Iron modulates phenotypic variation and phosphorylation of P270 in double-stranded RNA virus-infected Trichomonas vaginalis

Abstract

Trichomonas vaginalis infected with a double-stranded RNA virus undergoes phenotypic variation on the basis of surface versus cytoplasmic expression of the immunogenic protein P270. Examination of batch cultures by flow cytofluorometry with monoclonal antibody (MAb) to P270 yields both fluorescent and nonfluorescent trichomonads. Greater numbers and intensity of fluorescent organisms with surface P270 reactive with MAb were evident in parasites grown in medium depleted of iron. Placement of iron-limited organisms in medium supplemented with iron gave increased numbers of nonfluorescent trichomonads. Purified subpopulations of trichomonads with and without surface P270 obtained by fluorescence-activated cell sorting reverted to nonfluorescent and fluorescent phenotypes when placed in high- and low-iron media, respectively. No similar regulation by iron of P270 was evident among virus-negative T. vaginalis isolates or virus-negative progeny trichomonads derived from virus-infected isolates. Equal amounts of P270 were detectable by MAb on immunoblots of total proteins from identical numbers of parasites grown in low- and high-iron media. Finally, P270 was found to be highly phosphorylated in high-iron parasites. Iron, therefore, plays a role in modulating surface localization of P270 in virus-harboring parasites.

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 IgG_2a 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 (, –6) before being suspended in hybridoma supernatant containing C20A3 or irrelevant MAb. After incubation with MAb by using established protocols (–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 (–6). Flow cytofluorometry as shown here was performed on at least three separate occasions, with similar results.

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-α-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 M_r 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.