RESEARCH ARTICLE Immunoproteomics of the active degradome to identify biomarkers for Trichomonas vaginalis Lucero A. Ramo ´n-Luing 1 , Francisco J. Rendo ´n-Gandarilla 1 , Rosa E. Ca ´rdenas-Guerra 2 , Norma A. Rodrı´guez-Cabrera 2 , Jaime Ortega-Lo ´pez 2 , Leticia Avila-Gonza ´lez 1 , Claudia Angel-Ortiz 1 , Carmen N. Herrera-Sa ´nchez 3 , Manuela Mendoza-Garcı´a 3 and Rossana Arroyo 1 1 Departamento de Infecto ´ mica y Patoge ´ nesis Molecular, Centro de Investigacio ´ n y de Estudios Avanzados del IPN (CINVESTAV-IPN), Mexico City, Mexico 2 Departamento de Biotecnologı´a y Bioingenierı´a, CINVESTAV-IPN, Mexico City, Mexico 3 Laboratorio de Bacteriologı´a en el Laboratorio Central del Hospital General de Me ´ xico, Mexico City, Mexico Received: July 6, 2009 Revised: October 21, 2009 Accepted: November 9, 2009 Trichomonas vaginalis, a sexually transmitted parasite, has many cysteine proteinases (CPs); some are involved in trichomonal pathogenesis, express during infection, and antibodies against CPs have been detected in patient sera. The goal of this study was to identify the antigenic proteinases of T. vaginalis as potential biomarkers for trichomonosis. The proteases detected when T. vaginalis protein extracts are incubated without protease inhibitors, the trichomonad-active degradome, and the immunoproteome were obtained by using 2-DE, 2-D- zymograms, 2-D-Western blot (WB) assays with trichomonosis patient sera, and MS analysis. Forty-nine silver-stained spots were detected in the region of 200–21 kDa of parasite protease- resistant extracts. A similar proteolytic pattern was observed in the 2-D zymograms. Nine CPs were identified in the 30 kDa region (TvCP1, TvCP2, TvCP3, TvCP4, TvCP4-like, TvCP12, TvCPT, TvLEGU-1, and another legumain-like CP). The major reactive spots to T. vaginalis- positive patient sera by 2-D-WB corresponded to four papain-like (TvCP2, TvCP4, TvCP4-like, TvCPT), and one legumain-like (TvLEGU-1) CPs. The genes of TvCP4, TvCPT, and TvLEGU- 1 were cloned, sequenced, and expressed in Escherichia coli. Purified recombinant CPs were recognized by culture-positive patient sera in 1-D-WB assays. These data show that some CPs could be potential biomarkers for serodiagnosis of trichomonosis. Keywords: Cysteine proteinases / Degradome / Immunoproteomics / Microbiology / Trichomonas vaginalis / Trichomonosis biomarkers (serodiagnostic antigens) 1 Introduction Trichomonas vaginalis is a protozoan parasite responsible of trichomonosis, one of the most common sexually trans- mitted infections, affecting over 200 million people world- wide every year [1, 2]. This infection is associated with serious health complications for women, including atypical pelvic inflammatory disease, increased risk of human immunodeficiency virus 1 infection [3] and transmission [4, 5], infertility [6], low birth weight infants and preterm delivery [7], and predisposition to cervical neoplasia [8]. Trichomonosis can cause urethritis, prostatitis [9], and predisposition to prostate cancer in men [10]. T. vaginalis possesses multiple proteinases mainly of the cysteine type (CPs). Some of them are virulence factors involved in cytoadherence [11–14], hemolysis [15], comple- ment resistance [16], cytotoxicity [11, 12, 17–19], induction of apoptosis [20], nutrient acquisition [15, 21], and immune evasion [16, 22]. In vivo, some CPs are found in vaginal secretions of patients with trichomonosis and are Abbreviations: aa, amino acids; AE, asparaginyl endopeptidase; CNCD, ‘‘Centro Nacional de Clı´nicas de Displasias’’; CPs, cysteine proteinases; HGM, ‘‘Hospital General de Me ´ xico’’; HS, human sera; MW, molecular weight; PBS-T, PBS-Tween 20; TvCPs, T. vaginalis cysteine proteinases; TvLEGU, T. vaginalis legumain-like cysteine proteinases; WB, Western blot Correspondence: Dr. Rossana Arroyo, Departamento de Infec- to ´ mica y Patoge ´ nesis Molecular, Centro de Investigacio ´n y de Estudios Avanzados del IPN (CINVESTAV-IPN), Av. IPN ] 2508, Col. San Pedro Zacatenco, CP 07360, Mexico City, Mexico E-mail: rarroyo@cinvestav.mx Fax: 15255-5747-3377 & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com Proteomics 2010, 10, 435–444 435 DOI 10.1002/pmic.200900479