Characterization of the Trichomonas vaginalis surface-associated AP65 and binding domain interacting with trichomonads and host cells
© Garcia and Alderete; licensee BioMed Central Ltd. 2007
Received: 29 August 2007
Accepted: 25 December 2007
Published: 25 December 2007
AP65 is a prominent adhesin of Trichomonas vaginalis that mediates binding of parasites to host vaginal epithelial cells (VECs). AP65 with no secretion signal sequence, membrane targeting peptide, and anchoring motif was recently found to be secreted.
We first wanted to demonstrate surface association of AP65 to the parasite followed by the identification of the binding epitope interacting with both organisms and VECs. AP65 was found to bind to trichomonads, but not to trypsin-treated parasites, in an auto-ligand assay, suggesting the existence of a surface protein associating with AP65. Since rabbit antiserum IgG antibodies reactive with epitopes localized to the N-terminal region of AP65 inhibit the attachment of live parasites to VECs, we hypothesized that the binding domain was localized to this region. We subcloned five overlapping fragments of AP65 called c1 through c5, and expression of recombinant clones was confirmed with antibodies to AP65. Each purified recombinant protein was then tested for binding activity using an established ligand assay, and fragment c1 with the first twenty-five amino acids in the N-terminal domain was required for binding to VECs and, surprisingly, also to parasites. Importantly, c1 competed with the binding of AP65 to both cells types.
T. vaginalis AP65 is a secreted, surface-associated protein and a model is proposed to explain how this secreted protein functions as an adhesin.
Trichomonas vaginalis causes trichomonosis, the most common, non-viral sexually transmitted infection (STI) in humans . This STI poses a risk for adverse health consequences in both women and men. Adverse pregnancy outcomes, cervical neoplasia, atypical pelvic inflammatory disease are serious adverse outcomes for women [2–4]. Complications related to trichomonal infection in men are non-gonoccocal urethritis, prostatitis, epydidymitis, urethral disease, and infertility [4–8], and recently a relationship between trichomonosis and prostate cancer has been shown . Furthermore, increased risk for HIV acquisition and seroconversion has been well documented in both women and men [10–13].
T. vaginalis has increased ability to cytoadhere to epithelial versus fibroblast cells , and it is now accepted that preparatory to successful host infection and pathogenesis is adhesion of T. vaginalis to vaginal epithelial cells (VECs) [15–21]. Five different adhesin proteins (AP120, AP65, AP33, AP51, and AP23) mediate adherence to VECs, are members of multigene families, and except for one AP51 gene, are coordinately up-regulated by iron in medium, lactoferrin-iron, and heme-iron [15, 18, 19, 22–31]. Interestingly, laboratory-adapted T. vaginalis isolates synthesize lower amounts of adhesins and have lost the ability to up-regulate synthesis by iron . Fresh clinical isolate trichomonads immediately upon adherence to VECs but not HeLa cells display a dramatic change in morphology concomitant with synthesis and surface placement of adhesins , and this was more recently confirmed by analysis of the numerous trichomonad genes up-regulated upon contact with VECs . The adhesins AP120, AP65, AP33, and AP51 were found to have sequence identity to metabolic enzymes [19, 24–28, 31], which reside within the hydrogenosome .
AP65 is the hydrogenosomal NAD-dependent decarboxylating malic enzyme and is a prominent trichomonad adhesin . This conclusion is based on several lines of evidence. First, there is a direct relationship between the amount of AP65 bound to VECs and levels of adherence compared to other adhesins . Second, polyamine depletion increased levels of adherence up to 20-fold, and most of this increased adherence was abrogated by anti-AP65 antibody . Third, the genetic approaches involving antisense decreased expression of ap65  and heterologous expression of T. vaginalis AP65 in T. foetus  reaffirmed the role of AP65 in adherence. More recently analysis of the proteins secreted during growth of T. vaginalis parasites revealed numerous metabolic enzymes, among which included AP65. It is noteworthy that AP65 of the secreted protein preparation was found capable of being internalized by VECs, and this resulted in signaling of VECs for expression of genes, which included IL-8 and COX-2 . Episomal expression of AP65 within epithelial cells confirmed a role for AP65 within cells in up-regulating expression of genes . Therefore, as AP65 appears to play a role in establishment of infection and host response and given the significance of trichomonosis as a major STI, it is necessary to continue to characterize this adhesin and identify receptor-binding epitopes for possible future interference strategies.
In previous work, we have demonstrated that anti-AP65 serum IgG antibodies inhibit adherence of live parasites. Further, the mapping of the antibody-binding epitopes of AP65 revealed a cluster of epitopes at the amino terminus of AP65 . Therefore, we hypothesized that the receptor-binding epitope of AP65 was localized to the N-terminal region of the protein. In this study we confirmed that cytoadherence to VECs by T. vaginalis requires protein synthesis and surface placement of adhesins on trichomonads and showed that AP65 binds to trichomonads, but not trypsin-treated parasites, in an auto-ligand assay. Furthermore, purified recombinant overlapping fragments of AP65 tested for binding activity in a ligand assay showed that a complete N-terminal domain was required for binding to both VECs and parasites. A working model is proposed to explain how this secreted trichomonad protein associates with surface membranes and functions as an adhesin.
Binding to VECs requires protein synthesis and surface proteins
AP65 associates with fixed T. vaginalis organisms
AP65 recombinant fragments react with mAb and polyclonal antibodies to AP65
Recombinant subclone c1 binds to VECs and T. vaginalis
Recombinant subclone c1 competes with AP65 binding
Binding of T. vaginalis to host cells requires synthesis and surface expression of adhesins (Figure 1), in accordance with earlier studies [14, 17]. Recent data shows contact with VECs results in the up-regulation of various trichomonad proteins, among which include the trichomonad adhesins [17, 20]. The de novo synthesis of proteins might include not only the adhesins but chaperons or vesicle-associated proteins required to transport the adhesins to the surface. In fact, it has been shown that trichomonad disulfide isomerase (PDI), a protein with multiple functions and known to be a chaperon, is also up-regulated upon contact with VECs possibly helping in the compartmentalization of AP65 to various sites . Moreover, that AP65 was found to be secreted by T. vaginalis  requires that we reassess the manner in which this protein asserts its adhesive function. We hypothesized that AP65 and possibly other adhesins represent surface-associated enzymes bound after secretion and that possess alternative functions as has been shown for other microbial pathogens [34, 35]. It is noteworthy, however, that re-association of secreted AP65 for function need not exclude alternative mechanisms of surface placement of AP65. For example, two transmembrane domains have been identified , which may play a role for localizing AP65 onto the surface. If in fact there are multiple mechanisms at work for placing AP65 on the parasite surface, then this may represent an adaptation that enhances the overall functions of the adhesin-enzyme.
This data suggests the first 25 amino acids are essential to the binding domain of AP65 that is localized at the amino-terminus of the protein (Figure 5). Deletion of the first 25 amino acids resulted in the complete abolishment of binding to chemically-stabilized cells using a standardized ligand assay [15, 18, 29] of the recombinant clones (Figure 5). It is possible that a secondary structure formed by these amino acids is responsible for AP65 association. This hypothesis is supported by several observations. Two synthetic peptides (1–16 and 14–30) comprising the first 30 amino acids of AP65 did not inhibit adherence (Materials and Methods; data not shown), and bioinformatic analysis (The PSIPRED Protein Structure Prediction Server, University College London) of AP65 predicted two small helixes are formed by amino acids 8 to 11 and by amino acids 25 to 28. As a result, the recombinant AP65 subclones lacking the 25 amino acids would be missing the first helix, and this likely would yield improper folding of the putative binding motif. In addition, the hydrophobic nature of regions of AP65  may have no role in the binding of AP65 to host cells and parasites , and this is illustrated by the fact that all clones possess hydrophobic amino acids. Yet, only one recombinant clone bound to cell surfaces. Further, the recombinant clone c1 contains the 11–12 amino acid hydrogenosomal presequence, and our recent findings suggest based on electrophoretic mobility that the secreted AP65 is a preprotein with the presequence intact  and not processed as is the enzymatic form found within hydrogenosomes. This is a significant finding because it permits us understand for the first time the difference between the hydrogenosomal and the surface-associated AP65.
As mentioned above, housekeeping enzymes of pathogenic organisms are secreted and are anchorless, surface-associated proteins [34, 35]. Interestingly, the mechanism of secretion of some of these enzymes is unknown, and enzymes have no secretion peptide signal or LPXTG anchor motif [34, 35]. T. vaginalis in fact secretes numerous metabolic enzymes, including glyceraldehyde-3-phosphate dehydrogenase, α-enolase, fructose-1-6-biphosphate, and the adhesin AP65 into the extracellular environment . Like bacterial anchorless, surface proteins, T. vaginalis secreted metabolic enzymes do not possess any known secretion peptide signal(s) or anchor motif . These characteristics clearly categorize trichomonad AP65 and possibly other secreted enzymes as members of multifunctional proteins similar to those of other microorganisms.
The secretion and the absence of a covalent anchor motif suggest that AP65 is released extracellularly and binds to the surfaces of both organisms and VECs. Data suggests that the first 25 amino acids of the amino terminus are essential for the receptor-binding epitope. Once bound to a putative receptor on parasites, AP65 could form at least a dimer, since hydrogenosomal decarboxylating malic enzyme-AP65 is a dimer and a tretramer  possessing two amino-termini available for binding to respective parasite and host cell. This work permits us to generate a model for the association of trichomonad AP65 with surfaces of both trichomonad and VECs (Figure 7). This model is supported by previous data showing that polyclonal antibodies recognize surface AP65 and prevent cytoadherence . Furthermore, extracellular malic decarboxylase activity identical to AP65 is present . These data collectively support the idea of a protein motif, in both VECs and T. vaginalis, binding the N-terminal region of AP65. Further work will confirm the structure and orientation of the AP65 on the surface of T. vaginalis.
Parasites and MS74 cell cultures
T. vaginalis isolate T016 was grown at 37°C in trypticase-yeast-maltose (TYM) medium supplemented with 10% heat-inactivated horse serum (HS) . T016 isolate expressing AP65-Hemagglutinin (HA) fusion protein was generated by transfection with the pBS-ap65-3-HA-neo plasmid, as previously described [16, 23]. Stable transfectants were grown at 37°C overnight (o/n) in TYM-10% HS containing Geneticin (100 μg/lm) (GIBCO Invitrogen, Carlsbad, CA). Only mid-logarithmic phase parasites were used throughout.
Primary immortalized MS-74 vaginal epithelial cells (VECs)  were grown at 37°C in a 5% CO2 atmosphere in Dulbecco's Modified Eagle Medium (DMEM, GIBCO Invitrogen) supplemented with 10% HI fetal bovine serum (FBS), as before . For the adherence assay, 5 × 104 MS-74 cells were seeded into each well of 96-well black, clear bottom culture plates (Corning Incorporated Costar®, Corning, NY) and grown o/n in DMEM-10% FBS. Cells of a confluent monolayers were washed prior to fixation with 3% glutaraldehyde for 1 h at RT. Monolayers were washed twice and blocked o/n in 0.5 M glycine. Finally, wells were washed twice and maintained in 100 μl of RINGER buffer (0.12 M NaCl, 3.5 mM KCl, 2 mM CaCl2, 2.5 mM NaHCO3, pH 7.2–7.4) until use.
For adherence assays, T. vaginalis organisms were grown o/n in TYM-10% HS. Parasites were washed and suspended in TYM for labeling as before  prior to any treatment by adding calcein (2 μl ml-1) (Molecular Probes™ Invitrogen, Carlsbad, CA). Then, trichomonads were washed in RINGER buffer and suspended to 1 × 106 parasites ml-1 in TYM containing cycloheximide (20 μg ml-1) (Sigma-Aldrich, St. Louis, MO). Cycloheximide treatment of parasites was at 37°C for 30 min. Duplicate cultures of 1 × 106 parasites ml-1 in TYM were treated with bovine pancreatic type XI trypsin (1 mg ml-1) for 15 min at 37°C. Finally, a triplicate preparation of 1 × 106 organisms ml-1 was treated with cycloheximide (20 μg ml-1) for 30 min. However, at the 15 min incubation time point with cycloheximide, trypsin (1 mg ml-1) was added and parasites were incubated for the remaining 15 min at 37°C. Treated parasites were washed in TYM and resuspended in TYM to 2.5 × 106 cells ml-1. Then, 100 μl containing 2.5 × 105 parasites was added to individual wells of 96-well culture plates containing fixed confluent MS-74 VEC monolayers. After incubation for 30 min at 37°C, wells were washed 3 times in RINGER buffer followed by final addition of 200 μl of RINGER buffer. Fluorescence readings were taken at 485/528 nm (excitation/emission) in a Synergy HT plate reader (BioTek Instruments, Inc., Winooski, VT).
For experiments on inhibition of adherence by synthetic peptides, custom peptides were purchased from Sigma-Aldrich-Genosys. The peptides contained the amino-terminus of AP65-3 with the amino acids sequence MLASSVAAPVRNICRA and CRAKLPALKTGMTLLQD. A control peptide was also generated with random amino acids (RLAEVKGGPPHTSDMCNWI). Synthetic peptides were solubilized in 100% dimethylsulfoxide (DMSO) (Sigma-Aldrich), and the peptide concentration was quantified with BCA™ Protein Assay Kit (PIERCE, Rockford, IL). Synthetic peptides were diluted in 100 mM HEPES (Sigma-Aldrich), pH 7.5, to a stock concentration of 100 μg ml-1. Then 0.1 ng, 1 ng and 10 ng were added to individual wells of 96-well culture plates containing fixed 100% confluent MS-74 VECs monolayers. Each condition involved quadruplicate samples. Plates were incubated for 1 h at 37°C followed by addition of 100 μl of calcein-labeled parasites (2.5 × 105) to individual wells. After incubation for 20 min at 37°C, plates were washed 3 times in RINGER buffer and handle as above.
Cloning of trichomonad AP65 recombinant fragments
Primers1 used for cloning of ap65-3 gene
Primer sequence for AP65-3
5' c1 Bgl II
3' c1 Bgl II
5' c2 Bgl II
3' c2 Bgl II
5' c3 Bgl II
3' c3 Bgl II
5' c4 Bgl II
3' c4 Bgl II
5' c5 Bgl II
3' c5 Bgl II
5' FL NdeI
3' FL Asp718
Expression and purification of recombinant AP65 fragments
E. coli Tuner™ (DE3) competent cells (Novagen EMD Chemicals Inc) were transformed with individual AP65 construct plasmids and selected with kanamycin. Single colonies were used for culture and batch protein purification. One hundred ml of LB containing 50 μg ml-1 kanamycin were inoculated with 10 ml of o/n cultures of E. coli encoding each recombinant AP65 fragments. Cultures were grown for 3 h at 37°C with shaking. Induction of expression of recombinant protein was done by adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Fisher Scientific, Pittsburgh, PA), and the cultures were incubated for an additional 2.5 h at 37°C with shaking. Pellets were collected and bacteria lysed with 10 ml lysis buffer (50 mM sodium phosphate buffer, 50 mM Tris HCl, pH 8.0, 0.3 M NaCl, 1% Triton ×-100, 1% deoxycholic acid, and 8 M urea), sonicated for 2–5 min, and incubated for 30 min at 37°C. Debris in the lysate was clarified by centrifugation at 13,000 rpm for 30 min at 4°C in a Sorvall® SS-34 rotor (Thermo Fisher Scientific, Inc. Waltham, MA). Purification of recombinant proteins was done using HisLink™ Protein Purification Resin (Promega, Madison, WI), according to the manufacture's protocol. Briefly, lysate was added to 1 ml of resin and allowed to bind for 1 h at 4°C. Resin was allowed to precipitate and washed twice with 100 mM HEPES, pH 7.5, 25 mM imidazole, 8 M urea, and once with 100 mM HEPES, pH 7.5, 50 mM imidazole, and 8 M Urea. Finally, recombinant protein was obtained by placing the resin in a glass column. The bound recombinant proteins were eluted with 3 ml of 100 mM HEPES, pH 7.5, 750 mM imidazole, and 8 M urea. Purified recombinant proteins were dialyzed to remove the urea in Slide-A-Lyzer® dialysis cassettes (PIERCE,) with a 10,000 MW cutoff in a stepwise procedure. Cassettes were placed in a dialysis buffer (50 mM sodium bicarbonate, 1 mM DTT, 1 mM EDTA) containing decreasing concentrations of urea as follows: i) 500 ml dialysis buffer, pH 9.0, with 4 M urea, for 1 h at RT, ii) 500 ml dialysis buffer, pH 9.0, 2 M urea, for 2 h at 4°C, with change of buffer after an hour, iii) 200 ml dialysis buffer, pH 8.3, for 1 h at 4°C, without urea, and, iv) 500 ml dialysis buffer, pH 9.0, o/n at 4°C without urea. Protein concentration was quantified with the BCA™ Protein Assay Kit (PIERCE) and by SDS-PAGE.
Ligand assay and immunoblotting
This ligand assay has been extensively described [15, 18, 29]. MS-74 VECs were washed in RINGER buffer and fixed in 3% glutaraldehyde for 30 min at RT. Cells were washed twice and blocked o/n in 0.5 M glycine. For the ligand assay using recombinant AP65 subclones c1 through c5 (Figure 5, part A1), 10 μg of each purified recombinant protein was added to 2 × 105 fixed MS-74 VECs and allowed to bind for 1 h at RT.
For the auto-ligand assay, the experiment was performed using fixed trichomonads (Figure 5, part A2). In this case, 2 × 106 parasites were incubated with each clone identical to the ligand assay described with VECs. For trypsinized parasites (Figure 2, lane 2), 1 × 107 parasites ml-1 were first treated with trypsin (10 mg ml-1) for 15 min at 37°C before fixation. In addition, the auto-ligand assay was performed using lysate generated from 1 × 107 T016 parasites, as previously described . This detergent extract was incubated with 2 × 106 fixed T. vaginalis for 2 h at RT.
For competition studies with recombinant clones (Figure 6), fixed MS-74 VECs (2 × 105) and T. vaginalis (2 × 106) were incubated with 1 μg and 10 μg of recombinant AP65 clones c1 and c2 for 30 min at RT. Lysate from 1 × 107 T016 expressing the AP65-HA fusion protein was prepared as before , and protein concentration was determined with the BCA™ Protein Assay Kit (PIERCE). Then 10 μg of protein lysate were added to pretreated MS-74 VECs or T. vaginalis and incubated for an additional h at RT. After incubation with lysate or recombinant protein, cells were washed 3 times, and bound protein was solubilized by boiling for 3 min in 30 μl of 2× SDS electrophoresis sample buffer . Protein was separated on 8% or 10% acrylamide SDS-PAGE and transferred to nitrocellulose or PVDF membranes (BioRad Laboratories) for immunoblotting. Membranes were probed with mAb to AP65  and polyclonal anti-AP65 serum IgG antibodies, IgG mAb to Penta-His (Qiagen, Inc.), and IgG mAb to HA (Sigma-Aldrich). After o/n incubation with primary antibodies, the blots were extensively washed and incubated for 1 to 2 h in either secondary alkaline phosphatase- (AP) or horseradish peroxidase-conjugated goat anti-mouse IgG (Sigma-Aldrich) or AP-conjugated goat anti-rabbit IgG (BioRad Laboratories). Both primary and secondary antibodies were used at 1:1000 dilutions.
Reproducibility of experiments
Unless otherwise stated in the text, all experiments were performed numerous times and no less than on three different occasions. Statistical analysis using the t-test was performed as needed, and as indicated with asterisks, the p values were less than 0.05. Error bars represent standard deviations.
adhesin protein of molecular weight 65-kDa
fusion protein of AP65 and hemagglutinin (HA), HEPES, N-2-hydroxyethyl piperazine-N' -2-ethanesulfonic acid
sodium dodecylsulfate polyacrylamide gel electrophoresis
Trichomonas vaginalis VEC, vaginal epithelial cell
This work was supported by Public Health Service grant AI43940 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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