and Low-Virulence Phenotypes - American Chemical Society

Cysteine Peptidase Expression in Trichomonas vaginalis Isolates Displaying High- and Low-Virulence Phenotypes Jose Batista De Jesus,*,†,‡ Patrıćia Cuervo,§ Constança Britto,‡ Leonardo Sabo ´ ia-Vahia,‡,| ⊥,# † Fernando Costa e Silva-Filho, Andre Borges-Veloso, De´bora Barreiros Petro ´ polis,⊥ § | Elisa Cupolillo, and Gilberto Barbosa Domont Departamento de Cieˆncias Naturais, Universidade Federal de Saõ Joaõ del Rei, Saõ Joaõ del Rei, MG, Brazil, Laborato´rio de Biologia Molecular e Doenças Endeˆmicas, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil, Laborato´rio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz, FIOCRUZ, Rio de Janeiro, RJ, Brazil, Universidade Federal do Rio de Janeiro, Instituto de Quı´mica, Departamento de Bioquı´mica, Laborato´rio de Quı´mica de Proteıńas/Rede Proteoˆmica do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, Laborato´rio de Bioengenharia de Sistemas, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brazil, and Programa Institucional de Bioengenharia, Departamento de Engenharia de Biosistemas, Universidade Federal de São João del Rei, São João del Rei, MG, Brazil Received October 26, 2008

In the present study, we identified and characterized the cysteine peptidase (CP) profiles of Trichomonas vaginalis isolates exhibiting high- and low-virulence phenotypes using a combination of two-dimensional SDS-PAGE (2DE), tandem mass spectrometry (MS/MS), and data mining. Seven of the eight CPs identified belong to Clan CA, family C1, cathepsin L-like CP, and one belongs to Clan CD, family C13, asparaginyl endopeptidase-like CP. Quantitative and qualitative differences in CP expression were detected between the isolates. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of differentially expressed CPs showed identity or high homology to previously described CP cDNA clones CP1, CP3, CP4, and to a secreted CP fraction of 30 kDa involved in apoptosis of vaginal epithelial cells. One- and two-dimensional-substrate gel analyses revealed the differential CP profiles between the isolates, indicating that the combination of zymography with 2DE and MS/MS might be a powerful experimental approach to map and identify active peptidases in T. vaginalis. Toxicity exerted upon HeLa cells by high- and low-virulence isolates was 98.3% and 31%, respectively. Pretreatment of parasites with specific Clan CA papain-like CP inhibitor L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido(4guanidino)butane (E-64) drastically reduced the cytotoxic effect to 21.7% and 0.8%, respectively, suggesting that T. vaginalis papain-like CPs are the main factors involved in the cellular damage. Keywords: Trichomonas vaginalis • parasitic protozoan • cysteine peptidase • metallopeptidase • twodimensional electrophoresis • mass spectrometry • parasite cytotoxicity

Introduction The protozoan Trichomonas vaginalis is a cosmopolitan pathogen commonly found inhabiting the human urogenital tract which causes trichomoniasis, a sexually transmitted disease (STD) annually affecting near 180 million people throughout the world.1 Although infection with T. vaginalis may cause urethritis and prostatitis in men, it is more usual for male * To whom correspondence should be addressed. Dr. Jose B. De Jesus. Departamento de Cieˆncias Naturais, Universidade Federal de Saõ Joaõ del Rei, Saõ Joaõ del Rei, MG, Brazil. Phone: +55-32-3379-2483. E-mail: [email protected]. † Universidade Federal de Saõ Joaõ del Rei. ‡ Laborato´rio de Biologia Molecular e Doenças Endeˆmicas, Instituto Oswaldo Cruz. § Laborato´rio de Pesquisa em Leishmaniose, Instituto Oswaldo Cruz. | Universidade Federal do Rio de Janeiro, Instituto de Quı´mica. ⊥ Laborato´rio de Bioengenharia de Sistemas, Instituto de Biofı´sica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro. # Programa Institucional de Bioengenharia, Departamento de Engenharia de Biosistemas, Universidade Federal de São João del Rei. 10.1021/pr8009066 CCC: $40.75

 2009 American Chemical Society

patients to display no clinical symptoms.2 In contrast, clinical features of trichomoniasis in female may include dyspareunia, dysuria, pruritus, leucorrhea, and purulent vaginal discharge.3 Severe complications associated with T. vaginalis infection include premature delivery, low birth weight, atypical pelvic inflammatory disease, infertility, predisposition to developing invasive cervical cancer, and increased susceptibility to HIV infection.4-7 Although the mechanisms underlying T. vaginalis pathogenesis are not clearly understood, certain processes associated with virulence phenotypes, such as expression of specific genes encoding functional proteins involved in colonization of epithelial urogenital tract and expression of molecules related to immune evasion, cytoadhesion, and cytotoxicity, have been reported.8-11 Cysteine peptidases (CP) are the major proteolytic enzymes expressed by T. vaginalis.12,13 Such enzymes are detected in association with the cell surface and as secretion products of the parasite14-18 and seem to exert central roles in pathogenicJournal of Proteome Research 2009, 8, 1555–1564 1555 Published on Web 02/02/2009

research articles

De Jesus et al.

Figure 1. Magnified regions of the gels (A, B) showing soluble CPs identified in FF28JT-Rio and FMV-1 isolates. Proteins were separated over pH range 4-7 and 12.5% SDS-polyacrylamide gel (30% acrylamide, 0.8% bis-acrylamide). Protein spots were visualized by colloidal Coomassie Brilliant Blue G-250. Proteins differentially expressed are numbered, and details of their identification are showed in Table 1. The degree of differential expression is shown in the histogram, below the corresponding gel region, presented as a grouped bar chart with error bars. Each bar represents intensity averages ( SD of spots from 4 independent gel images from three different cellular suspensions.

ity as well as in critical biological processes of this protozoan. A 65 kDa CP (CP65) located at the cell surface19 and a secreted 39 kDa CP (CP39)20 have been described as participating in the cytotoxicity of T. vaginalis to HeLa cells. In addition, a cell surface 30 kDa CP and a secreted CP of 62 kDa are involved in the ability of the parasites to cytoadhere.21,22 A secreted 30 kDa fraction has been also associated with apoptosis of human vaginal epithelial cells.23 In a previous study, we identified several peptidases in the 2DE map of T. vaginalis;24 subsequently our group reported that T. vaginalis isolates exhibiting high- and low-virulence phenotypes differentially expressed several proteins and enzymes, including CPs.25 In the present work, we perform a further comparative analysis of peptidase expression between these parasite isolates using two-dimensional electrophoresis (2DE), mass spectrometry, 2DE-substrate gel, and data mining. This multimethodological approach allowed us to identify the active “cysteine peptidase fingerprint” expressed by each T. vaginalis isolate. Analysis of amino acid sequences of CPs differentially expressed by the isolates showed identity or high homology with the previously reported cDNA clones CP1, CP3, and CP4,16 as well as with the 30 kDa secreted fraction associated with apoptosis.23 The contribution of CPs to the toxicity exerted by each T. vaginalis isolate against HeLa cells was also demonstrated. 1556

Journal of Proteome Research • Vol. 8, No. 3, 2009

Experimental Procedures Chemicals. All reagents were purchased from Sigma (St. Louis, MO) and Merck (Saõ Paulo, SP, Brazil). MilliQ water (Millipore Corp., Bedford, MA) was used for all solutions. Microorganisms. T. vaginalis microorganisms of high (FMV1)26 and low (FF28JT-Rio) cytotoxicity27 were axenically cultivated for 24 h in TYM medium, pH 6.2, supplemented with 10% bovine serum (heat inactivated at 56 °C for 50 min).28,29 Parasites which reached the exponential phase of growth were collected by centrifugation at 1400g for 5 min at 4 °C, and washed twice with PBS (0.01 M phosphate-buffered 0.145 M NaCl) pH 7.2. Protein Solubilization and Quantification. Parasites (109 cells) collected as described above were lysed by 10 cycles of freezing and thawing in 1 mL of hypotonic PBS (13.6 mM NaCl, 0.27 mM KCl, 0.4 mM Na2HPO4, 0.15 mM KH2PO4) containing a cocktail of peptidase inhibitors (Peptidase Arrest Geno Technology, Inc.).30,31 The lysate was centrifuged at 14 000g for 15 min at 4 °C to remove insoluble material, and the proteins in the resulting supernatant were precipitated with 10% (v/v) TCA and washed with cold acetone. Finally, the pellet was resuspended in IEF buffer (9 M urea, 4% CHAPS, 40 mM dithiothreitol (DTT) and 2% Pharmalyte 3-10). Protein concentration was determined using the 2D

XP_001303267

XP_001316414

XP_001314419

XP_001326005

XP_001316414

AAV98582

XP_001327438

XP_001314419

5.95 4.85

23352

4.43

20609 43516.6

6.17

5.76

23065

33622

7.51

5.08

20502 35657.3

7.47

4.73

20579 33746.2

6.17

5.29

21278 33622

7.47

5.02

23152 33776.2

6.65

5.93

23118 34385.4

7.51

theor. pI/exp. pI

35657.3

theor. MW/exp. MW

8/4

9/2

10/5

10/3

8/3

7/3

3/2

12/3

matching pep./ pep. identified by MS/MS

264

119

-83 -71 -60 -37

166

340

120

-59 -48 -14 -8 -9 -17 -3 -3 -2 13 -21

-47 -41 -45 IILMC*YDDIVNDAENPFK DTWAVIFC*GSRc

-44

b

264

91

282

138

117

133

94

228

ion score

+

+

-

ND

-

-

expression in FF28JT-RIO strain

ND

-

+

+

+

+

expression in FMV-1 strain

In bold: peptides sequenced manually and blast

284

164

-50 -48 -62

-15

174

-53

-30

protein score

error ( ppm

SLDHLNVYPGR

NC*FFPITAC*YAGSVAK

VTGYVNVVEGDEKDLATK

GLWMLETDYPYTAR GLWM*LETDYPYTAR SYVRPTTTQNEDELK SC*SSTFLDHAVGLVGYGTENK VTGYVNVVEGDEK

FMTEADYPYTAR FM*TEADYPYTAR VDYWIVR

VTGYVNVVEGDEKDLATK FM*TEADYPYTAQDGSC*K NSWGTAWGEK

FMTEADYPYTAR FM*TEADYPYTAR VTGYVNVVEGDEK

NSWGTTWGEK

VAEPTVTGYITVTEGDEKDLMNK

SYVRPTTTQNEDELK SCSSTFLDHAVGLVGYGTENK VAEPTVTGYITVTEGDEK

VDYWIVR

pep. sequenceb

a C* ) cysteine modified by carbamidomethyl; M* ) Methionine modified by oxidation. (-) Decreased expression; (+) increased expression; ND, not detected. searched in T. vaginalis peptides database. Amino acid sequence manually deduced is underlined. c Fragment.

31

37

119

118

29

28

Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis] Clan CD, family C13, asparaginyl endopeptidase-like cysteine peptidase [Trichomonas vaginalis]

23

26

protein name

Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis] Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis] cathepsin L-like cysteine proteinase precursor [Trichomonas vaginalis] Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis] Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis] Clan CA, family C1, cathepsin L-like cysteine peptidase [Trichomonas vaginalis]

code

NCBI accesion no.

Table 1. Cysteine Peptidases Identified in T. vaginalis Isolates Displaying High- and Low-Virulence Phenotypesa

Cysteine Peptidase Expression in T. vaginalis Isolates

research articles

Journal of Proteome Research • Vol. 8, No. 3, 2009 1557

research articles

De Jesus et al.

Figure 2. Alignments of identified CPs using Clustal W (1.83) (http://www.ebi.ac.uk). Asterisks (*) designate identical residues between the sequences. Active site residues are in bold and peptide sequenced by MS/MS are underlined. The gray block denotes amino acid change. (A) Alignment of sequence XP_001314419 (protein spots 23 and 119) with CP3; (B) alignment of sequences AAV98582 and XP_001326005 (corresponding to spots 28 and 118, respectively) with CP4; (C) alignment of sequence XP_001316414 (corresponding to spots 29 and 37) with CP4; (D) alignment of sequence XP_001327438 (corresponding to spot 26) with CP1. Sequences of CP1, CP3, and CP4 were obtained from NCBInr database (accession numbers CAA54435, CAA54437.1, and CAA54438.1, respectively.16 1558



research articles

Figure 3. Manual sequencing of the tandem mass spectra (MS/MS) corresponding to single charged peptides (A) NSWGTTWGEK (m/z 1165.47) and (B) NSWGTAWGEK (m/z 1135.53) from tryptic digests of spots 28 and 118.

Quant Kit (GE Healthcare), and the extracts were stored as single-use samples of 500 µg (equivalent to 2 × 108 parasites, approximately) at -80 °C. 2DE. Electrophoresis was performed as previously described.31 Briefly, 500 µg of protein was diluted in 350 µL of rehydration solution (9 M urea, 2% CHAPS, 40 mM DTT, 0.5% IPG buffer pH 4-7, trace bromophenol blue) and applied to

the IPG-strip (18 cm, pH 4-7 linear; Amersham Biosciences) by in-gel rehydration. All isoelectric focusing procedures were carried out in an IPGphor system (GE Healthcare) as described previously.31 After reduction and alkylation of the proteins, the IPG strips were directly transferred to the second-dimension gel. Proteins were then separated on 12.5% SDS-PAGE gels (30% Acrylamide, 0.8% bis-acrylamide) using an Ettan Daltsix large Journal of Proteome Research • Vol. 8, No. 3, 2009 1559

research articles

Figure 4. One-dimensional-substrate gel electrophoresis showing the profiles of active CPs detected in whole extracts of FF28JTRio and FMV-1 parasites (A), and the effect of inhibitor E-64 (10 µM) on the cysteine peptidase activities (B).

vertical system (GE Healthcare) and standard Tris/glycine/SDS buffer until the tracking dye left the gel. Protein Visualization and Image Analysis. Gels were stained with colloidal Coomassie Brilliant Blue G-250,32 and imaged using an ImageScanner II densitometer (GE Healthcare). Image analyses were performed using the ImageMaster 2D Platinum software (GE Healthcare). Three independent parasite suspensions per sample were compared, and at least 4 representative gels for each sample were analyzed. To determine the experimental pI and Mr coordinates for each single protein spot, 2DE gels were calibrated using a select set of reliable identification landmarks distributed throughout the entire gel. Protein Digestion, Peptide Extraction, and Mass Spectrometric Analysis. Protein spots were manually excised and treated for digestion as previously described.30,31 Mass spectra were acquired using a 4700 Proteomics Analyzer mass spectrometer (MALDI-TOF/TOF) operating in delayed reflector mode with an accelerating voltage of 20 kV. MS/MS analysis was performed by precursor ion fragmentation of the five more intense peptides in a collision-induced dissociation (CID) cell using N2 at a pressure of 2.8 × 10-6 Torr. The mass spectrometer was calibrated using the Sequazyme Standard kit (Applied Biosystem). Database Search. A combined search against the National Center for Biotechnology nonredundant (NCBInr, Version 050623, 2 564 994 sequences) database using the MS and MS/ MS data was performed using the GPS Explorer Protein Analysis Software (Applied Biosystems) and Mascot database search engine. Mascot search parameters were tryptic peptides with 1 missed cleavage allowed; no taxonomic restrictions; fixed modifications: carbamidomethylation of Cys residues; variable modifications, Met-oxidation, N-terminal acetylation; and mass accuracy within 100 ppm in MS and 0.2 Da for MS/MS data. A global MASCOT score greater than 50 was considered significant (p < 0.05). In addition, a hit was positive when a combination of the number of matching peptides (>5) in MS mode and peptides identified by MS/MS (>2) was found. 1560


De Jesus et al. Finally, proteins automatically identified were confirmed by manual sequencing of at least one matching peptide. 1DE and 2DE Zymographic Analysis of FMV-1 and FF28JT-Rio Isolates. For 1DE zymographic analysis, 107 microorganisms/mL from both highly virulent FMV-1 and low virulent FF28JT-Rio isolates were disrupted in a lysis buffer containing 10% glycerol, 0.6% Triton X-100, 100 mM Tris-HCl, pH 6.8, and 150 mM NaCl. Thirty micrograms (per slot) of the resulting extracts was fractionated on SDS-PAGE (10%) and copolymerized with 0.1% porcine gelatin. After electrophoresis, the gels were washed twice for 30 min at 4 °C in 0.1 M sodium acetate buffer, pH 5.5, containing 2.5% Triton X-100. Peptidase activity was detected by incubating the gels in reaction buffer containing 0.1 M sodium acetate, 1 mM DTT, pH 5.5, at 37 °C for 2 h. Bands of gelatin degradation were visualized by staining the gels with 0.2% (w/v) Coomassie blue R-250, 50% (v/v) trichloroacetic acid, and then destaining with 10% (v/v) acetic acid. For inhibition assays, 30 µg of protein was first incubated with 10 µM of specific Clan CA papain-like CP inhibitor E-64 for 30 min. For 2DE zymographic analysis, 107 parasites were directly lysed in IEF buffer containing 2 U DNase and 2 U RNase (Promega), 30 µg of protein was applied to the IPG-strip (7 cm, pH 4-7; GE Healthcare) by in-gel rehydration, and samples were then resolved as described above. Effect of CP Inhibitor on Toxicity of T. vaginalis Isolates to HeLa Cells. HeLa cells (ATCC CCL-2.1 HeLa 229) were cultured in 24-well plates in RPMI medium supplemented with 10% bovine serum and maintained at 37 °C in an atmosphere of 5% CO2, until confluence. Before interaction with HeLa cell monolayers, parasites were preincubated for 30 min in serumfree RPMI medium with or without 100 µM E-64. Interaction of both nontreated and E-64-treated parasites (5 × 106 parasites/ well) with HeLa cell monolayers was conducted in serum-free RPMI medium for 3 h at 37 °C and 5% CO2. The interaction of HeLa cells with E-64-treated parasites was allowed to proceed in the presence of the same inhibitor concentration (100 µM), whereas for the interaction with nontreated parasites, the inhibitor was omitted of the interaction medium. As a control, HeLa cell monolayers, without parasites, were cultured in the presence or absence of E-64 for 3 h at 37 °C and 5% CO2. HeLa cell monolayers which did or did not interact with T. vaginalis were fixed in 2.5% glutaraldehyde, washed in PBS, and stained with 0.13% crystal violet solubilized in 0.1 M borate buffer, pH 8.7, as previously described.26 The resulting products were washed with PBS, and the incorporated stain was recovered by incubating the cells in 0.1 N HCl (30 min at 37 °C). The amount of crystal violet associated with the HeLa cells was then spectrophotometrically measured at 660 nm. Cytotoxicity was expressed as the percentage of recovered stain from HeLa cells that were or were not allowed to interact with T. vaginalis. Mean values were derived from five independent experiments carried out in triplicate. Viability of parasites and HeLa cells was not affected by the treatment with inhibitor.

Results and Discussion 2DE Mapping of Peptidases. Eight protein spots corresponding to CPs were identified in 2DE gels of T. vaginalis isolates (Figure 1, Table 1). Seven protein spots belong to Clan CA (papain-like), family C1, cathepsin L-like CPs, and one belongs to Clan CD (legumain-like), family C13, asparaginyl endopeptidase-like CP. Quantitative differences in the expression of Clan CA CPs (protein spots 23, 26, 29, 118) were detected in high- and low-virulence T. vaginalis isolates (Figure 1, Table


research articles

Figure 5. Two-dimensional-substrate gel electrophoresis showing the profiles of active CPs detected in whole extracts of FF28JT-Rio (A and B) and FMV-1 (C and D) parasites. Assays were performed in the absence (A and C) and presence (B and D) of CP inhibitor E-64 (10 µM).

1). Qualitative differences were also observed: one CP Clan CA (protein spot 119) was found exclusively expressed in lowvirulence FF28JT-Rio parasites, whereas one CP Clan CA (protein spot 28) was only detected in high-virulence FMV-1 parasites (Figure 1, Table 1). No differential expression could be detected between the T. vaginalis isolates regarding Clan CA protein spot 37 and Clan CD protein spot 31. To further investigate the protein spots differentially expressed by the T. vaginalis isolates, we performed a comparative analysis between the CP sequences we identified. BLAST analysis followed by CLUSTAL alignment of amino acid sequences of six protein spots corresponding to Clan CA CPs (spots 23, 26, 28, 29, 118 and 119) showed identity or high sequence homology with those previously reported for cDNA clones of CP1, CP3, CP4,16 as well as with a secreted CP fraction of 30 kDa, which may induce apoptosis in human vaginal epithelial cells23 (Figure 2). Through MS analysis of such a secreted fraction, Sommer et al.23 observed that this fraction was constituted by abundant signals of 23.6 and 23.8 kDa. In agreement, we previously reported that CP Clan CA spots are located at 22-25 kDa in 2D-maps,24,25 even though their theoretical masses are about 33-35 kDa (Table 1). Differences between experimental and predicted CP molecular masses

might be due to N-terminal processing of pro-enzymes implicated in CP activation.16,23 In addition, the differences observed between theoretical and experimental pI for all CPs (Table 1) are coincident with the removal of about 100 amino acids from the enzyme precursors. It has been reported that the occurrence of multiple forms of CP4 in T. vaginalis are due to the expression of multiple coding genes, whereas CP1 and CP3 are coded by single genes.16 We found that differentially expressed protein spots 23 and 119 (both corresponding to NCBInr entry XP_001314419) matched to CP3 (Figure 2A). Since (i) CP 119 was only detected in the low-virulence FF28JT-Rio isolate, and (ii) it includes the peptides VDYWIVR and SYVRPTTTQNEDELK, which identify CP3 of the apoptotic 30 kDa fraction described by Sommer and colleagues,23 it can be argued that CP 119 might play other relevant roles during the interaction between T. vaginalis and target cells. Still, such differential CP expression could also represent another example of the intriguing phenotypic diversity exhibited by T. vaginalis populations. In considering the differential expression of protein spots 28 and 118, which correspond to NCBInr entries AAV9858233 and XP_001326005,34 respectively, it was found that they present high homology to the CP4 sequence (Figure 2B). The FMV-1 isolate expresses Journal of Proteome Research • Vol. 8, No. 3, 2009 1561

research articles

De Jesus et al. Protein spot 26 (entry XP_001327438) corresponded to the CP1 sequence16 (Figure 2D).

Figure 6. Effect of E-64 on the cytotoxicity exerted by FF28JTRio and FMV-1 isolates on HeLa cell monolayers. (A) Monolayers were stained after parasite-host cell interaction. (B) Cytotoxicity was spectrophotometrically quantified by dye recovery at 660 nm. In the control assay, HeLa cell monolayers, without parasites, were cultured for 3 h in the presence or absence of E-64. The values represent the means ((SE) of five independent experiments performed in triplicate.

protein spots 28 and 118, whereas the FF28JT-Rio isolate only expresses the protein spot 118. The data shown in Figure 3B clearly show that protein sequences 28 and 118 differ only at one amino acid (T and A, respectively) located five residues after Asn-175, which is involved in the catalytic site of this CP. Manual sequencing of the MS/MS spectra corresponding to peptides NSWGTTWGEK and NSWGTAWGEK (m/z 1165.47 and m/z 1135.53, respectively) corroborated the existence of the amino acid difference between spots 28 and 118 (Figure 3). Accordingly, Sommer and colleagues23 reported the sequence of a prominent peptide (NSWGTTWGEK) identified in the 30 kDa secreted fraction of T. vaginalis which did not fully match the CP4 sequenced by Mallinson and colleagues16 due to one amino acid residue difference (NSWGTAWGEK). As pointed out by Sommer et al.,23 such an amino acid difference might be due to a sequencing error or the fact they had used a different T. vaginalis strain than that used by Mallinson et al.16 Since we detected the occurrence of both isoforms in the FMV-1 microorganisms, it is plausible to consider the latter hypothesis. T residue-containing CP4 isoforms might indeed be a virulence marker for T. vaginalis, which appears to be expressed only in isolates presenting high toxicity to vaginal epithelial cells. This hypothesis is strongly supported by the observation that CP4 containing the T residue was detected in fresh clinical highly virulent microorganisms,23 but not in long-term grown isolates. Protein spots 29 and 37 showed approximately 90% homology with CP4 (Figure 2C), and corresponded to a CP that was named CPT (NCBInr entry XP_001316414).23 CPT is part of the previously mentioned apoptotic 30 kDa secreted fraction. 1562


Proteolytic Profile of T. vaginalis Isolates as Revealed by Both 1D and 2D Substrate Gel Electrophoresis. We performed zymographic analysis using one- and two-dimensional substrategels to identify active peptidases in whole extracts of the two T. vaginalis isolates. A comparative 1D zymographic analysis of the protease activities found in the T. vaginalis extracts obtained from high- and low-virulence microorganisms resulted in the appearance of at least nine bands ranging from 18 to 98 kDa (Figure 4A). Remarkable differences in the migration pattern or band intensities of proteases were detected between the isolates. When whole extracts were analyzed by 2D gels, both qualitative and quantitative differences were observed between the isolates (Figure 5A,C). Proteolytic spots were detected between 20-98 kDa, and they displayed pI values between 4.2 and 6.5 (calculated according to a linear distribution of pH across the strip). Further, preincubation of the 1Dand 2D-substrate-gels with E-64 strongly inhibited the proteolytic activities observed in both isolates (Figures 4B and 5B,D). However, remaining enzymatic activities were observed both in 1D- and 2D-zymogram after inhibitor treatment. The lack of complete inhibition could be due to the following reasons: (i) E-64 specifically inhibits CPs from the papain-like family belonging to the Clan CA, whereas other CPs such as those of the Clan CD would not be affected; (ii) E-64 concentration used here (10 µM) is lower than others previously reported.21,35 Altogether, these data confirm previously published results on the marked occurrence of CPs in T. vaginalis.12,13,15 The detection of proteolytic spots between 20 and 30 kDa by 2Dzymography, and their inhibition by E-64, are in agreement with the 2DE and MS/MS analyses, and also confirm that the mapped CPs are active enzymes. In this work, use of 2Dzymography combined with 2DE and MS/MS showed to be a powerful experimental approach to both map and identify active peptidases, at least in T. vaginalis. Notably, 2D-zymography, in addition to allowing preliminary mapping of active forms of low-abundance CPs, which are not easily visualized in 2DE colloidal-stained gels, is also a valuable tool for the identification of novel active peptidase spots without using specific antibodies. In addition, copolymerization of polyacrylamide gels with fluorescent substrates might increase the sensitivity of the method allowing the detection of other peptidase classes.36,37 To our knowledge, the use of a combination of 2D-zymography and MS/MS analysis to investigate active peptidases is still restricted.36,37 The qualitative and quantitative differences observed in the zymographic pattern of CPs from T. vaginalis are in agreement with previously reported data for fresh and long-term isolates.15,38-40 Finally, the apparent contrast between the number of CP genes coded by T. vaginalis34 and the number of CPs observed in 2Dzymographies could mean that only a limited number of active enzymes are expressed in vitro. Nevertheless, the possibility that other CPs could be detected under different experimental conditions cannot be excluded. Cytotoxicity Assays. To examine the degree to which the toxicity exerted by T. vaginalis parasites on cultured cells could be related to the observed CP activities, each T. vaginalis isolate was or was not preincubated for 30 min with the specific CP inhibitor E-64 (100 µM), and then added to confluent HeLa cell monolayers. FMV-1 isolate exhibited higher toxicity to HeLa cells than FF28JT-Rio isolate (Figure 6A). Nevertheless, when

research articles

Cysteine Peptidase Expression in T. vaginalis Isolates parasites were pretreated with E-64 and allowed to interact with HeLa cells in the presence of this inhibitor, their original cytotoxicities were strongly diminished (Figure 6A). As shown in the control condition, any potential cell disruptions were observed when E-64 was added to HeLa cultures indicating that the cytolytic effect is only due to the cytotoxic activity of the parasites (Figure 6A). The spectrophotometric method used to quantify cytotoxicity showed that in the absence of E-64 98.3% (SE ( 1.5) and 31% (SE ( 2.5) of the HeLa cells monolayer was disrupted by FMV-1 and FF28JT-Rio isolates, respectively (Figure 6B). In contrast, cytotoxicity of FMV-1 and FF28JTRio isolates was drastically reduced to 21.7% (SE ( 2.6) and 0.8% (SD ( 0.1), respectively, in the presence of E-64 (Figure 6B). These results unequivocally showed that papain-like CPs of T. vaginalis are greatly involved in in vitro cytotoxicity. However, the remaining levels of cytotoxicity observed in the presence of E-64 indicate that factors other than the CPs could be involved to a lesser extent in cellular damage. Therefore, the in vitro cytotoxicity exerted by T. vaginalis is indeed a multifactorial phenomenon in which CPs play a relevant role.14,19-21,23,38,39,41-43 The quantitative differences observed in terms of the cytotoxicity exerted by each of the FMV-1 and FF28JT-Rio parasites might be attributed to the differential expression of specific CP isoforms between microorganisms of different isolates. This seems to be the case for the protein spot 28 (identified as CP4) which was only detected in the high cytotoxic FMV-1 microorganisms. Finally, regarding specificity of T. vaginalis toxicity, the cytopathic effect here observed on HeLa cells (an epithelial, nonvaginal human cellular linage) seems to be similar to that observed during the interaction of T. vaginalis with MDCK cells, which are epithelial (nonvaginal) canine cells.26 Such ability of T. vaginalis to disrupt different cell linage and tissues suggests that the mechanisms involved in these processes are neither cell type- nor species-specific.26,44-48

Conclusions The heterogeneity in peptidase expression that we observed reinforces the hypothesis that T. vaginalis species is constituted by phenotypically distinct subpopulations of parasites which would express, qualitatively and/or quantitatively, different proteins or enzymes involved in pathogenicity. Differences in the peptidase profile between low- and high-virulence isolates could also be associated, for instance, with regulation of the expression of a specific isoform whose activity would be required for T. vaginalis survival during host colonization. In fact, qualitative and quantitative differences in the CP patterns between fresh and long-term T. vaginalis isolates led some authors to suggest that (i) environmental conditions would modulate CP expression, and (ii) that a particular CP profile could be associated with cytoadherence or cytopathogenicity of the parasites.15,38,40,49 The genome sequencing project of T. vaginalis demonstrated that this parasite codes one of the most complex degradomes among protozoan parasites currently sequenced.34 The staggering number of protease coding genes undoubtedly suggests that such enzymes have an important role in the parasite’s biology. The use of approaches such as those developed in this study might be a shortcut in the identification of active T. vaginalis peptidases and provide new insights to understand the precise mechanism underlying the pathophysiology of trichomoniasis.

Abbreviations: E-64, L-3-carboxy-2,3-trans-epoxypropionylleucylamido(4-guanidino)butane; TYM, Trypticase Yeast extract Maltose.

Acknowledgment. We are grateful to Dr. Marcelo Alves Ferreira (DBBM-FIOCRUZ), Mr. Rodrigo Mexas, and Mr. Bruno Eschenazi (Laborato´rio de Produçaõ e Tratamento de Imagem-FIOCRUZ) for valuable technical advice. This work was supported by the following Brazilian agencies: FIOCRUZ/ CNPq-PAPES V (J.B.D.J.). References (1) World Health Organization. In Global Program on AIDS; World Health Organization: Geneva, 1995; pp 2-27. (2) Sen ˜ a, A. C.; Miller, W. C.; Hobbs, M. M.; Schwebke, J. R.; Leone, P. A.; Swygard, H.; Atashili, J.; Cohen, M. S. Trichomonas vaginalis infection in male sexual partners: implications for diagnosis, treatment, and prevention. Clin. Infect. Dis. 2007, 44, 13–22. (3) Petrin, D.; Delgaty, K.; Bhatt, R.; Garber, G. Clinical and microbiological aspects of Trichomonas vaginalis. Clin. Microbiol. Rev. 1998, 11, 300–317. (4) Cotch, M. F.; Pastorek, J. G., II.; Nugent, R. P.; Hillier, S. L.; Gibbs, R. S.; Martin, D. H.; Eschenbach, D. A.; Edelman, R.; Carey, J. C.; Regan, J. A.; Krohn, M. A.; Klebanoff, M. A.; Rao, A. V.; Rhoads, G. G. Trichomonas vaginalis associated with low birth weight and preterm delivery. The Vaginal Infections and Prematurity Study Group. Sex. Transm. Dis. 1997, 24, 353–360. (5) Moodley, P.; Wilkinson, D.; Connolly, C.; Moodley, J.; Sturm, A. W. Trichomonas vaginalis is associated with pelvic inflammatory disease in women infected with human immunodeficiency virus. Clin. Infect. Dis. 2002, 34, 519–522. (6) Zhang, Z. F.; Begg, C. B. Is Trichomonas vaginalis a cause of cervical neoplasia? Results from a combined analysis of 24 studies. Int. J. Epidemiol. 1994, 23, 682–690. (7) Johnston, V. J.; Mabey, D. C. Global epidemiology and control of Trichomonas vaginalis. Curr. Opin. Infect. Dis. 2008, 21, 56–64. (8) Alderete, J. F.; Garza, G. E. Identification and properties of Trichomonas vaginalis proteins involved in cytadherence. Infect. Immun. 1988, 56, 28–33. (9) Alderete, J. F.; Arroyo, R.; Dailey, D. C.; Engbring, J.; Khoshnan, M. A.; Lehker, M. W.; McKay, J. Molecular analysis of Trichomonas vaginalis surface protein repertoires. Mol. Cell. Biol. Hum. Dis. Ser. 1992, 1, 173–202. (10) Addis, M. F.; Rappelli, P.; Delogu, G.; Carta, F.; Cappuccinelli, P.; Fiori, P. L. Cloning and molecular characterization of a cDNA clone coding for Trichomonas vaginalis alpha-actinin and intracellular localization of the protein. Infect. Immun. 1998, 66, 4924–4931. (11) Kucknoor, A.; Mundodi, V.; Alderete, J. F. Trichomonas vaginalis adherence mediates differential gene expression in human vaginal epithelial cells. Cell. Microbiol. 2005, 7, 887–897. (12) Coombs, G. H.; North, M. J. An analysis of the proteinases of Trichomonas vaginalis by polyacrylamide gel electrophoresis. Parasitology 1983, 86, 1–6. (13) Lockwood, B. C.; North, M. J.; Scott, K. I.; Bremner, A. F.; Coombs, G. H. The use of a highly sensitive electrophoretic method to compare the proteinases of trichomonads. Mol. Biochem. Parasitol. 1987, 24, 89–95. (14) Arroyo, R.; Alderete, J. F. Trichomonas vaginalis surface proteinase activity is necessary for parasite adherence to epithelial cells. Infect. Immun. 1989, 57, 2991–2997. (15) Neale, K. A.; Alderete, J. F. Analysis of the proteinases of representative Trichomonas vaginalis isolates. Infect. Immun. 1990, 58, 157–62. (16) Mallinson, D. J.; Lockwood, B. C.; Coombs, G. H.; North, M. J. Identification and molecular cloning of four cysteine proteinase genes from the pathogenic protozoon Trichomonas vaginalis. Microbiology 1994, 140, 2725–2735. (17) Garber, G. E.; Lemchuk-Favel, L. T. Analysis of the extracellular proteases of Trichomonas vaginalis. Parasitol. Res. 1994, 80, 361– 365. (18) Scott, D. A.; North, M. J.; Coombs, G. H. The pathway of secretion of proteinases in Trichomonas vaginalis. Int. J. Parasitol. 1995, 25, 657–666. (19) Alvarez-Sańchez, M. E.; Avila-Gonza´lez, L.; Becerril-Garcıá, C.; Fattel-Facenda, L. V.; Ortega-Lo´pez, J.; Arroyo, R. A novel cysteine proteinase (CP65) of Trichomonas vaginalis involved in cytotoxicity. Microb. Pathog. 2000, 28, 193–202.

Journal of Proteome Research • Vol. 8, No. 3, 2009 1563

research articles (20) Hernańdez-Gutie´rrez, R.; Ortega-Lo´pez, J.; Arroyo, R. A 39-kDa cysteine proteinase CP39 from Trichomonas vaginalis, which is negatively affected by iron may be involved in trichomonal cytotoxicity. J. Eukaryotic Microbiol. 2003, 50, 696–698. (21) Mendoza-Lo´pez, M. R.; Becerril-Garcia, C.; Fattel-Facenda, L. V.; Avila-Gonzalez, L.; Ruı´z-Tachiquıń, M. E.; Ortega-Lopez, J.; Arroyo, R. CP30, a cysteine proteinase involved in Trichomonas vaginalis cytoadherence. Infect. Immun. 2000, 68, 4907–4912. (22) Hernańdez, H.; Sariego, I.; Garber, G.; Delgado, R.; Lo´pez, O.; Sarracent, J. Monoclonal antibodies against a 62 kDa proteinase of Trichomonas vaginalis decrease parasite cytoadherence to epithelial cells and confer protection in mice. Parasite Immunol. 2004, 26, 119–125. (23) Sommer, U.; Costello, C. E.; Hayes, G. R.; Beach, D. H.; Gilbert, R. O.; Lucas, J. J.; Singh, B. N. Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J. Biol. Chem. 2005, 280, 23853–23860. (24) De Jesus, J. B.; Cuervo, P.; Junqueira, M.; Britto, C.; Silva-Filho, F. C.; Saboía-Vahia, L.; Gonza´lez, L. J.; Barbosa Domont, G. Application of two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for proteomic analysis of the sexually transmitted parasite Trichomonas vaginalis. J. Mass Spectrom. 2007, 42, 1463–1473. (25) Cuervo, P.; Cupolillo, E.; Britto, C.; Gonza´lez, L. J.; E Silva-Filho, F. C.; Lopes, L. C.; Domont, G. B.; De Jesus, J. B. Differential soluble protein expression between Trichomonas vaginalis isolates exhibiting low and high virulence phenotypes. J. Proteomics 2008, 71, 109–122. (26) De Jesus, J. B.; Vannier-Santos, M. A.; Britto, C.; Godefroy, P.; SilvaFilho, F. C.; Pinheiro, A. A.; Rocha-Azevedo, B.; Lopes, A. H.; MeyerFernandes, J. R. Trichomonas vaginalis virulence against epithelial cells and morphological variability: the comparison between a well-established strain and a fresh isolate. Parasitol. Res. 2004, 93, 369–377. (27) Costa e Silva Filho, F.; Elias, C. A.; de Souza, W. Further studies on the surface charge of various strains of Trichomonas vaginalis and Tritrichomonas foetus. Cell. Biophys. 1986, 8, 161–176. (28) Diamond, L. S. The establishment of various trichomonads of animals and men in axenic culture. J. Parasitol. 1957, 43, 488– 490. (29) De Jesus, J. B.; Ferreira, M. A.; Cuervo, P.; Britto, C.; e Silva-Filho, F. C.; Meyer-Fernandes, J. R. Iron modulates ecto-phosphohydrolase activities in pathogenic trichomonads. Parasitol. Int. 2006, 55, 285–290. (30) Cuervo, P.; de Jesus, J. B.; Junqueira, M.; Mendonça-Lima, L.; Gonza´lez, L. J.; Betancourt, L.; Grimaldi, G., Jr.; Domont, G. B.; Fernandes, O.; Cupolillo, E. Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresis and mass spectrometry. Mol. Biochem. Parasitol. 2007, 154, 6–21. (31) De Jesus, J. B.; Cuervo, P.; Junqueira, M.; Britto, C.; Silva-Filho, F. C.; Soares, M. J.; Cupolillo, E.; Fernandes, O.; Domont, G. B. A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis. Proteomics 2007, 7, 1961–1972. (32) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255– 262. (33) Solano-Gonzalez, E.; Burrola-Barraza, E.; Leon-Sicairos, C.; AvilaGonzalez, L.; Gutierrez-Escolano, L.; Ortega-Lopez, J.; Arroyo, R.

1564


De Jesus et al.

(34)

(35) (36)

(37)

(38)

(39)

(40)

(41)

(42) (43) (44) (45)

(46) (47) (48) (49)

The trichomonad cysteine proteinase TVCP4 transcript contains an iron-responsive element. FEBS Lett. 2007, 581, 2919–2928. Carlton, J. M.; Hirt, R. P.; Silva, J. C.; Delcher, A. L.; Schatz, M.; Zhao, Q.; Wortman, J. R.; Bidwell, S. L.; Alsmark, U. C.; Besteiro, S.; et al. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 2007, 315, 207–212. Irvine, J. W.; North, M. J.; Coombs, G. H. Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis. FEMS Microbiol. Lett. 1997, 149, 45–50. Zhao, Z.; Russell, P. J. Trypsin activity assay in substrate-specific one- and two-dimensional gels: a powerful method to separate and characterize novel proteases in active form in biological samples. Electrophoresis 2003, 24, 3284–3288. Zhao, Z.; Raftery, M. J.; Niu, X. M.; Daja, M. M.; Russell, P. J. Application of in-gel protease assay in a biological sample: characterization and identification of urokinase-type plasminogen activator (uPA) in secreted proteins from a prostate cancer cell line PC-3. Electrophoresis 2004, 25, 1142–1148. Arroyo, R.; Alderete, J. F. Two Trichomonas vaginalis surface proteinases bind to host epithelial cells and are related to levels of cytoadherence and cytotoxicity. Arch. Med. Res. 1995, 26, 279– 285. Hernańdez-Gutie´rrez, R.; Avila-Gonza´lez, L.; Ortega-Lo´pez, J.; CruzTalonia, F.; Go´mez-Gutierrez, G.; Arroyo, R. Trichomonas vaginalis: characterization of a 39-kDa cysteine proteinase found in patient vaginal secretions. Exp. Parasitol. 2004, 107, 125–135. Yadav, M.; Dubey, M. L.; Gupta, I.; Bhatti, G.; Malla, N. Cysteine proteinase 30 in clinical isolates of T. vaginalis from symptomatic and asymptomatic infected women. Exp. Parasitol. 2007, 116, 399– 406. Fiori, P. L.; Rappelli, P.; Rocchigiani, A. M.; Cappuccinelli, P. Trichomonas vaginalis haemolysis: evidence of functional pores formation on red cell membranes. FEMS Microbiol. Lett. 1993, 109, 13–18. Pindak, F. F.; Mora de Pindak, M.; Gardner, J. Contact independent cytotoxicity of Trichomonas vaginalis. Genitourin. Med. 1993, 59, 35–40. Lehker, M. W.; Sweeney, D. Trichomonad invasion of the mucous layer requires adhesins, mucinases, and motility. Sex. Transm. Infect. 1999, 75, 231–238. Alderete, J. F.; Pearlman, E. Pathogenic Trichomonas vaginalis cytotoxicity to cell culture monolayers. Br. J. Vener. Dis. 1984, 60, 99–105. Escario, J. A.; Go´mez Barrio, A.; Martıńez Fernańdez, A. R. The relationship of experimental pathogenicity in vivo with in vitro cytoadherence and cytotocity of 6 different isolates of Trichomonas vaginalis. Int. J. Parasitol. 1995, 25, 999–1000. Heath, J. P. Behavior and pathogenicity of Trichomonas vaginalis in epithelial cell cultures. A study by light and scanning electron microscopy. Br. J. Vener. Dis. 1981, 57, 106–117. Pindak, F. F.; Gardner, W. A., Jr.; Mora de Pindak, M. Growth and cytopathogenicity of Trichomanas vaginalis in tissue cultures. J. Clin. Microbiol. 1986, 23, 672–678. Silva-Filho, F. C.; De Souza, W. The interaction of Trichomonas vaginalis and Tritrichomonas foetus with epithelial cells in vitro. Cell Struct. Funct. 1988, 13, 301–310. Provenzano, D.; Alderete, J. F. Analysis of human immunoglobulindegrading cysteine proteinases of Trichomonas vaginalis. Infect. Immun. 1995, 63, 3388–3395.

PR8009066

and Low-Virulence Phenotypes - American Chemical Society

Recommend Documents