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J. Biol. Chem., Vol. 280, Issue 25, 23853-23860, June 24, 2005

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Identification of Trichomonas vaginalis Cysteine Proteases That Induce Apoptosis in Human Vaginal Epithelial Cells^*

Ulf Sommer, Catherine E. Costello, Gary R. Hayes, David H. Beach¶, Robert O. Gilbert||, John J. Lucas, and Bibhuti N. Singh**

From the Mass Spectrometry Resource, Boston University School of Medicine, Boston, Massachusetts 02118, the Departments of Biochemistry and Molecular Biology and ^¶Microbiology and Immunology, State University of New York (SUNY) Upstate Medical University, Syracuse, New York 13210, and the ^||Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853

A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA^PLUS assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.

Received for publication, February 15, 2005 , and in revised form, April 14, 2005.

^* This work was supported by National Institutes of Health NIAID Grant AI47334, National Research Initiative of the USDA-Cooperative State Research Education and Extension Service Grants 97-2615 and 2003-321517, and grants from the SUNY Upstate Medical University Intramural Research and Women's Health Fund, SUNY Upstate Medical Foundation (to B. N. S.). The Boston University School of Medicine Mass Spectrometry Resource is supported by National Institutes of Health National Center of Research Resources Grants P41-RR10888 and S10-RR015942 (to C. E. C.). Sequencing of Trichomonas vaginalis was accomplished with support from the National Institutes of Health NIAID. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY 13210. Tel.: 315-464-5398; Fax: 315-464-8750; E-mail: singhb{at}upstate.edu.

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