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PubMed2000

Characterization, cloning and immunolocalization of a coronin homologue in Trichomonas vaginalis.

On adhesion to host cells the flagellate Trichomonas vaginalis switches to an amoeboid form rich in actin microfilaments. We have undertaken the identification of actin-associated proteins that regulate actin dynamics. A monoclonal antibody 4C12 raised against a cytoskeletal fraction of T. vaginalis labeled a protein doublet at circa 50 kDa. These two bands were recognized by the antibody against Dictyostelium discoideum coronin. During cell extraction and actin polymerization, T. vaginalis coronin cosedimented with F-actin. By two-dimensional gel electrophoresis, the protein doublet was separated into two sets of isoforms covering two Ip zones around 6 and 7. By screening a T. vaginalis library with 4C12, two clones Cor 1 and Cor 2 were isolated. This gene duplicity is a particularity among unicellular organisms examined. The complete sequence of the gene Cor 1 encodes a 435-residue protein with a calculated molecular mass of 48 kDa and Ip of 5.58. The incomplete sequence Cor 2 was very similar but with a more basic calculated Ip than Cor 1 on the same region. T. vaginalis coronin had 50% similarity with the coronin family, possessing the five WD-repeats and a leucine zipper in its C-terminal part. Double immunofluorescence labeling showed that coronin mainly colocalized with actin at the periphery of the adherent amoeboid cells. However, coronin labeling displayed patches within a reticular array. Immunogold electron microscopy confirmed the coronin labeling in the actin-rich microfilamentous fringe beneath the plasma membrane, with accumulation in phagocytic zones and pseudopodial extensions. In T. vaginalis, one of the first emerging lineage of eukaryotes, coronin seems to play an important role in actin dynamics and may be a downstream target of a signaling mechanism for the cytoskeleton reorganization.

Actins↗
PubMed2001

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

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

Acid Phosphatase↗
PubMed2004

Prevalence of HIV, syphilis, Chlamydia trachomatis, Neisseria gonorrhoea, Trichomonas vaginalis and candidiasis among pregnant women attending an antenatal clinic in Khartoum, Sudan.

Antenatal screening and treatment for sexually transmitted infections (STIs) has shown some benefits in countries where the practice is adopted. To date the prevalence of STIs in the Sudan is unknown. Our aims were to establish the prevalence of STIs among pregnant women in Khartoum (Sudan), to identify any risk factors and to suggest management. One hundred and fifty-one pregnant women were recruited for the study in an antenatal clinic in Khartoum in 1999. The prevalence of Chlamydia trachomatis, HIV, Trichomonas vaginalis, syphilis, Neisseria gonorrhoea and vulvo-vaginal candidiasis among the study population was established; 7.3% of patients suffered multiple infections. No risk factors for acquiring an STI were identified apart from genital ulcer disease. These findings suggest that a syndromic approach for treating STIs during pregnancy might not be possible. We conclude that STIs have a high prevalence among pregnant Sudanese women in Khartoum. The providers of health care in the Sudan need to revise their priority list.

Adult↗
PubMed1984

Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors.

Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

Animals↗
PubMed1984

The deoxyribonucleoside phosphotransferase of Trichomonas vaginalis. A potential target for anti-trichomonial chemotherapy.

Trichomonas vaginalis, a human protozoan parasite known to lack the capability of synthesizing purine and pyrimidine nucleotides de novo, was found also incapable of converting its ribonucleotides to deoxyribonucleotides. The only apparent means of providing deoxyribonucleotides for DNA synthesis relies on salvaging exogenous deoxyribonucleosides by a deoxyribonucleoside phosphotransferase activity in the T. vaginalis 10(5) g pelletable fraction. The activity, constituted by at least two isozymes I and II, can be solubilized by Triton X-100, has a pH optimum of 5.0-6.0, and recognizes only thymidine, deoxyadenosine, deoxyguanosine, and deoxycytidine as the phosphate acceptor. TMP, dAMP, dGMP, dCMP, dUMP, FdUMP, and p-nitrophenylphosphate can serve as phosphate donors. Enzyme I has been purified 10-fold by DEAE-Sepharose chromatography and Sephacryl 200 filtration, and is totally freed of the acid phosphatase of T. vaginalis. It has an estimated molecular weight of 200,000 and Km values of 2-3 mM for the four deoxyribonucleosides, which act on each other as competitive inhibitors. It also possesses phosphatase activity capable of hydrolyzing p-nitrophenylphosphate with a Michaelis constant of 0.74 mM. The rates of hydrolysis are enhanced by thymidine, which suggests that the latter may be the preferred phosphate acceptor, and Enzyme I may be, thus, more a transferase than a phosphatase. This enzyme could be a potential target for antitrichomonial chemotherapy.

Animals↗
PubMed1984

Trichomonas vaginalis is dependent on uptake and degradation of human low density lipoproteins.

Human plasma low density lipoprotein uptake by the urogenital pathogen, Trichomonas vaginalis, was examined. Rapid binding and internalization of 125I-labeled low density lipoproteins by live T. vaginalis was observed at 37 degrees C. Data showing parasite degradation of the internalized apoproteins and lipid accumulation following low density lipoprotein uptake was obtained. Acquisition of low density lipoproteins was by a trichomonad surface protein that possessed a molecular weight of greater than 250,000. The receptor is specific for apolipoprotein CIII, a component of high, low, and very low density lipoprotein subfractions. Low density lipoproteins in a semi-defined medium of trypticase, nucleic acid precursors, vitamins, and maltose promoted T. vaginalis growth and multiplication at rates and levels equal to the yeast extract-trypticase-serum complex medium routinely used for culture of trichomonads. HeLa cell membranes as a source of lipids were unable to sustain T. vaginalis organisms. These data demonstrate host lipoprotein internalization by T. vaginalis via a specific uptake mechanism.

Animals↗
PubMed1987

Trichomonas vaginalis phenotypic variation occurs only among trichomonads infected with the double-stranded RNA virus.

Trichomonas vaginalis isolates were examined for the presence of viral double-stranded RNA (dsRNA) and the property of phenotypic variation. Only the heterogeneous isolates composed of mAb-reactive and -nonreactive organisms, as determined by indirect immunofluorescence and flow cytofluorometry, and capable of phenotypic variation possessed the dsRNA. Both the positive and negative phenotype subpopulations separated from the heterogeneous parent contained equal amounts of the dsRNA. Loss of the dsRNA upon prolonged in vitro cultivation always correlated with the lack of expression of the major immunogen. The data indicate a relationship between the presence of the dsRNA and the ability of the pathogenic human trichomonads to express immunogens on their surfaces and to undergo phenotypic variation.

Animals↗
PubMed1990

Specific erythrocyte binding is an additional nutrient acquisition system for Trichomonas vaginalis.

Specific receptor-mediated binding by Trichomonas vaginalis of human erythrocytes was demonstrated. The ability of live parasites to internalize erythrocytes was also documented. In vitro growth assays during lipid-free and iron-limiting conditions that do not support the survival of T. vaginalis organisms showed that purified erythrocyte lipids and hemoglobin were each able to provide lipids and/or hemoglobin iron for trichomonal growth and multiplication. Parasites bound hemoglobin in a highly specific receptor-mediated fashion, and only the homologous unlabeled hemoglobin, but not lactoferrin and transferrin, competed with iodinated hemoglobin binding. Two antibody-crossreactive surface proteins of the parasites were identified as adhesins, and antibody to the individual adhesins inhibited T. vaginalis recognition and binding of erythrocytes. Finally, patient sera possessed antibody to the adhesins, showing the immunogenic nature and in vivo relevance of the trichomonad proteins during infection.

Animals↗
PubMed1991

The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis.

Levels of adherence of Trichomonas vaginalis to epithelial cells was found to be modulated by iron. Cytoadherence values were greater than or equal to twofold higher for trichomonads grown in a complex cultivation medium supplemented with iron. This increase in adherence levels was specifically mediated by iron; parasites cultured in a low-iron medium in the presence of salts other than iron were unresponsive to changes in adherence levels. Expression of the higher adherence property, by parasites grown first in low-iron medium followed by supplementation with iron, was a function of time, and the extent of cytoadherence was proportional to the concentration of iron added to the medium. Lactoferrin, an important iron source for trichomonads at the site of infection, elevated adherence of the parasite to epithelial cells, demonstrating the likely in vivo modulation of adherence by iron. The alteration of levels of adherence caused by iron was determined to be a reflection of gene expression of previously characterized trichomonad adhesins. Parasites grown under iron-replete conditions had higher quantities of surface-exposed adhesins, and this was a result of increased synthesis of adhesins. Actinomycin D and alpha-amanitin prevented expression of adhesin molecules, which resulted in decreased cytoadherence, showing that adhesin synthesis was dependent on gene transcription. Data indicated that genes encoding the four trichomonad adhesins are coordinately regulated by iron.

Amanitins↗
PubMedDate not supplied

Alternating phenotypic expression of two classes of Trichomonas vaginalis surface markers.

The antigenic heterogeneity of Trichomonas vaginalis is due in part to the membrane disposition of immunogens (repertoire A) among some but not all isolates and among subpopulations of trichomonads of certain isolates. Heterogeneous T. vaginalis isolates undergo phenotypic variation for the A repertoire of immunogens. The presence of immunogens on (A+ phenotype) or the absence of them from (A- phenotype) trichomonal surfaces clearly influences the virulence traits of the pathogenic human trichomonads. A- parasites, for example, possess an enhanced ability to cause cytadherence-dependent killing of HeLa cells as compared with A+ parasites. A relation between phenotype of repertoire A and adherence was further substantiated by fractionating parasites of a parent T. vaginalis isolate, yielding adherent and nonadherent subpopulations. Trichomonad proteins were identified as putative adhesin candidates (repertoire B); adhesins were synthesized only by adherent (B+ parasites). Flow cytofluorometry of B+ and B- (nonadherent) subpopulations with antibody to proteins of the A repertoire demonstrated corresponding A- B+ and A+ B- phenotypes, respectively. Data support the hypothesis that trichomonads of some isolates of T. vaginalis undergo alternating expression of at least two classes of surface markers.

Animals↗
PubMed1993

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

Two major ethanolamine phosphate-substituted inositol phosphosphingolipids have been identified in the unsaponifiable acidic lipid fractions of Tritrichomonas foetus and Trichomonas vaginalis. The compounds were radiolabelled and purified by high-performance thin-layer chromatography followed by high-performance liquid chromatography. The structures were determined by a combination of tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) experiments, and gas-liquid chromatography of components obtained by degradation and derivatization. Inositol in the T. foetus component was 1-linked to the phosphosphingolipid, had the phosphoethanolamine group at the 3-position and a fucosyl residue at the 4-position. The T. vaginalis component lacked the fucosyl moiety. Both organisms also produced inositol phosphosphingolipids having the same long-chain base (sphingosine or dihydrosphingosine) and the same fatty acyl distribution as the inositol diphosphate compounds. These glycosphingolipids may represent metabolic intermediates for new types of membrane anchors for surface glycopeptides or glycolipids that mediate the host-parasite relationship of these trichomonads. The MS/MS and NMR spectroscopic data should provide reference information for structural determinations of other phosphorylated inositol derivatives.

Animals↗
PubMed1982

Zinc sensitivity of Trichomonas vaginalis: in vitro studies and clinical implications.

The high zinc concentration (2.3-15.3 mM) in human prostatic secretions may be an important defense of the male lower urinary tract against infection by Trichomonas vaginalis. Trichomonads were rapidly killed by zinc salts at concentrations similar to those in prostatic fluid of normal men. There was some variation in the zinc sensitivity of 15 clinical isolates of T. vaginalis (minimal inhibitory concentration, 0.8-6.4 mM). A time-kill technique showed subtle differences in the kinetics of zinc killing of trichomonad strains. It was possible to select relatively zinc-resistant substrains of T. vaginalis from a zinc-sensitive population. Zinc resistance was a stable characteristic after multiple passages of substrains in growth medium without supplemental zinc. Variations in the zinc sensitivity of infecting T. vaginalis strains or in the zinc content of host prostatic secretions may be significant determinants of the natural history of T. vaginalis infection in men.

Animals↗
PubMed1983

Phagocytosis and killing of Neisseria gonorrhoeae by Trichomonas vaginalis.

Type 2 and 4 transparent and opaque Neisseria gonorrhoeae demonstrated a logarithmic loss of viability with a half life of approximately 10-30 min when incubated in the presence of Trichomonas vaginalis. Although this effect was observed in the absence of serum for most types of gonococci tested, it was consistently enhanced by the addition of human serum. Only for type 4 transparent gonococci did this process show an absolute serum requirement. Cytochalasin B inhibited the loss of viability. The nonphagocytic cattle parasite Tritrichomonas foetus did not ingest or kill N. gonorrhoeae. Electron microscopy revealed phagocytic uptake and degradation of N. gonorrhoeae in T. vaginalis, indicating that the loss of viability of N. gonorrhoeae was the result of phagocytosis followed by intracellular killing of gonococci by T. vaginalis.

Animals↗
PubMed1987

The effects of inhibitors of sulphur-containing amino acid metabolism on the growth of Trichomonas vaginalis in vitro.

A range of inhibitors of enzymes catalysing the metabolism of sulphur-containing amino acids were tested for efficacy against Trichomonas vaginalis in vitro. Sinefungin, tubercidin, ara A, bithionol, hexachlorophene, dichlorophene and 5-azacytidine were found to be effective antitrichomonal agents. Combinations of any two of these inhibitors were, in most cases, no more effective than one inhibitor used alone, but marked synergy was apparent with monothioglycerol and methionine. None of the inhibitors investigated was as potent as metronidazole, the drug of choice for the treatment of trichomoniasis.

Amino Acids, Sulfur↗
PubMed1987

Activities of metronidazole and niridazole against Trichomonas vaginalis clinical isolates.

The activities of niridazole and metronidazole against Trichomonas vaginalis C1-NIH and six isolates of clinical origin were compared using the in-vitro assay technique. Under the standard anaerobic assay conditions both metronidazole and niridazole were highly effective at low concentrations against all the strains used. However niridazole, in contrast to metronidazole, was equally effective in the aerobic assay, a feature which may be exploited in the chemotherapy of patients with refractory trichomoniasis.

Animals↗
PubMed1988

Treatment failure in Trichomonas vaginalis infections in females. I. Concentrations of metronidazole in plasma and vaginal content during normal and high dosage.

Concurrent estimation by high pressure liquid chromatography (HPLC) of the concentrations of metronidazole in plasma and vaginal content in 12 patients with infections due to Trichomonas vaginalis who did not respond to normal and high-dose treatment has shown that the two levels are closely related to each other and to the dose. Individual idiosyncrasies in absorption of drug from the gut lumen or in its transfer into the vaginal content are unlikely to be the cause of treatment failure.

Adolescent↗
PubMed1988

Treatment failure in Trichomonas vaginalis infections in females. II. In-vitro estimation of the sensitivity of the organism to metronidazole.

We have developed a standard procedure to measure the susceptibility of Trichomonas vaginalis to metronidazole in vitro, by controlling the state of oxygenation of the test cultures, the size of the inocula in terms of infectivity, and providing multiple tests at each drug concentration. Estimates were made of the in-vitro sensitivity to metronidazole of T. vaginalis isolated from 11 randomly selected patients, all, except for three defaulters, known to have been treated successfully, and from six 'treatment failure' patients. Stocks isolated from 'successfully treated' patients, were all highly sensitive to metronidazole. Eighteen stocks isolated from the 'treatment failure' patients fell into two groups: (a) those clearly separable from sensitive stocks, with ED50 values (the concentration of drug at which growth is prevented in 50% of test wells) in N2 of up to 0.51 mg/l and 3.5 to 11 mg/l in N2/1%O2; (b) those giving intermediate ED50 values of up to 0.20 mg/l in N2 and 0.7 to 1.8 mg/l in N2/1%O2. The current procedure demonstrates clearly that repeated treatment failure in patients without other complication is associated with enhanced resistance to metronidazole of T. vaginalis, and it may discriminate between T. vaginalis infections that are likely to respond to higher dosage and those that are not.

Adult↗
PubMed1995

Cytotoxicity of hamycin for Trichomonas vaginalis, HeLa and BHK-21.

Hamycin, a polyene antibiotic related to amphotericin B, has been used topically to treat fungal and protozoan infections in India. We assessed the cytotoxic activity of hamycin on nine metronidazole resistant or susceptible Trichomonas vaginalis strains isolated from symptomatic or asymptomatic patients. Cytotoxic activity of hamycin against two mammalian cell lines, BHK-21 and HeLa, was also determined. Tritiated thymidine pulse labeling after drug-washout and a recovery period was used to distinguish death of target cells from temporary static effects of the drug. Liposomal hamycin and hamycin formulated with dimethyl sulphoxide, deoxycholate or glycerin were compared. Although all hamycin preparations were trichomonacidal at approximately 1 mg/L, hamycin-dimethyl sulphoxide was stable for only 24 h, and hamycin glycerin was incompletely solubilized. Hamycin-deoxycholate remained a stable gel for 2 months at room temperature, but its activity was reduced four-fold when compared to the fresh preparation. The mammalian tissue culture cell lines HeLa and BHK-21 were killed by trichomonacidal concentrations of hamycin-deoxycholate. This cytotoxicity of hamycin for mammalian cells is a concern, but new forms of the drug are under development that may allow more widespread use of this drug.

Animals↗