Identification and properties of Trichomonas vaginalis proteins involved in cytadherence.

Trichomonas vaginalis NYH286 surface proteins which are candidates for mediating parasite cytadherence (adhesins) were identified. At least four trichomonad protein ligands ranging in relative molecular mass from 65 to less than or equal to 21 kilodaltons were found to selectively bind to chemically stabilized HeLa cells. The proteins were present on the surfaces of 10 different isolates of T. vaginalis examined; however, the nonpathogenic trichomonad T. tenax did not possess similar HeLa cell-binding proteins under identical experimental conditions, suggesting that these proteins are unique to the pathogenic human trichomonads. The surface nature of the candidate adhesins was confirmed by the ability of the proteins on intact, live organisms to be radioiodinated and to be removed with trypsin treatment. Rabbit antiserum (immunoglobulin G fraction) generated against adhesin proteins electroeluted from acrylamide preparations inhibited cytadherence compared with control immunoglobulin G. An adherence-negative subpopulation of T. vaginalis NYH286 organisms was also isolated. These nonadherent trichomonads did not synthesize the adhesin proteins. Interestingly, absence of adhesins from these parasites paralleled expression of a major immunogen known to undergo phenotypic variation. Revertant organisms derived from the adherence-minus subpopulation synthesized the adhesins and attached to HeLa cells. The emergence of revertant adherent T. vaginalis organisms also corresponded with the appearance of parasites which were without the major immunogen on their surface. Finally, it was determined that only those parasites lacking the major surface immunogen were capable of adherence and toxicity to HeLa cells.

Phenotypes and protein-epitope phenotypic variation among fresh isolates of Trichomonas vaginalis.

Fresh isolates of Trichomonas vaginalis were examined for reactions to a panel of five monoclonal antibodies (MAbs). Four MAbs (C20A3, DM126, DM116, and C55) were to distinct surface immunogens and one MAb (L64) was to a cytoplasmic component. The fresh isolates were evaluated by indirect immunofluorescence (IF), immunoblotting, and radioimmunoprecipitation. IF assay with C20A3 MAb gave isolates which were homogeneous nonstaining (negative [Neg] phenotype) and isolates which were heterogeneous staining and nonstaining (positive [Pos] and Neg phenotype, respectively) organisms. Immunoblotting and radioimmunoprecipitation assays revealed that surface phenotypic heterogeneity among isolates with C20A3 MAb was due to the presence or absence of the immunogen from the parasite surface. IF assay with DM126 MAb also gave Pos and Neg phenotypes among parasites of some isolates. All of the isolates were always Neg phenotype with DM116 and C55 MAbs. The occurrence of Neg phenotype organisms with DM126, DM116, and C55 was due to epitope inaccessibility to their respective MAbs and not to the absence of the immunogen from trichomonal membranes. All isolates possessed the cytoplasmic protein recognized by L64 MAb. Paired isolates (taken 5 to 6 days apart) from 24 women were also studied. Four of the 24 paired isolates (16%) had different phenotype distributions at the two timepoints for C20A3. Fresh isolates also underwent phenotypic variation during in vitro growth and multiplication, as determined with C20A3. Also, 7 of the 24 paired isolates demonstrated dramatic changes in the accessibility of DM126 MAb to epitope binding. Lastly, 55 (90%) of 60 serum samples from patients with trichomoniasis evaluated in this study possessed antibody to the C20A3 reactive molecule. The data show that the fresh T. vaginalis isolates were predominantly Neg phenotype and confirm the occurrence of protein and epitope phenotypic variation for major immunogens among fresh isolates of the pathogenic human trichomonads.

Phenotypic variation and diversity among Trichomonas vaginalis isolates and correlation of phenotype with trichomonal virulence determinants.

The extent and nature of heterogeneity among representative Trichomonas vaginalis isolates were evaluated by flow cytofluorometry analysis. Monoclonal antibody and monospecific antiserum to an immunodominant trichomonad surface glycoprotein with a molecular weight of 267,000 (267K glycoprotein) were used to evaluate fresh isolates (JHH and RU375) and long-term grown isolates (NYH286, IR78, and JH31A) of T. vaginalis. Isolates NYH286, JH31A, and JHH were made up of heterogeneous staining (positive [pos] phenotype) and nonstaining (negative [neg] phenotype) populations of trichomonads, whereas RU375 and IR78 were all neg phenotype parasites. Flow cytofluorometric patterns of agar clones derived from single organisms of heterogeneous isolates such as NYH286 showed populations which were either homogeneous pos or neg and also showed clones which were heterogeneous in nature containing both phenotypes. Fluorescence-activated cell sorting was also accomplished, and subpopulations of defined pos or neg phenotype were purified. Flow cytofluorometry evaluation of all isolates for an extended period revealed a phenotypic variation among all heterogeneous isolates and also for all clones and subpopulations derived from the heterogeneous isolates. On the other hand, IR78 and RU375 did not undergo phenotypic variation and have remained neg for greater than 4 years. Parasites which were nonreactive with either monoclonal antibody or monospecific antiserum to the 267K glycoprotein in flow cytofluorometry did not possess the antigen on their surface. This was determined by radioimmunoprecipitation assays using extracts of iodinated trichomonads. Finally, neg phenotype parasites yielded enhanced rates of contact-dependent cytotoxicity of host cell monolayers as compared with the pos phenotype trichomonads.

Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface proteins in a specific receptor-ligand fashion. Furthermore, parasitism of epithelial cells appears to render this cell type more susceptible than fibroblast cell types to contact-dependent cytotoxicity.

Soluble Trichomonas vaginalis antigens in cell-free culture supernatants.

Trichomonad proteins present in cell-free supernatants from logarithmic phase cultures of a pathogenic strain of Trichomonas vaginalis were detected with enzyme-linked immunosorbent assay employing antibody from mice and rabbit antisera. Numerous immunogenic membrane proteins of T. vaginalis were identified by immunoblotting of culture filtrate with rabbit antisera. Released membrane antigens ranging in molecular weight from 30 000 to 300 000 under reducing conditions were also detected in radioimmunoprecipitation assays using growth medium from mid-logarithmic phase organisms which were extrinsically labeled. Parallel experiments using [3H]thymidine-labeled T. vaginalis demonstrated that lysis was not solely responsible for the presence of parasite proteins in growth medium.