IDENTIFICATION & CHARACTERIZATION OF TRICHOMONAD CYSTEINE PROTEASES

这个子项目是许多利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 滴虫寄生虫阴道毛滴虫引起人类最常见的非病毒性传播感染之一，滴虫病。寄生虫，以及分泌的半胱氨酸蛋白酶（CP）部分分离的亲和层析，然后由生物凝胶P-60柱层析，显示诱导人阴道上皮细胞（HVEC）的细胞凋亡，细胞死亡检测ELISA（PLUS）测定和膜联蛋白V-异硫氰酸荧光素流式细胞术分析证明。细胞凋亡的启动与蛋白酶活性相关，因为特异性CP抑制剂E-64抑制这两种活性。从T.通过SDS-PAGE和质谱分析显示诱导HVEC凋亡的迷走神经肽。CP组分的SDS-PAGE分析显示约30 kDa的三重带，基质辅助激光解吸/电离飞行时间MS显示分子量为23.6和23.8 kDa的两个密切相关的峰。蛋白水解消化的CP的质谱肽测序导致与先前报道的cDNA克隆CP 2、CP 3和CP 4（Mallinson，D. J.，洛克伍德，B。C.的方法，Coombs，G. H、北，M。J.（1994）Microbiology 140，2725-2735），以及与CP 4具有高度同源性的另一序列（www.tigr.org）。这最后两个物种是最丰富的CP部分的组成部分。这些结果提示CP诱导的程序性细胞死亡可能参与了T。体内迷走神经感染可能对治疗干预具有重要意义。研究结果最近发表在Infect上。Immun. (72，4151-8，2004）和J. Biol. Chem.（280，23853-60，2005）。相关的手稿最近发表在《感染与免疫》杂志上。

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. The results were recently published in Infect. Immun. (72, 4151-8, 2004 ) and J. Biol. Chem. (280, 23853-60, 2005). A related manuscript has recently been published in Infection and Immunity.