TvMP50 is an Immunogenic Metalloproteinase during Male Trichomoniasis*

Trichomonas vaginalis, a human urogenital tract parasite, is capable of surviving in the male microenvironment, despite of the presence of Zn2+. Concentrations > 1.6 mm of Zn2+ have a trichomonacidal effect; however, in the presence of ≤1.6 mm Zn2+, several trichomonad proteins are up- or down-regulated. Herein, we analyzed the proteome of a T. vaginalis male isolate (HGMN01) grown in the presence of Zn2+ and found 32 protein spots that were immunorecognized by male trichomoniasis patient serum. Using mass spectrometry (MS), the proteins were identified and compared with 23 spots that were immunorecognized in the proteome of a female isolate using the same serum. Interestingly, we found a 50-kDa metallopeptidase (TvMP50). Unexpectedly, this proteinase was immunodetected by the serum of male trichomoniasis patients but not by the female patient serum or sera from healthy men and women. We analyzed the T. vaginalis genome and localized the mp50 gene in locus TVAG_403460. Using an RT-PCR assay, we amplified a 1320-bp mp50 mRNA transcript that was expressed in the presence of Zn2+ in the HGMN01 and CNCD147 T. vaginalis isolates. According to a Western blot assay, native TvMP50 was differentially expressed in the presence of Zn2+. The TvMP50 proteolytic activity increased in the presence of Zn2+ in both isolates and was inhibited by EDTA but not by ptosyl-L-lysine chloromethyl ketone (TLCK), E64, leupeptin, or phenylmethane sulfonyl fluoride. Furthermore, the recombinant TvMP50 had proteolytic activity that was inhibited by EDTA. These data suggested that TvMP50 is immunogenic during male trichomoniasis, and Zn2+ induces its expression.

Trichomoniasis is the most common nonviral sexually transmitted infection. The infection is caused by Trichomonas vagi-nalis with ϳ174 million new cases per year worldwide (1). This infection is implicated as a cofactor in the transmission of the human immunodeficiency virus (2). Infected women develop vaginitis, cervicitis, urethritis, and malodourous seropurulent vaginal discharge with serious health consequences such as infertility (3)(4), preterm delivery, low-birth weight infants (5), and a predisposition to cervical neoplasia (6). In men, the infection is mainly asymptomatic (7) and self-limiting, although there are complications such as urethritis, prostatitis, and an association with prostate cancer and infertility (8 -11).
The primary antimicrobial defenses of the male genitourinary tract are prostatic and seminal fluids with high levels of Zn 2ϩ (4.5 to 7 mM) (12). In particular, Zn 2ϩ is the major prostate defense due to its ability to prevent pathogens from establishing in the reproductive male tract (12). Indeed, it has an antimicrobial spectrum that includes bacteria, viruses, chlamydiae and fungi (13). T. vaginalis is sensitive to relatively low concentrations of Zn 2ϩ and has a minimum trichomonacidal concentration of 1.6 mM (12). However, the Zn 2ϩ concentration (ϳ0.8 mM) found in patients with chronic bacterial prostatitis is not trichomonacidal (14) and T. vaginalis may persist for longer periods of time in the male genitourinary tract. Parasites may possibly progress to the prostate, where they have been observed to infect the prostatic epithelium causing chronic prostatitis and elicit an inflammatory immune response (15). Indeed, T. vaginalis has been observed in prostate tissue near inflammation areas and epithelial hyperplasia, suggesting that this parasite might be involved in prostate carcinogenesis (9).
Recently, we reported that the variation in trichomonal pathogenesis between the sexes might imply different hostparasite molecular interactions and changes in the parasite proteome (16). At least five trichomonad proteinases (CP70, TvCP65, CP39, CP25, and CP20) are involved in the interaction between parasites and prostatic DU-145 cells. Interestingly, the trichomonal cytotoxicity toward DU-145 cells is 80% lower than it is in HeLa cells. Furthermore, parasites display an oval form and pseudopods forming upon contact with DU-145 cells, but amoeboid trophozoites were not observed (16). Interestingly, proteolytic activity and the amount of TvCP65, a cysteine proteinase involved in trichomonal cytotoxicity toward cervical cells (17)(18) and its transcript are diminished by 80% in the presence of Zn 2ϩ (16). In a previous study, we identified a 50-kDa metalloproteinase that was highly expressed in the presence of Zn 2ϩ in the CNCD147 female isolate (16).
This work examines the immunoproteome of the T. vaginalis HGMN01 male isolate compared with the CNCD147 female isolate. This work also reports on the characterization of a metalloproteinase that is differentially immunodetected by male trichomoniasis serum in the presence of Zn 2ϩ . This difference could be a starting point for the diagnosis of this infection.
RNA Extraction and cDNA Synthesis-For RNA extraction, we used 2 ϫ 10 7 parasites from both T. vaginalis isolates grown with or without 1.6 mM of Zn 2ϩ and treated as previously described (19). Briefly, 1 g of total RNA was reverse-transcribed using the Superscript Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA) and an oligo-dT (dT18) (10 pmol/l) primer. The cDNA obtained was stored at Ϫ20°C until use.
Analysis of mp50 mRNA Expression by Semiquantitative RT-PCR-PCR was performed in 50 l reactions containing 50 ng of cDNA, as previously reported (16). We used the following primer pairs to amplify the 1320-bp mp50 gene: forward, 5Ј-CCGGATCCATGTCAGGTGAC-GAATTC-3Ј (BamHI recognition site is underlined) and reverse, 5Ј-CCAAGCTTTCAAAGTACTCTTGAAGC-3Ј (HindIII recognition site underlined). In addition, a 112-bp fragment of the ␤-tubulin gene was used as a loading control (20). The amplified products were analyzed on 1% agarose gels and visualized by ethidium bromide staining. Gene expression densitometry analyses were performed with Quantity One Software (Bio-Rad, Hercules, CA). Data from the densitometry quantification of the housekeeping gene (␤-tubulin) were used to normalize the results.
Two-dimensional Electrophoresis-For proteomic maps, we used a previously reported protocol (16) with modifications (for details see the supplemental experimental procedures in the Supplemental Data file). Briefly, 70 g of protein was applied to an IPG strip (17 cm, pH 4 -7 linear; Bio-Rad) for passive rehydration for 12 h. Then, the equilibrated IPG strips were separated in 12% SDS-PAGE gels (20 cm ϫ 20 cm ϫ 1.0 mm) and stained with SYPRO® Ruby Protein Gel Stain (Invitrogen), following procedures described by the manufacturer. Finally, the gels were documented using Gel Doc EQ (Bio-Rad) system. Image analysis was performed using PDQuest software (Bio-Rad). Three independent protein preparations, each obtained from an independent parasite culture, were performed for comparisons of the two-dimensional electrophoresis (2DE) 1 maps.
Human Biological Samples-The serum samples from trichomoniasis patients were donated by Rossana Arroyo PhD, obtained as previously reported (21). The serum samples were obtained from trichomoniasis patients attending the "Centro Nacional de Clinicas de Displasias (CNCD)" and "Laboratorio Central" from the "Hospital General de Mexico" (HGM) at Mexico City. All patients agreed to participate in this study via written informed consent and the study was approved by the Bioethics Standard Committee (21). In this discovery stage study, either male (HGMN01) or female (HGM124) sera were used (22). Healthy sera from males and females were used as negative controls (for details see the supplemental experimental procedures in the Supplemental Data file).
Immunoblot Analysis of Two-dimensional Electrophoresis-The 2DE-PAGE separated proteins were electroblotted onto nitrocellulose membranes (GE Healthcare, USA) as previously reported (23). After being blocked, the membranes were incubated with a 1:500 dilution of HGMN01 or HGM124 serum from trichomoniasis patients at 4°C overnight. Next, the membranes were further incubated with a 1:3000 dilution of peroxidase-labeled rabbit anti-human IgG (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at room temperature and developed using an enhanced chemiluminescence ECL Plus Western blotting Detection System (GE Healthcare), according to the manufacturer's instructions (for details see the supplemental experimental procedures in the Supplemental Data file).
Liquid Chromatography Electrospray Ionization Quadrupole Time of Flight Mass Spectrometry-The MS analysis of each fraction obtained from the offline separation steps was conducted according to a previously reported method (16) (for details see the supplemental experimental procedures in the Supplemental Data file).
Data Processing and Search Parameters-Peaklists were generated using Mascot Distiller v2.1. For MS/MS data analysis, the MASCOT server (Matrix Science, London, UK, available at http://www. matrixscience.com, version 2.2; Matrix Science, London, UK) was used to search against the NCBI database nr 2011.07.09 (14652852 sequences, 5012444178 residues). The default search parameters were such that a Mascot threshold score of 5% indicated that the protein identification was likely incorrect. All samples were searched with the taxonomy filter "Other Eukaryota" (379307 sequences).The database search parameters were set as follows: enzyme (trypsin with a maximum of one missed cleavages); fixed modifications (carbamidomethyl (ϩ57.021 Da at cysteine residue)); variable modifications (deamidated (ϩ1 Da at cysteine residue)) and methionine oxidation (ϩ15.995 Da at methionine residue); peptide mass tolerance (Ϯ 1.2 Da); fragment mass tolerance (Ϯ 0.6 Da); mass tolerance for precursor ions (100 ppm); and mass tolerance for fragment ions (0.6 Da). The protein identification reporting criteria included at least two MS/MS spectrum matched at the 95% level of confidence (Mowse score ϭ 25) and the presence of a consecutive ␥ and/or ␤ ion series of three or more amino acids.
The functional classification of the T. vaginalis immunoproteome was performed according to the Gene Ontology index (http://www. geneontology.org/).
Proteolytic Activity of Native TvMP50 -A proteinase extract was obtained as previously reported (17). The supernatant was loaded into Bio-gel P6 Micro Bio-Spin chromatography columns (Bio-Rad) and eluted in 10 mM Tris-HCl, pH 7.5, according to the manufacturer's instructions (for details see the supplemental experimental procedures in the Supplemental Data file). Metallopeptidase activity was analyzed by one-or two-dimensional gelatin zymography under nonreducing conditions, using 10% SDS-PAGE copolymerized with 0.2% gelatin as previously reported (24). The cysteine peptidase activity was also tested under reducing conditions as previously described (17). The gels were stained with 0.25% Coomassie Brilliant Blue with clear bands against a black background corresponding to zones of digestion.
Samples containing 20 g of proteinase extract were incubated at 4°C for 20 min in the presence or absence of several proteinase inhibitors 0.25 mM E64, 1.0 mM ptosyl-L-lysine chloromethyl ketone (TLCK) and 1.0 mM leupeptin for cysteine proteinases, 1.0 mM phenylmethane sulfonyl fluoride (PMSF) for serine proteinases and 0.2 mM EDTA for metalloproteinase (all purchased from Sigma Chemical Co) and analyzed as described above.
Expression, Purification, and Proteolytic Activity of Recombinant TvMP50 -The 1320-bp mp50 gene was digested using BamHI and HindIII (both from New England BioLabs, Ipswitch, MA) and ligated with T4 DNA ligase into the pQE80L expression vector (Qiagen, Valencia, CA) according to the manufacturerЈs instructions. Positive recombinant clones were identified by sequencing (3130 Genetic Analyzer, Applied Biosystems, Foster City, CA). The recombinant TvMP50 (rTvMP50) was expressed using the M15 E. coli strain by adding 1 mM IPTG (isopropyl-1-thio-␤-D-galactopyranoside). Purification of 6xHis-tagged rTvMP50 was performed by nickel affinity chromatography according to the manufacturer's instructions using a 1-ml HisTRap HP precast column (GE Healthcare) under native conditions. The identity of rTvMP50 was corroborated by Western blot analysis using an anti-His-Tag antibody as previously described (25). Anti-rTvMP50 polyclonal antibody was obtained using a standard protocol (for details see the supplemental experimental procedures in the Supplemental Data file). Anti-rTvMP50 was used to perform Western blot analysis with the T. vaginalis total protein extract from HGMN01 and CNCD147 isolates grown in the presence or absence of Zn 2ϩ obtained by TCA precipitation as previously reported (17) (for details see the supplemental experimental procedures in the Supplemental Data file). The proteolytic activity of rTvMP50 was evaluated as described above.

Immunoproteome of T. vaginalis Isolates HGMN01 and
CNCD147-The proteomic analyses were performed using two T. vaginalis isolates, CNCD147 and HGMN01 from an infected woman and man, respectively.
For the comparative proteomic analysis, images of three representative 2-DE gels obtained from three independent experiments using parasites grown in the presence and absence of 1.6 mM Zn 2ϩ from the different isolates were analyzed with PDQuest software (data not shown). The proteomic profiles from either the T. vaginalis HGMN01 or CNCD147 isolates were highly reproducible in terms of the total number of protein spots and their relative positions and intensities. SYPRO® Ruby-stained gels from the T. vaginalis HGMN01 isolate displayed ϳ609 spots in the control (absence of Zn 2ϩ ) (Fig. 1A), whereas 540 spots were obtained in the presence of Zn 2ϩ (Fig. 1C). In contrast, the CNCD147 isolate displayed 447 ( Fig. 2A) spots in the control, whereas 361 spots were observed in the presence of Zn 2ϩ (Fig. 2C). Proteins detected in pI range of 4 -7 had molecular masses between 10 and 250 kDa. The gels displayed remarkable changes in protein profiles when parasites were grown in the presence of Zn 2ϩ . In addition, duplicate of these gels were transferred onto nitrocellulose membranes for Western blot analyses using serum from a male trichomoniasis patient (Figs. 1B and 1D and Figs. 2B and 2D).
Interestingly, 52 spots were immunodetected in the HGMN01 isolate using the male patient serum and the control parasites ( Fig. 1B), whereas 32 spots were detected in the total protein extract from parasites grown in the presence of Zn 2ϩ (Fig. 1D). Moreover, this serum immunodetected 29 spots from the control parasites of the CNCD147 isolate ( Fig.  2B) and 23 spots from parasites grown in the presence of Zn 2ϩ (Fig. 2D). These spots were identified by LC-ESI-QUAD-TOF. The accession numbers, protein scores, theoretical and experimental pI and molecular weights and sequence coverage of all identified proteins in the T. vaginalis male immunoproteome are shown in supplemental Tables S1 to S3. (For specific details of Peptide Mass Fingerprint (PMF) see Supplemental Tables I to VI).
The proteins identified in our immunoproteome discovery study are mostly hypothetical, cytoskeleton proteins and proteins involved in glycolysis and carbohydrate metabolism (supplemental Fig. S1 in Supplemental Data file). Interestingly, proteins involved in redox homeostasis, fermentative metabolism, adhesion, protein synthesis, and amino acid catabolism were immunogenic. Proteins from secretion pathways, phagocytosis, ATP synthesis, chaperons, and heat shock proteins were less abundant but also immunogenic. Furthermore, putative cysteine and metalloproteinases were identified as immunogenic molecules.
Surprisingly, protein spots 6 and 66, which corresponded to the same aminopeptidase P-like metallopeptidase from Clan MG and family M24 (supplemental Table S1), were overexpressed in the presence of Zn 2ϩ in the two isolates according to 3D view analysis using the Melanie software (Figs. 1E and 2E, panels b and h). Interestingly, in the presence of Zn 2ϩ , only the spot corresponding to TvMP50 was found in both isolates (supplemental Fig. S2 in the Supplemental Data file). In contrast, in the absence of this cation, we found no overlapping proteins (Supplemental Fig. S2 in Supplemental Data file).
Interestingly, in a comparative analysis of the close-ups of these spots, immunodetection by the male trichomoniasis patient serum (Figs. 1E, panels c, i and 2E, panel i) but not by the female patient serum (Figs. 1E and 2E, panels d and j) or by the healthy male or female people serum (Figs. 1E and 2E, panels e, f, k and l) was observed. Unexpectedly, this metallopeptidase was undetectable in the CNCD147 isolate in the control trichomonads. This observation correlated with the fact that this molecule was not detected by the female patient serum.
T. vaginalis Contains a 50-kDa Metallopeptidase Gene Differentially Expressed in the Presence of Zn 2ϩ -We found that the immunogenic aminopeptidase P-like metalloproteinase is encoded by a 1320-bp ORF termed mp50 (GenBank accession number JF263458), located in the locus TVAG_403460 (supplemental Fig. S3 in Supplemental Data file), which contains another two ORFs on the opposite strand that encode a calcium-dependent lipid-binding protein and a conserved hypothetical protein (supplemental Fig. S3 in the Supplemental Data file). The mp50 gene encodes a 439-amino acid protein with a predicted molecular weight of 50,416 Da.
The deduced amino acid sequence of TvMP50 was aligned with metallopeptidases representative of subfamilies A (data not shown) and B (supplemental Fig. S3 in Supplemental Data file) from family M24 of clan MG. The primary structure of TvMP50 is highly conserved at the amino acid residues that interact with the metal ion and the residues from the catalytic site when compared with the subfamily B peptidases. In this clan, three histidine residues form the catalytic triad. Interestingly, TvMP50 contains these catalytic residues located in similar positions. Both the amino acid residues that bind to the metal ion in this clan (D, D, H, E) and also the amino acids surrounding the metal-binding residues are conserved in TvMP50, suggesting that this molecule might be a member of this clan.
According to RT-PCR semiquantitative analyses, mp50 mRNA displayed a differential expression level in the presence of Zn 2ϩ in both the CNCD147 and HGMN01 T. vaginalis isolates (Fig. 3A). In the CNCD147 isolate, the mp50 FIG. 1. Zn 2؉ proteomic map of T. vaginalis isolate HGMN01. The total proteins of T. vaginalis grown in the absence (A and B) or the presence (C and D) of 1.6 mM Zn 2ϩ were separated in the first dimension by isoelectric focusing over a pH range from 4.0 to 7.0 followed by 12% SDS-polyacrylamide gel electrophoresis (A and C) or transferred to a nitrocellulose membrane for Western blot analysis using serum from a trichomoniasis male patient (B and D). The spots immunodetected by the serum from a male trichomoniasis patient are indicated with squares. E, HGMN01 isolate cultivated in the absence (control) (panels a-f) or presence of Zn ϩ2 (panels g-l). Close-up of the 50-kDa metalloproteinase region of the 2 DE gels (panels a and g), 3D view of spots in panel a or panel g (panels b and h, respectively) and 2DE-WB (panels c-f and i-l) using male (panels c and i) or female (panels d and j) trichomoniasis patients sera and male (panels e and k) or female (panels f and l) healthy people sera. The white squares show spot 6, which corresponds with the Zn 2ϩ -differential expressed TvMP50 in the HGMN01 isolate (panel g and h) and the TvMP50 immunorecognition by male sera with trichomoniasis (panels c and i). mRNA was almost undetectable (Fig. 3A, mp50, lane 1) in the absence of Zn 2ϩ . However, in the presence of Zn 2ϩ , mp50 mRNA expression was detected (Fig. 3A, mp50, lane  2). Moreover, in the HGMN01 isolate, the mp50 transcript was detected even in the absence of Zn 2ϩ (Fig. 3A, mp50,  lane 3); however, its expression increased in the presence of this cation (Fig. 3A, mp50, lane 4). In contrast, no differences were found in the expression of the ␤ tubulin tran-script used as a loading control (Fig. 3A, ␤ tub). The normalized intensity of the bands used for densitometric analyses indicated that in the presence of Zn 2ϩ and a 100% mp50 mRNA level was found in the female isolate (CNDC147) (supplemental Fig. S4 in the Supplemental Data file), whereas a 50% mp50 mRNA level was found in the male isolate (HGMN01), confirming the differential expression of the mp50 transcript.  A and B) or the presence (C and D) of 1.6 mM Zn 2ϩ were separated in the first dimension by isoelectric focusing over a pH range from 4.0 to 7.0 followed by 12% SDS-polyacrylamide gel electrophoresis (A and C) or transferred to a nitrocellulose membrane for Western blot analysis using serum from a trichomoniasis male patient (B and D). The spots immunodetected by the serum from a male trichomoniasis patient are indicated with squares. E, CNCD147 isolate cultivated in the absence (control) (panels a-f) or presence of Zn ϩ2 (panels g-l). Close-up of the 50-kDa metalloproteinase region of the 2DE gels (panels a and g), 3D view of spots in panel a or panel g (panels b and h, respectively) and 2DE-WB (panels c-f and i-l) using male (panels c and i) or female (panels d and j) trichomoniasis patient sera and male (panels e and k) or female (panels f and l) healthy people sera. The white squares show spot 66, which corresponds with Zn 2ϩ -differential expressed TvMP50 in the CNCD147 isolate (panel g and h) and the TvMP50 immunorecognition by male trichomoniasis patient sera with (panel i). Furthermore, the native TvMP50 was Zn 2ϩ differentially expressed in both isolates (Fig. 3B). The expression of TvMP50 in the CNCD147 isolate was not observed in the absence of Zn 2ϩ (Fig. 3B, TvMP50, lane 1), whereas in the presence of Zn 2ϩ the band corresponding to native TvMP50 was observed (Fig. 3B, TvMP50, lane 2). On the other hand, in the HGMN01 isolate, the native TvMP50 was observed even in the absence of Zn 2ϩ (Fig. 3B, TvMP50, lane 3) and its expression was increased in the presence of this cation (Fig.  3B, TvMP50, lane 4). The expression of ␣-TUB was not changed by the presence or absence of Zn 2ϩ (Fig. 3B, ␣-TUB,  lanes 1 to 4).
TvMP50 is a 50-kDa Metalloproteinase-To determine if TvMP50 has proteolytic activity, we generated zymograms using total protein extract obtained from parasites of CNCD147 and HGMN01 isolates grown in the presence or absence of 1.6 mM Zn 2ϩ (supplemental Fig. S5 in the Supplemental Data file). As a control, we searched for cysteine proteinase activity and found that the proteolytic activity profiles in reducing conditions in the presence or absence of Zn 2ϩ (supplemental Fig. S5 in Supplemental Data file) are in congruence with previous reports (16 -17). The proteolytic activity profile under nonreducing conditions was performed at several pH activation values, although the highest proteolytic activity was observed at pH 7.5. The proteolytic activity of proteinases from male and female T. vaginalis isolates in nonreducing conditions has not yet been reported. In the CNCD147 isolate, three proteolytic activity bands were observed in the absence (Fig. 3C lane 1) or presence of Zn 2ϩ (Fig. 3C, lane 3). The same proteolytic activity bands were observed in the HGMN01 isolate in the absence (Fig. 3C lane 5) or presence of Zn 2ϩ (Fig. 3C, lane 7). Interestingly, the intensity of the ϳ50 kDa proteolytic activity band that might correspond to the TvMP50 activity increased in the presence of Zn 2ϩ in both isolates.
The inhibition profile of the proteinase extract was tested with zymograms (Fig. 3C). Interestingly, the three proteolytic activity bands described above were observed even in the presence of specific cysteine and serine proteinase inhibitors (TLCK, E64, leupeptin, and PMSF) (Fig. 3C, lanes 1, 3, 5, and  7), but in the presence of EDTA, a metalloproteinase inhibitor, the activity bands were no longer observed (Fig. 3C, lanes 2,  4, 6, and 8), suggesting that the three proteolytic bands, including the 50-kDa band, correspond to the metallopeptidase activity.
In addition, the metallopeptidase activity profile was analyzed with 2DE zymograms. Fig. 3D shows a representative zymogram of the metallopeptidase activity profile from the T. vaginalis HGMN01. Seven activity spots were detected, three of the spots with a molecular weight (MW) Ͼ150 kDa, one of the spots with a MW Ͼ50 kDa, two of the spots with a MW of ϳ50 kDa, and one spot with a MW of ϳ37 kDa (Fig. 3D). Interestingly one of the two spots with a MW of ϳ50 kDa had a pI of ϳ5.3, which corresponds to the predicted pI of TvMP50 (5.34).
Furthermore, IPTG-induced E. coli harboring the construct pQE80L-mp50 displayed a band of ϳ51 KDa (Fig. 3E, lane 2), corresponding to rTvMP50, which was not detected in the noninduced control culture (Fig. 3E, lane 1). The rTvMP50 was expressed in both the soluble (Fig. 3E, lane 3) and insoluble form (Fig. 3E, lane 4) and it was purified from the soluble fraction (SF) by nickel affinity chromatography (Fig. 3E, lane  5). The protein's identity was confirmed by Western blot analysis using an anti-His Tag antibody (supplemental Fig. S6 in the Supplemental Data file). According to the zymograms, the purified rTvMP50 has proteolytic activity (Fig. 3E, lane 6), but it was inhibited in the presence of EDTA (Fig. 3E, lane 7). DISCUSSION The molecular events during male trichomoniasis remain an area of great interest but are poorly understood. Because iron plays a critical role in host-parasite relationships and in the general physiology of the parasite (26) in the female microenvironment, modulating several pathogenic properties, such as adhesin synthesis (27), protease expression (28), and the expression of crucial metabolic enzymes (26), it seems plausible to infer that in the male microenvironment, Zn 2ϩ might has a crucial role as well. Because men are the reservoir of trichomonads, there is an urgent need to identify novel molecular targets for the diagnosis of trichomoniasis in men.
We previously reported that Zn 2ϩ has a negative effect on trichomonal cytotoxicity, most likely because of the downregulation at the protein and transcriptional levels of TvCP65 (16). We also reported at least 27 differentially expressed proteins in the presence of Zn 2ϩ in the CNCD147 isolate proteome using 7-cm IPG strips (16). Among these proteins, we found an aminopeptidase P-like metallopeptidase from Clan MG that was up-regulated in the presence of Zn 2ϩ (16).
Here, we present a comparative proteomic approach to identify the immunogenic molecules that might be candidates for the development of a male trichomoniasis diagnostic method. To study the influence of the source of the isolate, we used a male (HGMN01) and female (CNCD147) T. vaginalis isolate for proteomic analysis. A total of 52 and 29 immunogenic proteins were observed in the HGMN01 and CNCD147 isolates, respectively. Gene Ontology analyses revealed that these immunogenic proteins were involved in diverse biological processes, particularly cytoskeleton organization (20%), glycolysis (7.5%), and carbohydrate metabolism (7.5%).
A proteome reference map of T. vaginalis of ϳ500 protein spots was previously generated with silver-stained 2-DE gels. Although 353 protein spots were excised for MS identification and successfully identified, these spots only represented 64 unique proteins (29). In this study, we excised 52 protein spots that represented 40 unique proteins. The most abundant proteins found in our study were cytoskeleton proteins and glycolytic proteins. These findings are consistent with the previously mentioned proteome study (29).
The protein profile reported in our study is similar to the protein profile of T. vaginalis grown in iron-rich and irondepleted medium (26). The enzymes involved in energetic metabolism, proteolysis and hydrogenosomal iron-sulfur (Fe-S) proteins were down-regulated or even suppressed in iron-depleted parasites. In contrast, six isoforms of actin, phosphoenolpyruvate carboxykinase, putative lactate dehydrogenase, and putative adenosine triphosphatase were upregulated (26).
Among the immunogenic proteins, we identified a 50-kDa metallopeptidase of clan MG (TvMP50), which presented an expression level increase of 0.5-fold in the HGMN01 isolate in the presence of Zn 2ϩ whereas it was 1.0-fold in the CNCD147 isolate. These data are in agreement with a previous reports (16). Unexpectedly, this metallopeptidase was immunorecognized by the male trichomoniasis patient serum but not by the female patient serum. Although there are many commercial kits for the diagnosis of trichomoniasis, none of them include a specific molecule up-regulated in the male microenvironment. Thus, we propose that TvMP50 might be a promising candidate for male trichomoniasis diagnosis.
The protein and transcript levels of TvMP50 were up-regulated by the presence of Zn 2ϩ . This is not the first protease discovered that is up-regulated by the presence of certain cations from the host. For example, Fe 2ϩ and polyamines, present in the female microenvironment, down-regulate the protein expression levels and the transcript of TvCP65 (41). In contrast, the amount of mRNA and protein of TvMP50 were up-regulated in the presence of Zn 2ϩ .
According to the T. vaginalis genome, this parasite contains 13 families of metallopeptidases (30). However, until now, only four metalloproteinases from T. vaginalis had been reported (16,42). Two of them (142 kDa and Ͼ220 kDa) were reported by Bozner and Demes, and the activity of these metallopeptidases is inhibited in the presence of 2 mM EDTA (42). Another reported metalloproteinase is a 47-kDa peptidase from the trichomonad hydrogenosome, the hydrogenosomal processing peptidase (HPP). HPP contains an active site motif (HXXEHX 76 E) characteristic of the ␤ subunit of the mitochondrial processing peptidase. Interestingly, HPP forms a homodimer (100 kDa) in vitro and in vivo (43). The fourth metallopeptidase reported is a 50-kDa proteinase that was up-regulated in the presence of Zn 2ϩ in a female T. vaginalis isolate (16). However, in the present study, we demonstrate the presence of this metallopeptidase in a male T. vaginalis isolate. Interestingly, we found a 50-kDa-proteolytic activity band in the HGMN01 and CNCD147 T. vaginalis isolates that might correspond to the previously reported metallopeptidase (16). According to 2DE zymograms, two activity spots of ϳ50 kDa were observed and the pI of one of the spots corresponded to the predicted pI of TvMP50, suggesting that the proteolytic activity band observed in the one-dimension zymograms corresponds at least to TvMP50. TvMP50 was inhibited by EDTA but not by cysteine or serine proteinase inhibitors such as TLCK, E-64, leupeptin, or PMSF. This proteolytic activity was not detected by Bozner and Demes, possibly because of pH activation. Bozner and Demes activated the proteinases at pH 8.2 (42), whereas we activated them at pH 7.5 because at the basic pH of 8.2, the activity of TvMP50 was not observed. A pH of 7.5 has previously been reported for the activation of metallopeptidases (24). In this study, we performed proteinase activation at several pH values, although the highest proteolytic activity was observed at pH 7.5.
Peptidases with at least two metal ions in the active site are a structurally heterogeneous group. Based on their folds, their active site architectures and the identity of the metal ions, they have been divided into clans MF, MG, and MH in the MEROPS database. Clan MG metallopeptidases, such as the methionine aminopeptidases, have the pita-bread fold and contain two cobalt or two manganese ions in their active centers (44).To identify the residues involved in catalysis, the TvMP50 sequence was aligned with other members of the peptidase clan MG, including aminopeptidase P (Ec_ MER001244) and Xaa-Pro dipeptidase (Ec_MER001250) from Escherichia coli. The sequence analysis revealed that His215, His324, and His335 are the catalytic residues, whereas Asp232, Asp243, His328, Glu364, and Glu407 are predicted to serve as metal ion ligands in the active site. These results suggest that TvMP50 has an active-site configuration similar to other members of the peptidase clan MG. According to the alignment analyses, TvMP50 might be classified as an aminopeptidase of the clan MG. However, its proteolytic activity and structural characterization remain unknown. A study is currently in progress to elucidate these features.
According to the crystal structures of E. coli methionyl aminopeptidase and aminopeptidase P (APP) (45)(46), TvMP50 might have diverse metal requirements. The cytosolic and membrane-bound APPs maximum catalytic activities depends on two manganese (Mn 2ϩ ) or Zn 2ϩ ions per subunit, respectively (47). Because TvMP50 was found to be regulated in the presence of Zn 2ϩ , we suggest that this molecule might require this cation for its catalytic activity. Currently, we are determining the metal requirements of TvMP50.
The Role of Some Immunogenic Proteins-To successfully establish infection, pathogenic organisms depend on an arsenal of virulence factors that facilitate host colonization, including adhesins, which are involved in cell binding but also participate in host cell signaling and cellular invasion (48). Five adhesins (AP120, AP65, AP51, AP33, and AP23) were previously reported in T. vaginalis (27, 49 -53); however, we observed that AP51 was recognized by male trichomoniasis patient serum. Our findings are in agreement with a previous report that demonstrated another molecule involved in adhesion (AP65) was an antigenic protein in T. vaginalis (54). Interestingly, AP65 has been used for the diagnosis of trichomoniasis (55).
In general, the expression of surface adhesins and adhesion are iron-dependent phenomena in the female microenvironment (52)(53). Despite AP51 being down-regulated by Zn 2ϩ , it still unknown how Zn 2ϩ directly affects parasite adhesion. A study is in progress to determine the responsible mechanism.
Enolase, a key glycolytic enzyme, is a multifunctional protein that has the ability to serve as a plasminogen receptor on the surface of a variety of hematopoietic, epithelial and endothelial cells. Moreover, ␣-enolase functions as a heat shock protein (HSP) and binds cytoskeletal and chromatin structures, suggesting a crucial role in transcription and pathophysiological processes (56). When T. vaginalis is in contact with vaginal epithelial cells (VECs), the parasite releases ␣-enolase (tvENO1) but at the same time, it is present on the parasite surface, most likely as an adhesion molecule, suggesting its role as a virulence factor (57). Previously, it had been reported as a T. vaginalis enolase family protein (including putative enolase 3 and enolase 4) (58); however, enolase 2 was not included. Although enolase 2 is down-regulated by Zn 2ϩ , this molecule was immunorecognized by the male trichomoniasis patient serum in the present study. Furthermore, Whiting and colleagues reported that 15 of 22 patients with proven Streptococcus pneumoniae infection have antibodies to ␣-enolase, suggesting its important role in natural immunity to S. pneumonia (59). Elucidating the relationship between antibodies and enolase 2 in male trichomoniasis infection will require further study.
HSPs participate in cellular protection, limiting protein aggregation and facilitating protein folding (60). Moreover, the immunization of BALB/c mice with ⌬HSP70-II null mutant promastigotes leads to an effective immune response able to protect these mice against infection with Leishmania major (61). Because the ⌬hsp70-II line could be useful as a platform for introduction of immunoprotective antigens relevant to leishmaniasis (61) or even to other diseases, we propose that the immunogenicity of T. vaginalis HSP70 might be used in a trichomoniasis diagnostic method. Surprisingly, several actin molecules were immunorecognized by the sera of trichomoniasis patients. Identification of a structural protein, such as actin, as an immunogenic molecule has important implications because it is present in all isolates. Although Western blotting analysis revealed that 94% of the IgG-positive sera reacted against T. vaginalis ␣-actinin, also involved in the structure organization (62), the analyzed samples corresponded to infected women. In contrast, our evidence indicated that actin was immunogenic in male trichomoniasis patients. Interestingly, in another study, Histomonas meleagridis ␣-actinin strongly contributed to immune-reaction with hosts, namely turkeys and chickens, and these molecules displayed amino acid identities between 54% and 82.5% (63). The actin genes present in T. vaginalis would provide the parasite with a mechanism that allows it to rapidly change its morphology when exposed to different stimuli such as Fe 2ϩ or Zn 2ϩ . Work is in progress to determine the influence of Zn 2ϩ on the morphological appearance of T. vaginalis.
Interestingly, we found a C2 domain containing protein as an immunogenic molecule. Moreover, no significant changes in the amount of this protein were found. The C2 domain is a Ca 2ϩ -dependent membrane-targeting module found in many cellular proteins involved in signal transduction or membrane trafficking (64).
The energy metabolism of parasites may include the activity of alcohol dehydrogenase, such as Entamoeba histolytica in which the pyruvate, the end-product of carbohydrate catabolism by glycolysis, is oxidatively decarboxylated by pyruvate: ferredoxin oxidoreductase (PFOR) (65), transferring the electrons produced during pyruvate oxidation to ferredoxin, whereas acetyl-CoA is consecutively reduced to acetaldehyde and ethanol (under microaerophilic conditions), mainly via the activity of a bifunctional NADH-dependent aldehydealcohol dehydrogenase (EhADH2) or to ethanol and acetate (under aerobic conditions) via the latter and acetyl-CoA synthetase (ADP-forming) (65)(66)(67)(68). Interestingly, the expression levels of alcohol dehydrogenase 2 (EhADH2) and alcohol dehydrogenase 3 (EhADH3) are higher in a virulent versus nonvirulent amoebae (69). Although EhADH3 is present on the plasma membrane surface of E. histolytica, the role of EhADH3 in the virulence of amoeba is still unknown (69). On the other hand, EhADH2 is a major adhesion factor only in pathogenic strains and is involved in the internalization of human transfer by E. histolytica (70). In T. vaginalis, the alcohol dehydrogenase activity is related to metronidazole-resistance. In this case, the trichomonads compensate for the hydrogenosomal deficiency by an increased rate of glycolysis and by changes in their cytosolic pathways (71). T. vaginalis enhances lactate fermentation, whereas T. foetus activates pyruvate conversion to ethanol. Drug-resistant T. foetus also increases activity of the cytosolic NADP-dependent malic enzyme to enhance the pyruvate producing bypass and provide the NADPH required by alcohol dehydrogenase (71). Interestingly, two alcohol dehydrogenases were immunorec-ognized by the male trichomoniasis patient serum in the present study, but their expression levels did not change in the presence of Zn 2ϩ . Because the alcohol acetaldehyde dehydrogenase from Listeria monocytogenes (LAP) is a pathogenic factor, promoting bacterial adhesion to intestinal epithelial cells by interacting with mammalian receptor Hsp60 (72)(73)(74)(75), it could be possible that this type of enzymes might induce a host immune response.
Currently, the male trichomoniasis diagnosis methods such as APTIMA require a male urethral swab sample (76), which is a painful procedure for the patients. Therefore, the availability of several immunogenic molecules, as shown herein, might facilitate the male immunodiagnosis of T. vaginalis. CONCLUSION The Zn 2ϩ present in the male microenvironment might regulate the expression of several proteins and several of them are immunogenic. The T. vaginalis proteome is influenced by the isolate source but also by the presence of Zn 2ϩ . The proteome of T. vaginalis HGMN01 isolated from a male host consisted of 609 spots, whereas the CNCD147 isolated from a female host consisted of 447. In the presence of Zn 2ϩ , 540 spots were found in the HGMN01 isolate and 361 spots were found in the CNCD17 isolate. The male trichomoniasis patient serum recognized 52 and 32 proteins in the HGMN01 or CNCD147 isolate, respectively. These immunogenic proteins are involved in diverse biological processes, particularly cytoskeleton organization, glycolysis, and carbohydrate metabolism.
The 50-kDa metalloproteinase from T. vaginalis was differentially expressed in the presence of Zn 2ϩ independent of the source of the trichomonads isolate. This molecule was only immunorecognized by the male trichomoniasis patient serum but not by female patient serum, suggesting this molecule as a possible target for the diagnosis of male trichomoniasis.