IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

Boston University Medical Campus, Boston MA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The trichomonad parasite Trichomonas 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, were shown to induce apoptosis in human vaginal epithelial cells (HVEC), as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis was correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. The secreted cysteine protease (CP) fraction from T. vaginalis that was shown to induce apoptosis in HVEC was analyzed by SDS-PAGE and mass spectrometry. SDS-PAGE analysis of the CP fraction revealed triplet bands around 30 kDa, and matrix-assisted laser desorption/ionization time-of-flight MS indicated two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs resulted 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. These 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 and prevention of AIDS. The results have been published in Infect. Immun. (72, 4151-8, 2004 ) and J. Biol. Chem. (280, 23853-60, 2005). A related manuscripts was published in Infection and Immunity and another has been submitted for publication in Glycoconjugate Journal.