Heterologous expression in Tritrichomonas foetus of functional Trichomonas vaginalis AP65 adhesin
© Kucknoor et al; licensee BioMed Central Ltd. 2005
Received: 01 November 2004
Accepted: 04 March 2005
Published: 04 March 2005
Trichomonosis, caused by Trichomonas vaginalis, is the number one, nonviral sexually transmitted infection that has adverse consequences for the health of women and children. The interaction of T. vaginalis with vaginal epithelial cells (VECs), a step preparatory to infection, is mediated in part by the prominent surface protein AP65. The bovine trichomonad, Tritrichomonas foetus, adheres poorly to human VECs. Thus, we established a transfection system for heterologous expression of the T. vaginalis AP65 in T. foetus, as an alternative approach to confirm adhesin function for this virulence factor.
In this study, we show stable transfection and expression of the T. vaginalis ap65 gene in T. foetus from an episomal pBS-ap65-neo plasmid. Expression of the gene and protein was confirmed by RT-PCR and immunoblots, respectively. AP65 in transformed T. foetus bound to host cells. Specific mAbs revealed episomally-expressed AP65 targeted to the parasite surface and hydrogenosome organelles. Importantly, surface-expression of AP65 in T. foetus paralleled increased levels of adherence of transfected bovine trichomonads to human VECs.
The T. vaginalis AP65 adhesin was stably expressed in T. foetus, and the data obtained using this heterologous system strongly supports the role of AP65 as a prominent adhesin for T. vaginalis. In addition, the heterologous expression in T. foetus of a T. vaginalis gene offers an important, new approach for confirming and characterizing virulence factors.
The colonization of the urogenital tract of humans by the protozoan parasite Trichomonas vaginalis is responsible for trichomonosis , the most prevalent, non-viral sexually transmitted infection worldwide. Despite an estimated 8 million new cases per year in the United States alone , this health disparities disease  remains poorly studied. T. vaginalis infection is associated with adverse health consequences to both men and women, including infertility [4, 5], atypical pelvic inflammatory disease , and increased HIV transmission [7, 8]. Trichomonosis is also associated with preterm birth, low birth weight infants , predisposition to development of cervical neoplasia  in women and non-gonococcal urethritis  and chronic prostatitis  in men. T. vaginalis adherence to host VECs, a step preparatory to infection , is complex and involves four surface protein adhesins. Three adhesins studied to date at the molecular level share identity with metabolic enzymes of the hydrogenosome organelle [14, 15]. Thus, the proteins exhibit functional diversity based on cellular location. AP65 is a prominent T. vaginalis adhesin that is targeted both to the surface and hydrogenosomes, and this compartmentalization is in part modulated by iron . Further, the surface placement of AP65 is increased upon contact with host cells . Recently, silencing of ap65 gene expression in T. vaginalis with antisense reinforced the role of AP65 as a major adhesion .
The related Tritrichomonas foetus is a causal agent of bovine fetal wastage [17, 18]. Adherence by T. foetus to bovine VECs appears equally complex as that seen for T. vaginalis except that in this case, adherence involves glycoconjugates [19, 20]. Interestingly, the interaction between T. foetus organisms and VECs is both cell- and species-specific [21, 22]. For example, T. foetus attaches to bovine VECs, MDCK and HeLa epithelial cells, which have been used previously for studies on the host-parasite interaction. However, the bovine trichomonads have low background levels of adherence to human VECs. This may suggest that the phylogenetically-related T. vaginalis and T. foetus trichomonads differ in mechanisms of host parasitism and cytopathogenicity. We hypothesized that, given the different mechanisms of host VEC adherence by T. foetus and T. vaginalis, the heterologous expression in T. foetus of the T. vaginalis AP65 could be an alternative approach to confirm the function of AP65 as an adhesin.
Importantly, no cross-hybridization and immuno-crossreactivity with ap65 and mAbs to AP65, respectively, were evident in T. foetus. We now show the stable expression in T. foetus of a single copy of the T. vaginalis ap65 gene encoding an ~65-kDa protein . The stable transfectants expressed functional AP65 on the T. foetus surface. These findings importantly illustrate the utility of a stable transfection system for heterologus expression in T. foetus of T. vaginalis virulence genes.
Low levels of adherence to immortalized human MS-74 VECs and lack of ap65 by T. foetus
We next wanted to know if T. foetus had mRNA that hybridized to ap65 used as a probe. As shown in Figure 1B, no RNA band was detected by Northern analysis for T. foetus (lane labeled Tf). In contrast, a band of ~2-kb was readily hybridized by total RNA of T. vaginalis. MR100, the drug-resistant T. vaginalis that is unable to adhere to host cells as shown recently also had no detectable ap65 transcript, as a control . The equivalent amounts of rRNA bands seen in Figure 1C illustrate that similar amounts of RNA were added to the lanes used in the Northern blots (Figure 1B).
Episomal expression in T. foetus of ap65
We then checked for the episomal gene expression by means of RT-PCR. The PCR products following reverse transcription were separated on 1% agarose gels. Figure 2B confirms the presence of an ap65 band corresponding to 580-bp amplified from the transfectants with pBS-ap65-neo (lane 3), confirming the expression of ap65 from the plasmid. The same band is also seen in T. vaginalis (lane 1). As an internal control in these experiments, the common band of 650-bp seen in all lanes corresponds to the a-tubulin gene. This control was important to show the similar amounts of RNA used for RT-PCR in all samples. These results demonstrate that the episomal plasmid with the T. vaginalis ap65 gene is successfully expressed in transfected T. foetus.
T. vaginalis AP65 and AP65-HA are expressed in T. foetus, and episomal AP65 has function
Importantly, we further tested for the ability of episomal AP65 in T. foetus to bind to host cells using a ligand assay [15, 25]. Figure 3B (lane 4) shows a typical result of a ligand assay using T. vaginalis, and mAb 12G4 used as a probe detected AP65 on blots of proteins that bound MS-74 VECs that were solubilized for electrophoresis and blotting. Likewise, AP65 from transfected parasites bound to MS-74 VECs (lane 2) and gave a band with the same Mr. There were no detectable protein bands for wild-type T. foetus (lane 1) and T. foetus transfected with control plasmid (lane 3) handled identically in the ligand assay. These results show the ability of T. foetus transfectants to stably express functional AP65. Further, the lack of any immuno-crossreactivity with mAb is consistent with the absence of transcript (Figure 1B) and illustrates the absence of an AP65-like protein reactive with mAbs in T. foetus.
AP65 expressed in T. foetus is compartmentalized to the surface and hydrogenosomes
Immunofluorescence detects AP65 in transfectants on the surface and in hydrogenosomes
Episomal expression of fusion AP65-HA and sorting to the parasite surface
Finally, as shown in Figure 6B, immunofluorescence microscopy gave a positive reaction with anti-HA mAb with AP65-HA protein on the surface using non-permeabilized and in hydrogenosomes with permeabilized transfected (Tf-pBS-ap65-neo) trichomonads. The T. foetus transfected with the control plasmid (Tf-pBS-neo) was unreactive with anti-HA mAb and gave results identical to those shown for the wild type T. foetus. Finally, in data not shown, no fluorescence with anti-HA mAb was obtained using T. vaginalis organisms as controls under identical experimental conditions. These data using a different mAb to the fusion protein construct independently demonstrated the surface placement of episomal AP65 and AP65-HA on T. foetus.
Episomally-expressed AP65 in T. foetus is related to enhanced adherence to VECs
We have shown that trichomonads possess surface adhesins with functional diversity . The adhesins are also enzymes in hydrogenosome organelles involved in energy generation. Although complex and multifactorial, the process of T. vaginalis adherence to human VECs is mediated in part by AP65, the surface protein that plays a major role in adherence [15, 16]. In this study, we established the utility of the T. foetus bovine trichomonad as a heterologous expression model system. We feel that among the noteworthy findings of this report are the following: 1) We show the stable episomal expression of the T. vaginalis prominent ap65 gene (Figure 2). 2) The transcript is translated to form functional AP65 adhesin (Figure 3). 3) Episomal AP65 and fusion AP65-HA are compartmentalized to the surface and hydrogenosomes and are accessible to recognition by specific anti-AP65 and anti-HA mAbs (Figures 4 through 6). 4) AP65 on transfected T. foetus increased adherence to VECs compared to parasites transfected with control plasmid (Figure 7), and the enhanced binding was reduced with anti-AP65 antibodies, showing specific AP65 mediated VEC attachment.
The stable expression of T. vaginalis AP65 in transfected T. foetus allowed us to characterize via cell fractionation experiments the cellular location of the episomal protein. Remarkably, AP65 fractionated to the plasma membrane and hydrogenosomes (Figure 4). This fractionation data was reaffirmed by fluorescence experiments (Figure 5), and the ability to detect both surface AP65 and surface AP65-HA using distinct mAbs provides strong evidence for the trafficking of T. vaginalis proteins to distinct cellular compartments in the bovine trichomonad. These results indicate that machinery for recognition of the surface and organelle targeting sequences is similar among these phylogenetically-related trichomonad species. Importantly, the expression of AP65-HA now will permit future subclone analysis for identification of the protein region within AP65 directing surface channeling.
That surface AP65 on transfected T. foetus elevated levels of adherence to human VECs (Figure 7) is significant because human VECs are not the natural host cell for T. foetus [27, 28]. The net increase in binding levels above T. foetus wild type controls were specific, as evidenced by the fact that anti-AP65 IgG reduced adherence to original control values. It is not inconceivable that the experimental conditions used here would not increase adherence by transfected T. foetus to those seen by T. vaginalis. One possible explanation is the fact that efficient and optimal adherence to VECs requires at least three additional adhesins, something that has been experimentally verified [15, 16]. Alternatively, the copy number of episomal AP65 on T. foetus may not be optimal for production of amounts of adhesin needed for elevated adherence levels. Such a difference in copy number between AP65 on T. vaginalis and T. foetus (Fig. 3) may not be obvious based solely on fluorescence intensity, as this is not quantitative. In this regard, it may be that the equivalent protein of T. foetus decarboxylating malic enzyme is also surface expressed thereby interfering with sequestration of sufficient molecules of episomal AP65 for adherence. The future availability of specific antibodies to the T. foetus decarboxylating malic enzyme will be useful for testing this possibility. It is noteworthy that the ap65 gene and mAbs to AP65 did not cross-hybridize and immunoreact, respectively, with the equivalent decarboxylating malic enzyme gene and protein known to reside in hydrogenosomes of T. foetus [29, 30]. These data also now indicate that there is significant nucleotide and amino acid sequence divergence between T. vaginalis and T. foetus in these equivalent genes and proteins. Importantly, except for the N-terminal sequence submitted to the GenBank (Accession number AAK55143.1), to our knowledge the entire decarboxylating malic enzyme gene of T. foetus has not been characterized . Finally, not all episomal, surface-expressed AP65 molecules may be in the proper orientation to have adhesive function. Nonetheless, the significant increase in adherence attributable to episomal AP65 by transfected T. foetus once again underscores the role of AP65 in adherence by T. vaginalis.
This paper now provides evidence for the functional expression of a heterologous virulence factor in a related trichomonad species. This ability to express T. vaginalis or other foreign proteins using T. foetus as a model is particularly noteworthy, especially if an identical and/or equivalent virulence factor and property are not detectable within the bovine trichomonad. We feel that this approach is innovative and represents a significant advance for understanding the contribution of specific T. vaginalis virulence factors to overall mechanisms of trichomonal pathogenesis.
Parasite and cell cultures
Trichomonas vaginalis isolate T016 and Tritrichomonas foetus (02–97) were grown in Trypticase-yeast extract-maltose (TYM) medium supplemented with 10% heat-inactivated horse serum . Immortalized human MS-74 VECs were grown as detailed recently by us [15, 16] in D-MEM supplemented with 10% fetal bovine serum. VECs were grown at 37°C in a 5% CO2 atmosphere.
The plasmid pBS-ap65-neo was constructed by cloning the coding region of AP65-3 in pBS-FdHAHA-neo (forward primer 5' GTCCAGCATATGATGCTCGCATCTTCA GTC-3' and reverse primer 5'-GTCCACGGTACCTTAGTAGAGTTGCTCGTATTC-3'). The parent plasmid pBS-FdHAHA-neo  was used by us recently . The parent plasmid was partially digested and the 1.7-kb ap65-3 gene was cloned into the NdeI and Asp718 sites giving rise to pBS-ap65-neo. In order to generate AP65 with the HA tag, the stop codon of AP65 was mutated and cloned into pBS-FdHAHA-neo resulting in pBS-ap65-HA-neo. The new plasmids were confirmed by sequencing. Plasmid DNA for transfection was purified using maxi prep columns (Qiagen, Inc., Valencia, CA).
Stable transfection and selection for G418 resistance
Transfection of T. foetus cells was carried out by electroporation . Parasites at early logarithmic phase of growth were used for transfection. Briefly, 4 × 107 parasites were centrifuged at 1,800 rpm at 4°C, and the pellet was resuspended in 400 μl fresh TYM before transferring into a 4-mm gap cuvette (BTX®, Genetronics, Inc., San Diego, CA) with 25 μg of plasmid DNA. Electroporation was performed at 320 V, 1000 microfarads and 725 ohms using the ECM 630 Electro cell manipulator (BTX®). Following the pulse, cells were placed on ice for 10 min and transferred into two T25 flasks with 50 ml of fresh TYM-serum medium. The cells were grown free of drug for 24 h followed by the addition of Geneticin (Invitrogen-Life Technologies, Carlsbad, CA) at 100 μg ml-1. Single cells were cloned using soft-agar plates . The DNA was isolated from single cell cultures using DNAZol (Invitrogen) and further purified by phenol-chloroform extraction. The presence of plasmid in single cell clones was confirmed by PCR amplification of the neo gene. The sense 5' GATCGGTACCATGATTGATTGAACA AGATGGATTG-3', and antisense 5'-CTTTAGACCAAGTTCGTGTCAGAAGAACT CGTCAAG-3' primers were used in the PCR reaction.
RNA isolation and RT-PCR analysis
Total RNA was isolated using the Trizol reagent (Invitrogen). Briefly, 1 μg of total RNA was reverse transcribed using SuperScript II RNase H- Reverse Transcriptase (Invitrogen) in a reaction volume of 20 μl. One μl of the reverse transcribed cDNA was used as template for the PCR amplification. The ap65 primer sequences used for amplifications were ap65-sense 5'-CAGTCAGTCGACCAGTTAGATATGGGTACAGAC-3' and ap65-antisense 5'-GTGACAGGATCCCGCTCGCAGTTAGCGCATGTAG-3'. The α-tubulin gene primers were sense 5'-ACTCTGCTGCCTCGAGCACGGTATC-3' and antisense 5'-GAAATGACTGGTGCATAAGAGC-3'. The common annealing temperature for the primers of both genes allowed for the ap65 and α-tubulin primers to be included in the same PCR reaction.
Immunoblot detection of episomally-expressed AP65
Total protein from parasites was obtained as before using trichloroacetic acid (TCA) precipitation  and separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE)  prior to blotting onto Hybond-P membranes (Amersham Pharmacia Biotech, Pisscataway, NJ) for immunoblot detection with mAb 12G4 to AP65 and anti-HA mAb. The mAbs and epitope reactivity were described recently . Following blot reactivity with the mAbs, the bands were visualized by the chemiluminescence assay using horseradish peroxidase as the color developer (BioRad Laboratories, Hercules, CA).
The hydrogenosomes were isolated as recently detailed . Briefly, 1 × 108 trichomonads were homogenized in buffer (250 mM sucrose, 20 mM KCL, 10 mM KH2PO4, 5 mM MgCl2 and 20 mM Tris-HCl, pH 7.0). The homogenate was centrifuged to sediment nuclei and costae at 1,500 g for 10 min at 4°C. The supernatant was then transferred to a new tube and centrifuged at 12,000 × g for 10 min. The sediment was then washed once more with homogenization buffer and was highly enriched for hydrogenosomes, which was confirmed biochemically as recently described . The sediment was resuspended in the buffer and stored at -70°C. Membrane isolation was also carried out as described recently . T. foetus (1 × 108) were resuspended in phosphate-buffered saline (PBS), sonicated, and subjected to low-speed centrifugation at 500 × g for 15 min at 4°C. The resulting supernatant was further fractionated by sequential differential centrifugations of 1,500 × g, 10,000 × g, and 100,000 × g for 30 min at 4°C. The supernatant was used for protein analysis.
Ligand assay to determine the amounts of adhesins on the surface
The ligand assay to detect adhesins that bind to host cell surfaces was carried out as before . Briefly, after fixation of the MS-74 VECs with glutaraldehyde, 106 epithelial cells were incubated with a trichomonal detergent extract derived from 2 × 107 solubilized parasites. After incubation, cells were vigorously washed to remove unbound and loosely-associated trichomonad proteins. Cells were boiled in electrophoresis dissolving buffer to elute the proteins bound to the MS-74 VEC followed by SDS-PAGE. The gels were further stained with Coomassie Brilliant blue for visualization, and duplicate gels were blotted onto Hybond-P membranes for immunoblot analysis using the mAbs as probes.
AP65 from the membranes and hydrogenosomes derived from the fractionation experiments was immunoprecipitated using mAb DM116 . A standard immunoprecipitation protocol was used . Briefly, the fractions (0.5 mg) prepared as mentioned above, were mixed with 5.0 μg of mAb DM116 for 4 h at 4°C. The extract and mAb were mixed on a rotating platform for 3 h with Protein A-Sepharose beads. The beads were washed three times with PBS, and samples were eluted by boiling for 10 min in SDS dissolving buffer. Eluted proteins were resolved by SDS-PAGE, and immunoblot analyses and ligand assays were performed as described above.
Immunofluorescence detection of AP65 on the surface
Immunofluorescence of AP65 on the surface and in hydrogenosomes of trichomonads was carried out using a modification of a recently-described procedure . Briefly, 1 × 106 logarithmic-phase organisms were washed twice with cold PBS and fixed with 4 % paraformaldehyde for 10 min at RT. Fixed cells were washed in PBS and permeabilized with 1% NP-40 for 45 min at RT. Trichomonads were then blocked with 5% BSA for 1 h at RT prior to incubation for an additional 1 h at RT with hybridoma supernatants of mAb 12G4 (1:100) and anti-HA mAb F11 (1:1000). Parasites were washed with PBS and incubated for 1 h at 37°C with fluorescein isothiocyanate-conjugated anti-mouse IgG (Sigma) diluted 1:100. Finally, parasites were washed twice with PBS and observed under 1000× magnification using the Olympus BX41 microscope.
Immortalized human MS-74 VECs  were used for the adherence assay as recently described . Briefly, 2 × 104 MS-74 VECs were seeded onto individual wells of 96-well Costar flat bottom plates (Corning Inc., Corning, NY) and grown for 24 h in DMEM supplemented with 10 % fetal bovine serum (FBS). VECs were then washed twice with a medium mixture of DMEM:TYM (2:1; v/v) without serum. Trichomonads were labeled with [3H] thymidine for 18 h, washed three times with DMEM-TYM and resuspended. Then, 4 × 105 tritium-labeled parasites were added to the individual wells of the 96-well plate with a confluent MS-74 VEC monolayer and incubated for 30 min at 37°C. Cells were then washed thoroughly with the DMEM-TYM. Individual wells were placed in mini-scintillation vials and radioactivity was measured. The assay was performed with quadruplicate samples.
Reproducibility of experiments
Unless otherwise stated in the text, all experiments were performed numerous times and no less than on four different occasions.
adhesin protein Mr 65-kDa
Dulbecco's minimum essential medium
relative molecular weight
plasmid construct with the ap65 and neomycin genes
vaginal epithelial cells.
This work was supported by Public Health Service grants AI43940 and AI45429 from the National Institutes of Health. Members of the laboratory are also acknowledged for their suggestions and discussion of our work.
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