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.


Introduction
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][4][5][6][7]. It was discovered by indirect immunofluorescence (IF) that some only T. vaginalis isolates infected with a dsRNA virus (called TVV) is related to the phenotypic variation of P270 [7,8], and the presence of TVV upregulated expression of the p270 gene concomitant with increased amounts of P270 transcripts and protein [8]. The presence of TVV and the property of phenotypic variation among some isolates further delineated two distinct isolate types among naturally-occurring clinical isolates [6]. Type I-designated isolates of T. vaginalis were TVV¯ and only expressed P270 cytoplasmically.
Type II isolates were TVV + that had heterogeneous subpopulations of surface and non-surface-expressing organisms that underwent phenotypic variation [6].
Therefore, in this study it was important to visualize and provide immuno-cytochemical evidence on the role of TVV with respect to the surface localization of P270 among Type II TVV + T. vaginalis parasites when compared with Type I TVV¯ isolate trichomonads. It was then found that long-term batch culture of TVV + isolate organisms resulted in the loss of TVV [6][7][8]. These isogenic TVV¯ progeny trichomonads were analyzed and compared to the original TVV + parental parasites [8]. Finally, as the function for P270 is unknown and no data exist regarding the intracellular location of P270, immuno-cytochemical localization experiments were performed. Among Type II TVV + organisms, P270 was seen throughout the external region of the membrane, in vesicles peripheral to the Golgi complex, and within vacuoles distinct from the electron dense hydrogenosomes involved in energy metabolism [9,10]. No surface P270 was detected among Type I TVV¯ trichomonads and P270 was detected in the cytoplasm within a subpopulation of organisms. These results reinforce important roles for Type II TVV + in the synthesis [8] and surface localization of P270.

Quantitation of P270 in trichomonads
It was established that Type I TVV¯ isolate trichomonads synthesize lower amounts of P270 when compared to the phenotypically varying Type II TVV + isolate organisms [3,7,8].
Therefore, it was important to quantitate amounts of P270 among the three virus-harboring T. vaginalis isolates used in this study as well as TVV¯ trichomonads derived from isolate 347 that lost the dsRNA virus after extended batch cultivation. This quantitation would then permit establishing the amounts of P270 within trichomonads with the IF and immune-cytochemistry observations.
The amounts of P270 were quantitated using an established previously published protocol [3]. Further, as an alternative method of quantitation, ultrathin sections of trichomonads collected in nickel grids as detailed below were treated with hybridoma supernatant with MAb C20A3 and secondary gold-conjugated antimouse IgG. One hundred randomly selected grids of Type II TVV + 347 isolate organisms and of the TVV¯ progeny trichomonads were analyzed for the number of detectable P270.

Transmission electron microscopy (TEM)
Organisms were first washed three times in PBS as above at 37°C prior to fixation overnight at RT in a solution of 0.4% glutaraldehyde, 4% paraformaldehyde, 0.5% sucrose, 1% picric acid, and 2mM CaCl 2 prepared in 100mM sodium cacodylate buffer.

Cryo-ultramicrotomy
After overnight chemical fixation at 4°C with 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1M cacodylate buffer, pH 7.2, the specimens were infused in a mixture of 25% (w/v) polyvinylpyrrolidone (PVP) and 2.3M sucrose for 30min, submerged in liquid nitrogen and transferred to a cryoultramicrotome (Leica Ultracuts UCT). Cryosections were obtained at the temperature range of -80°C to -100°C, collected on formvarcoated nickel grids and washed in PBS. This was followed by processing of the sections for treatment with MAb C20A3, as above.
Finally, the grids containing cryosections were thinly embedded in a mixture of 9:1 (v:v) of 3% polyvinyl alcohol and uranyl acetate [11] and observed in a JEOL 1210 electron microscope.

Immuno-cytochemistry
Ultrathin sections of trichomonads processed as above were collected in nickel grids and were quenched using 50 mM ammonium chloride with 3% BSA. The organisms were then washed in a PBS-2% BSA buffer, pH 8.0, for 30-min. Grids were then incubated with MAb C20A3 hybridoma supernatant. After incubation for 3 h at RT, the grids were washed prior to addition of secondary 5nm or 10nm gold-conjugated goat antimouse IgG (Sigma). The grids were finally stained in the dark for 20-min at RT with 5% uranyl acetate followed by lead citrate. Sections were then visualized by TEM using a JEOL 1210 model microscope.

Pre-embedding
Pre-embedding of samples was performed to avoid loss of protein antigen detected by MAb C20A3. Parasites were fixed as above for TEM. After 3 washes in PBS, trichomonads were quenched for 30min using 50mM ammonium chloride with 3% BSA and incubated with MAb for 3-h at RT under constant agitation. After several washes in PBS over a 30-min period, the organisms were incubated with 10nm gold-conjugated goat anti-mouse antibody for 1h at RT with constant stirring. The cells were then washed 3 times in PBS followed by re-fixation in 2.5% glutaraldehyde in 100mM sodium cacodylate buffer and routinely processed for TEM. Postfixation of parasites was in 1% osmium tetroxide and potassium ferricyanide, after which samples were dehydrated in acetone and embedded in Epon.

Reproducibility
Experiments on the NYH 286, T068-II and 347 T. vaginalis isolates were performed on at least four occasions under identical conditions and gave similar data.

Detection of P270 among dsRNA virus-positive and negative T. vaginalis isolates
We first performed IF experiments with hybridoma supernatant containing MAb C20A3 on a Type II TVV + (virusharboring) phenotypically-varying T. vaginalis isolate 347 that was heterogeneous for surface and non-surface expressing P270 [6][7][8].

Detection of P270 among TVV + T. vaginalis organisms and TVV¯ progeny trichomonads.
It has been established that Type II TVV + isolate organisms spontaneously abort the TVV during extended in vitro batch cultivation [7,8]. Comparative IF was performed using a parental surface-expressing P270 organism of isolate 347 with that of TVV¯ progeny trichomonads derived from the parental parasites. trichomonads [6,7]. Non-permeabilized TVV¯ progeny organisms gave 100% negative fluorescence for surface P270 equal to the negative, non-fluorescent trichomonads for part a. Therefore, IF was performed using permeabilized parasites, and not surprisingly, part b shows the detection of P270 for a single organism, which is consistent with previously established down-regulation of expression of P270 for TVV¯ trichomonads without surface P270, as shown below (Table 1) [3,8]. among Type II TVV + , dsRNA virus-harboring T. vaginalis isolate 347. Panels a and b were non-permeabilized organisms, and panel c were permeabilized parasites. Column 3 presents pictures of trichomonads visualized by Nomarsky differential interference microscopy to show the integrity of organisms during the experiments. Identical results were obtained with the other two TVV + isolates NYH 286 and T068-II. Negative controls performed simultaneously included control hybridoma supernatant without antibody, secondary gold-conjugated anti-mouse IgG antibody alone, and irrelevant MAb of the same IgG2a to a Mycoplasma pneumoniae protein, which is unreactive with T. vaginalis.

Figure 2:
Comparative IF using MAb C20A3 to P270 as probe. Panel a shows non-permeabilized parental TVV + T. vaginalis isolate 347 with a heterogeneous population of surface versus non-surface P270-expressing trichomonads, as before [6]. The parasites without surface P270 synthesize lower amounts of P270 compared with surface expressing organisms [8]. Panel b shows the cytoplasmic P270 of a permeabilized progeny TVVvirus-negative 347 parasites derived from extended batch cultivation of the parental TVV + 347 isolate trichomonads. The virus-minus T. vaginalis isolates and progeny organisms have down-regulated expression of the p270 gene and synthesize lower amounts of P270 [6][7][8], which is also evidenced by Table 1. The majority of the TVVvirus-negative 347 progeny organisms have no detectable P270 by IF. The same negative controls were used as in Figure 1 and gave no detectable P270.

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Quantitation of P270 among TVV + T. vaginalis organisms and TVV¯ progeny trichomonads
It was important to quantitate the amount of P270 among representative Type II TVV + isolate organisms compared to the progeny TVV¯ trichomonads.

Immuno-electron microscopy (IEM) localization of P270
The P270 protein was then visualized on the long-term grown TVV + T. vaginalis isolate NYH 286 organisms that were expressing surface P270. The property of phenotypic variation and heterogeneous populations with fluorescent and non-fluorescent trichomonads using the MAb C20A3 was first observed with this isolate [4]. As can be seen in Figure 3

Discussion
Evidence has shown that P270 is a member of a repertoire of high Mr protein immunogens that undergo coordinated phenotypic variation among trichomonads of T. vaginalis isolates that harbor a dsRNA virus (TVV) [6,7,16]. This property of phenotypic variation was originally discovered by FACS analyses and the experimental demonstration that trichomonads without surface P270 reverted to surface-positive for P270 during batch culture [6]. Similarly, purified parasites with surface P270 gave rise to a subpopulation of organisms without surface P270 [6].  (Figure 2), as before [4]. It is noteworthy that the MAb is directed to the DREGRD-epitope element that is tandemly repeated within P270 regardless of the presence or absence of TVV among isolates [5,13,14]. Earlier work on the molecular biology of P270 revealed a 17 amino acid transmembrane domain at the carboxy terminus [5,13,14], showing why MAb C20A3 strongly detects P270 by interacting with the exposed and high copy number of DREGRD  5). These data are also consistent with the known strong agglutination of surface-expressing parasites with MAb C20A3 [5]. This is the first time that P270 was detected on the flagella by IEM (Figure 3 suggest a mechanism for surface targeting heretofore unknown for T. vaginalis. Finally, as it has been shown that the MAb C20A3 is cytotoxic to trichomonads with surface P270 [15], the property of phenotypic variation for the Type II TVV + isolate organisms may represent an immune evasion strategy.
These findings on the immuno-localization of P270 contribute to our understanding of this unique and important protein of T. vaginalis organisms. I hope that this approach and results will provide new information that may shed light regarding the function of the molecule. Clearly, these studies establish a knowledge base that will be important for the future when compartmentalization analysis of P270 modified through alteration of the p270 gene will be performed.