimastigotes is almost totally blocked by addition of inhibitors of either protein synthesis (puromycin) or N-glycosidically linked glycoprotein synthesis (tunicamycin). The recovering process of trypomastigotes is not totally inhibited by either puromycin or tunicamycin. Treatment of T. cruzi with trypsin reduces by 11 and 40% the mean electrophoretic mobility of epimastigote and bloodstream trypomastigote forms. Trypsin-treated cells recover their normal electrophoretic mobility if incubated for 4 h in fresh culture medium. The recovering process of trypomastigotes is partially inhibited by puromycin. The results obtained indicate that sialoglycoproteins and sialoglycolipids exist on the surface of T. cruzi, the latter being predominant on the surface of trypomastigotes.     

Effect of Colchicine,Vinblastine, and Cytochalasin B on Cell Surface Anionic Sites of Tritrichomonas foetus1

ABSTRACT. Drugs that interact with microtubules (colchicine and vinblastine) and microfilaments (cytochalasin B) partially inhibited cell growth and motility of Tritrichomonas foetus. Parasites incubated with these substances became rounded and cell division was blocked. Neither colchicine nor vinblastine disrupted the microtubules that form the peltar-axostylar system. Any one of these drugs interfered with the net negative surface charge of T. foetus as evaluated by determination of the cellular electrophoretic mobility (EPM). The decrease in the EPM of cytochalasin B-treated cells was caused by dimethylsulfoxide, which was used as solvent. Untreated cells as well as cytochalasin B-treated cells showed a uniform distribution of anionic sites on the plasma membrane as seen with cationized ferritin particles. In cells treated with colchicine or vinblastine the anionic sites were distributed in patches. These results are discussed in terms of participation of labile cytoplasmic microtubules and microfilaments in the control of the distribution of anionic sitecontaining macromolecules located on the cell surface of T. foetus.     

Structural characterization of novel inositol phosphosphingolipids of Tritrichomonas foetus and Trichomonas vaginalis

Two major ethanolamine phosphate-substituted inositol phosphosphingolipidshave been identified in the unsaponifiable acidic lipid fractionsof Tritrichomonas foetus and Trichomonas vaginalis. The compoundswere radiolabelled and purified by high-performance thin-layerchromatography followed by high-performance liquid chromatography.The structures were determined by a combination of tandem massspectrometry (MS/MS) and nuclear magnetic resonance (NMR) experiments,and gas—liquid chromatography of components obtained bydegradation and derivatization. Inositol in the T.foetus componentwas 1-linked to the phosphosphingolipid, had the phospho-ethanolaminegroup at the 3-position and a fucosyl residue at the 4-position.The T.vaginalis component lacked the fucosyl moiety. Both organismsalso produced inositol phosphosphingolipids having the samelong-chain base (sphingosine or dihydrosphingosine) and thesame fatty acyl distribution as the inositol diphosphate compounds.These glycosphingolipids may represent metabolic intermediatesfor new types of membrane anchors for surface glycopeptidesor glycolipids that mediate the host—parasite relationshipof these trichomonads. The MS/MS and NMR spectroscopic datashould provide reference information for structural determinationsof other phosphorylated inositol derivatives. inositol phosphosphingolipids NMR tandem MS T.foetus T. vaginalis     

Clonal and Geographic Distribution of a Surface Antigen of Tritrichomonas foetus1

ABSTRACT. Geographically diverse strains and clones of Tritrichomonas foetus have been examined with respect to their expression of a major surface antigen of approximately 150,000 relative molecular weight (Mr), designated the 150 Ag. Radioiodination and ¹³S-methionine labeling of T. foetus followed by immunoprecipitation with monoclonal antibodies (MAbs), separation of polypeptides by SDS-PAGE, and autoradiography or fluorography confirmed the parasite origin of the 150 Ag. The results of flow cytometry analysis employing a panel of MAbs against live T. foetus parasites revealed that from 5 to 84% of individuals in a given population of T. foetus expressed a particular epitope of the 150 Ag. All strains and clones were positive for surface expression of epitopes of this antigen. These results show that the 150 Ag is widely distributed in populations of T. foetus, confirm the surface location of this antigen, and suggest its importance as a target for protective immune responses.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

The influence of natural enemies on brood production inIps typographus (Col. scolytidae) with special reference to egg-laying and predation byThanasimus formicarius (Col.: Cleridae)

The predatorThanasimus formicarius (L.) (Coleoptera, Cleridae) and its preyIps typographus (L.) (Coleoptera, Scolytidae) were studied in the laboratory and the field. In the laboratory, 11T. formicarius laid 71–132 eggs (mean=162) during 66–123 days. During this time they ate 66–132I. typographus adults per pair (male +female). The number of eggs laid per female was not correlated with life span or the number ofIps eaten. In the field, predation byT. formicarius larvae and other natural enemies onI. typographus brood was studied in the last year of an outbreak. Caged and uncaged spruce bolts attacked byI. typographus were used, and pairs ofT. formicarius were released in the cages. The treatments were: uncaged bolts, caged bolts withoutT. formicarius, caged bolts with 4T. formicarius pairs, and caged bolts with 8T. formicarius pairs. The productivity ofI. typographus was highest in the caged bolts withoutT. formicarius (mean=4.5 offspring/female) and lowest in the uncaged bolts (mean=0.9 offspring/female). The density ofI. typographus galleries was similar in the different treatments. Hence, the variation in productivity between treatments could not have been due to differences in the levels of intraspecific competition. There was no difference in bark beetle productivity or density ofT. formicarius larvae between bolts with 4 pairs ofT. formicarius and bolts with 8 pairs (mean=2.5 offspring/female). This indicates that some kind of interference occurred betweenT. formicarius individuals (e.g. cannibalism) and that a maximum level of predation was reached. Predation by larvae ofMedetera spp. (Diptera, Dolichopodidae),Thanasimus spp. and other beetles, and parasitism by wasps (Hymenoptera, Pteromalidae) probably caused the low productivity in the uncaged bolts.      

Hydrogen peroxide induces caspase activation and programmed cell death in the amitochondrial Tritrichomonas foetus

Tritrichomonas foetus is an amitochondrial parasite protist which lacks typical eukaryote organelles such as mitochondria and peroxisomes, but possesses the hydrogenosome, a double-membrane-bound organelle that produces ATP. The cell death of amitochondrial organisms is poorly studied. In the present work, the cytotoxic effects of hydrogen peroxide on T. foetus and its participation on cell death were analyzed. We took advantage of several microscopy techniques, including videomicroscopy, light microscopy immunocytochemistry for detection of caspase activation, and scanning and transmission electron microscopy. We report here that in T. foetus: (1) H₂O₂ leads to loss of motility and induces cell death, (2) the dying cells exhibit some characteristics similar to those found during the death of other organisms, and (3) a caspase-like protein seems to be activated during the death process. Thus, we propose that, although T. foetus does not present mitochondria nor any known pathways of cell death, it is likely that it bears mechanisms of cell demise. T. foetus exhibits morphological and physiological alterations in response to H₂O₂ treatment. The hydrogenosome, a unique organelle which is supposed to share a common ancestral origin with mitochondria and has an important role in oxidative responses in trichomonads, is a candidate for participating in this event.Abbreviations TUNEL Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick-end labeling - PARP Poly (ADP-ribose) polymerase - DAPI 4,6-Diamidino-2-phenylindole dihydrochloride     

Expression of antigens in virulent and avirulent Indian strains ofLeishmania donovani

Indo-Gen mediated surface labelling with¹²⁵I demonstrated differences in surface oriented antigens between virulent and virulent promastigote ofLeishniania donovani, In case of virulent strains, surface polypeptides with molecular masses of 63, 53, 42 and 38 kDa were found to be labelled with¹²⁵I whereas in the case of aviralent stains 68, 55, 50, 46, 42 and 33 Da, components were iodinated. Further studies by immunoblot assay using different subcellular fractions of virulent and avirulent parasites demonstrated that antibody raised against gp63 cross-reacted with the 63 and 60 kDa antigen of the virulent and avirulentLeishmania donovani strains of Indian origin respectively. It indicates that these two polypeptides are antigenically similar. When virulent and avirulent cells were grown in the presence of varying concentration of tunicarnycin and immunoblot with anti gp63, it was observed that with increasing concentration of tunicamycin the 63 kDa polypeptide of the virulent cells shifted to approximately 58–57 kDa and the 60 kDa polypoptide of the aviruleni cells shifted to 57 kDa. This suggests that glycosylation may play an important role in antigenic variation between virulent and avirulent parasites.     

The dsRNA ofTrichomonas vaginalis is associated with virus-like particles and does not correlate with metornidazole resistance

Twelve metronidazole-resistant and twelve metronidazole-susceptible strains ofTrichomonas vaginalis were tested for the presence of dsRNA. Three resistant and five susceptible strains were found to contain dsRNA which indicated that metronidazole resistance does not correlate with the absence of dsRNA. Electron microscopy showed the homogenates of all dsRNA -positive strains to contain virus-like particles 32 –38 nm in diameter, while no such particles were found in the dsRNA-negative strains. A mutual relationship between the dsRNA and virus-like particles seems to exist. After this paper had been accepted for publication the occurrence of virus-like particles in dsRNA-positive trichomonads was reported by others (Wang A.L., Wang C.C.: The double stranded RNA inTrichomonas vaginalis may originate from virus-like particles.Proc. Nat. Acad. Sci. USA 83, 7956–7961, 1986).     

Rearing of two predators,Thanasimus dubius andTemnochila virescens, for the biological control ofIps grandicollis in Australia

Rearing methods for two coleopterous predators,Thanasimus dubius andTemnochila virescens, imported into Australia for the biological control ofIps grandicollis, were developed. Bionomic data obtained from laboratory rearings between 1982–1987 showed thatT. dubius eggs took about 7 days to hatch and that duration of the larval stage was about 42 days. Observations showed thatT. dubius had a prolonged prepupal stage (x=56.4 days, range 14–274 days), which was probably non-diapausal in nature. Mean adult longevity was 50 days (range 1–358 days).Temnochila virescens eggs took almost 9 days to hatch, and a lengthy larval stage (x=155.4 days, range 73–333 days) was observed. Mean duration of the pupal stage was 14 days (range 7–28 days). A long preoviposition period (x=141 days, range 47–206 days) was observed, and adults were very long-lived (x=232.7 days, range 14–667 days). Capacity for increase (r_c) calculated from rearing data suggested that numbers ofT. dubius could be increased faster thanT. virescens. Mortality between 1982–1987 averaged about 70% for both species. However, mortality ofT. dubius in 1987 increased significantly, suggesting that inbreeding or other methodological factors could be responsible. A mass-rearing method usingIps-infested pine billets was developed as a cheaper alternative to laboratory rearing, and was shown to be effective in producing large numbers of insects for release.     

Subcellular Localization of the Enzymes of the Arginine Dihydrolase Pathway in Trichomonas vaginalis and Tritrichomonas foetus

The enzymes of the arginine dihydrolase pathway were demonstrated in Tritrichomonas foetus and their subcellular localization determined for both T. foetus and Trichomonas vaginalis. Ornithine carbamyltransferase (anabolic and catabolic activities), ornithine decarboxylase and carbamate kinase activity were localized predominately (56–80%) in the non sedimentable fraction of both species. A large proportion (35–40%) of the arginine deiminase was, however, recovered in the large granular fraction, and this distribution was unchanged by increasing the ionic strength of the buffer. Upon density gradient centrifugation the particles containing arginine deiminase activity had an isopycnic density of 1.09 g/ml in percoll, and separated from hydrogenosomes (1.18 g/ml) and lysosomes (1.12 g/ml). Arginine deiminase was also the only enzyme of the dihydrolase pathway which demonstrated latency upon treatment of the 1.09 g/ml fraction with non-ionic detergents. The results demonstrate the presence of the arginine dihydrolase pathway in T. foetus and indicate that at least a portion of the arginine deiminase in trichomonads is membrane associated.     

Entry and intracellular location of Mycoplasma hominis in Trichomonas vaginalis

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

Development of eggs, larvae and juveniles of laboratory-reared blue whiting,Sillago parvisquamis (Percoidei: Sillaginidae)

The embryonic, larval and juvenile development of blue whiting,Sillago parvisquamis Gill, are described from a series of laboratory-reared specimens. Mean egg diameter and mean total length (TL) of newly-hatched larvae were 0.71 mm and 1.58 mm, respectively. The eggs were non-adhesive, buoyant and spherical with an oil globule (mean diameter 0.18 mm). Hatching occurred about 20 hours after fertilization at a temperature of 24.0–25.0°C, newly-hatched larvae having 38–40 myomeres. The yolk and oil globule were completely absorbed 3 days after hatching at 2.8–3.2 (mean 3.0) mm TL. Notochord flexion was completed by 7.2–8.2 (7.7) mm TL, and pectoral and caudal fin rays fully developed by approximately 10 mm and 8.5 mm TL, respectively. Completion of fin development occurred in the following sequence: caudal, pectoral, anal and second dorsal, first dorsal and pelvic, the last-mentioned by approximately 11 mm TL. The larvae ofS. parvisquamis andS. japonica, which closely resemble each other in general morphology and pigmentation, could be distinguished as follows. Newly-hatchedS. parvisquamis larvae had more myomeres thanS. japonica (38–40 vs. 32–34) and more melanophores on the dorsal surface of the body (19–28 vs. about 40).Sillago japonica had a vertical band of melanophores on the caudal peduncle, which was lacking in postflexionS. parvisquamis larvae. In addition, juveniles ofS. parvisquamis (larger than 23 mm TL) had melanophores on the body extending anteriorly to below the lateral line to form a midlateral band, whereas no obvious band occurred on similarly-sizedS. japonica juveniles.