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Trichomonosis (14, 17) is the most common nonviral sexually transmitted disease (vaginitis) and is caused by infection with the protist Trichomonas vaginalis (28). Trichomonosis has major health consequences for women, as it is associated with adverse pregnancy outcomes (10), enhanced susceptibility to human immunodeficiency virus (20, 27) and possibly cervical neoplasia (29).

Experiments aimed at understanding the reported extensive antigenic heterogeneity among T. vaginalis isolates (9, 13, 18, 24) led to the discovery of the property of phenotypic variation (6). This was defined on the basis of surface versus cytoplasmic expression of a repertoire of high-M_r immunogens (1, 2, 4, 5). The monoclonal antibody (MAb) C20A3 recognized an epitope tandemly repeated within the highly immunogenic surface protein termed P270 (6-8). Analyses by flow cytofluorometry and fluorescence-activated cell sorting (FACS) of fresh clinical isolates revealed heterogeneous immunoreactivity, such as fluorescent and nonfluorescent subpopulations by indirect immunofluorescence with MAb (4, 6). These reactivities with MAb were similar to those reported for isolates with MAb, with polyclonal experimental sera, or with sera from patients with trichomonosis (8, 9, 13, 18, 24). Based on flow cytofluorometry with MAb, it became evident that two types of isolates occur naturally during infections with T. vaginalis (4). Type I isolates were homogeneous nonfluorescent (negative phenotype) trichomonads that synthesize and express P270 in the cytoplasm. In contrast, type II isolates were heterogeneous and comprised both fluorescent and nonfluorescent subpopulations (positive and negative phenotypes) that were then purified by FACS (6). Each purified subpopulation reverted to the opposite phenotype but only upon long-term daily passage in batch culture (6). It was further demonstrated that both MAb and polyclonal Ab from patients reactive with P270 were lytic for trichomonads with surface P270 in a complement-independent fashion (5, 11). In vivo, among type II isolates from patients, the percent of trichomonads with surface P270 ranged from 0 to 10% (4), suggesting that the host environment either eliminates parasites with surface P270 or favors cytoplasmic expression. Finally, the identification of the double-stranded RNA (dsRNA) virus within T. vaginalis organisms established a relationship between virus infection and phenotypic variation (26). The virus is multisegmented (15), and loss of virus from parental type II isolate organisms by batch culture (16, 26) produced virus-negative progeny such as type I isolate parasites that were incapable of surface placement of P270.

The complete sequence of a p270 gene of a fresh clinical isolate was recently reported (23). Furthermore, it has been shown that, except for the number of tandemly repeated units (23), the gene was highly conserved among type I and type II isolates (3). The repeated domain was flanked by 69 bp (23 amino acids) of upstream and 1,185 bp (395 amino acids) of downstream nonrepeat, coding regions (23). The sequences of the repeats within the p270 gene were identical (23). Furthermore, recent analyses revealed that the amino- and carboxy-terminal, nonrepeated regions were identical for P270s of different isolates (4), showing that protein sequences were not responsible for surface versus nonsurface placement of P270 during phenotypic variation and among isolates.

A relationship was established between iron and levels of cytoadherence and amounts of adhesins synthesized by T. vaginalis (21). Insofar as the cytoadherent type II trichomonads synthesizing adhesins were known to lack surface P270 (6, 21), our group hypothesized that iron directly modulated surface placement of P270. In this report I show that growth in low-iron medium promotes surface placement of P270 for virus-infected but not virus-negative parasites. Conversely, growth of virus-positive organisms in high-iron medium, which induces expression of trichomonad adhesins (21), yields parasites without surface P270. It is also shown that high-iron trichomonads highly phosphorylate P270 compared to organisms grown in low-iron medium.

Relationship between iron levels in medium and P270 surface expression among type II isolate trichomonads. Indirect immunofluorescence with live trichomonads was performed by using established conditions with the MAb C20A3. As seen for two representative experiments, whose results are given in Table 1, the type II clinical isolates T068-II, T066, and AL8 were heterogeneous for surface reactivity with MAb. The numbers of fluorescent trichomonads were always lower when parasites were grown overnight in the complex medium supplemented with iron. In medium depleted of iron, increased numbers of organisms with surface P270 were evident. This dramatic change in the percentage of the opposite phenotype in just three to four generations was unusual; earlier studies involving batch culture required weeks to produce a similar change in fluorescence patterns (6). Importantly, the dsRNA virus is lost among some isolates by daily passage in batch culture (6, 16, 26). An agar clone derived from a single parasite from the virus-positive parental organisms that lost the dsRNA virus (16) was also examined. These virus-negative AL8 progeny and the type I isolate T076 trichomonads were uniformly unreactive with MAb C20A3, as expected (16). As a control to affirm the absence of P270 from the surface of nonfluorescent organisms, a radioimmunoprecipitation (RIP) assay (6, 8) was performed by using MAb and detergent extracts of surface-iodinated type I isolate and virus-negative progeny trichomonads (AL8-N [Table 1]). Only type II isolate trichomonads had ¹²⁵I-labeled P270 readily immunoprecipitated and detected in autoradiograms after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (6, 8, 19).

Modulation of P270 surface expression by iron. Both subpopulations reactive and unreactive with MAb were purified by FACS (6) of isolate T068-II. Figure 1 presents cytofluorometric patterns of purified subpopulations grown in high-iron medium (panel A1) and low-iron medium (panel B1) with C20A3 MAb (solid lines) and an irrelevant MAb (dotted lines) over a 24-h period (three to four generations). These same trichomonads were then washed and placed in different media and monitored throughout another 24-h period. As seen in Fig. 1A2 and B2, within 6 h, a time period shorter than that required for purified subpopulations in batch culture (12), increased numbers of organisms changed to the opposite phenotype. Importantly, other divalent cations were added to iron-depleted medium, as was done previously by this laboratory (21). No similar change from surface to cytoplasmic expression of P270 as seen in Fig. 1B1 was observed, even after batch cultures were maintained over a period of several days (data not shown). No fluorescence was detected with an irrelevant MAb.

View larger version (17K): [in this window] [in a new window]   FIG. 1.   Flow cytofluorometry monitoring surface expression of P270 of purified nonfluorescent (A1) and fluorescent (B1) subpopulations of T. vaginalis isolate T068-II grown in media with different levels of iron. FACS to enrich for each phenotype was performed on the heterogeneous trichomonads grown in either low- or high-iron medium, as shown in Table 1. Purified subpopulations were then grown in medium described in Table 1 either supplemented with (A1) or depleted of (B1) iron. Indirect immunofluorescence was performed on live trichomonads with MAb C20A3 that binds to the DREGRD epitope contained within each repeat of the tandemly repeated unit (11) prior to examination by flow cytofluorometry. Solid lines refer to the incubation of parasites with the MAb C20A3, and dotted lines represent the negative control with an irrelevant MAb of the same IgG2a isotype (6, 8). Duplicate cultures of trichomonads shown in panels A1 and B1, grown identically, were then washed and incubated in low-iron (A2) or high-iron (B2) medium for up to 24 h, followed by flow cytofluorometry. Briefly, for FACS, 2 × 106 organisms were washed in phosphate-buffered saline (1, 4-6) before being suspended in hybridoma supernatant containing C20A3 or irrelevant MAb. After incubation with MAb by using established protocols (4-6), FACS was performed on trichomonads by using a Becton Dickinson FACS-IV. Data are presented on the basis of log fluorescence intensity versus parasite number. In this case, the data reflect the use of 2 × 103 cells in the analysis. Similar results were obtained when up to 105 organisms were used, as before (4-6). Flow cytofluorometry as shown here was performed on at least three separate occasions, with similar results.

Phosphorylation of P270 and cytoplasmic expression occurs in high iron. Figure 2A shows P270 bands from autoradiograms after SDS-PAGE (8, 19) of immunoprecipitated P270 from a RIP assay. Detergent extracts were prepared from 10⁷ T. vaginalis T068-II organisms labeled overnight with 1 mCi of [³²P]orthophosphate in 15 ml of high- versus low-iron medium. P270 is readily phosphorylated in parasites grown overnight in high- compared to low-iron medium. Similarly, greater intensities of phosphorylated proteins were seen in autoradiograms (Fig. 2B) of the total trichomonad protein gels (Fig. 2D) after SDS-PAGE. Gel lanes contained proteins from equal numbers of parasites. Interestingly, as seen in Fig. 2C, immunoblotting (8, 25) performed on duplicate gels such as those seen in Fig. 2D gave similar intensities of bands immunoreactive with MAb C20A3, indicating that iron levels did not affect the overall relative amounts of P270 within organisms. As a control, fluorograms of ³⁵S[methionine]-labeled trichomonads grown in high- and low-iron media were examined. Similar overall protein patterns were seen, showing that the differences in phosphorylation were not due to toxic effects from the various levels of iron in the medium. As additional controls, duplicate samples handled identically, except that they were not radiolabeled, were monitored by flow cytofluorometry. Patterns of fluorescence for parasites grown in low- and high-iron media were similar to those in Fig. 1A1 and B1 and Table 1. Finally, under identical conditions, phosphorylation was never evident for the adhesins mediating cytoadherence, regardless of the level of iron in the medium (Table 2) (21), which served as an internal control during these assays.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Higher levels of phosphorylation of P270 in T. vaginalis organisms grown in high-iron (H) versus low-iron (L) medium. (A) Autoradiograms from RIP assay performed as detailed before (4, 6, 8) by using detergent extracts of [32P]orthophosphate-labeled trichomonads incubated with MAb C20A3 or irrelevant control MAb. Immune complexes were precipitated by using protein A-bearing Staphylococcus aureus (6, 8). All extracts contained N--p-tosyl-L-lysine chloromethyl ketone to inhibit cysteine proteinases released upon solubilization of parasites. Immunoprecipitated 32P-labeled proteins were then solubilized by boiling S. aureus for 3 min. After bacteria were pelleted, the supernatant was subjected to SDS-PAGE, and gels were dried for autoradiography, as before (6, 8). (B) SDS-PAGE-autoradiography showing 32P-labeled protein bands of total proteins obtained from high- and low-iron parasites. Total proteins were precipitated by 10% trichloroacetic acid and processed as before prior to electrophoresis (8). (C) SDS-PAGE of total proteins as in panel B was then blotted onto nitrocellulose for probing with MAb C20A3 by using established protocols (8, 25). Control irrelevant MAbs of the same isotype were used as controls and did not give any reactivity with trichomonad proteins on the nitrocellulose blots. (D) Coomassie brilliant blue-stained gels of total proteins after SDS-PAGE show the complex patterns for both high- and low-iron medium-grown trichomonads. Changes in patterns, as evidenced in the Mr region between size markers 97.4 (in kilodaltons) and 116.5, are representative of high- and low-iron parasites, respectively. These serve as internal controls to monitor the iron status of trichomonads.