Localization of the Phenotypically Varying P270 Protein on dsRNA Virus-positive and Negative Trichomonas vaginalis Isolates

Trichomonas vaginalis causes trichomonosis, the number one, non-viral STI, with adverse consequences to women’s reproductive health [1,2]. One of the most immunogenic proteins of T. vaginalis is called P270 of ~270-kDa in size. This P270 protein has tandemly repeated immunogenic DREGRD-epitopes that are detected by the monoclonal antibody (MAb) C20A3 [3-7]. It was discovered by indirect immunofluorescence (IF) that some naturally-occurring isolates had subpopulations of parasites that were heterogeneous in the surface expression of P270 [4]. These isolates had both fluorescent and non-fluorescent organisms. Fluorescence activated cell sorting (FACS) [6] showed that 100 percent fluorescent trichomonads reflecting surface P270 became non-fluorescent. Likewise, sorted non-fluorescent parasites with cytoplasmic P270 reverted to fluorescent organisms with surface P270. It was hypothesized that parasite factors affect P270 synthesis and surface expression. Subsequently it was found that Abstract Trichomonas vaginalis is a flagellated protist and causal agent for trichomonosis, the number one, nonviral sexually transmitted infection (STI). The parasites of all naturally-occurring isolates of T. vaginalis synthesize the immunogenic protein called P270, which is encoded by a single copy gene with numerous tandemly-repeated elements encoding the sequence DREGRD detected by the monoclonal antibody (MAb) C20A3. Isolates have been defined based on the absence (Type I) or presence (Type II) of T. vaginalis dsRNA virus (TVV). Type II TVV+ isolate organisms grown in batch culture have previously been shown to undergo phenotypic variation between surface and non-surface (cytoplasmic) expression of P270 using the MAb C20A3 in immunofluorescence (IF) assays. Type I TVVtrichomonads synthesize lower amounts of P270 that is only found in the cytoplasm. In this study both IF and immunoelectron microscopy (IEM) analysis were performed for the first time to localize P270 on and within both types of trichomonal isolates. IF experiments of Type II TVV+ parasites show whole surface labeling of intact, non-permeabilized organisms and surface and cytoplasmic fluorescence for permeabilized trichomonads. Immunocytochemistry of Type II trichomonads treated with MAb C20A3 presented gold-labeling of P270 throughout the external membrane as well as within peripheral vacuoles. Further, there was no P270 detected within the electron dense hydrogenosome organelles. On the other hand, for Type I TVVpermeabilized organisms, P270 is detected by IF only in the cytoplasm of some parasites, and immunocytochemistry confirmed that no trichomonads had surface P270. These data provide evidence for the first time of compartmentalization and surface localization of P270 among phenotypically varying, Type II TVV+ T. vaginalis compared to the Type I TVVorganisms.