PARASITE CYSTEINE PROTEASES AND HOST CELL APOPTOSIS

描述(申请人提供)：阴道毛滴虫，一种寄生虫，导致人类最常见的非病毒性性传播疾病之一，滴虫病。滴虫病是一种严重的疾病，会导致女性阴道炎和男性尿道炎。它与不孕不育、早产、低出生体重婴儿和宫颈癌有关。阴道毛滴虫可能正在成为放大艾滋病毒传播的最重要的辅助因素之一，特别是在美国的非裔美国人社区。这项建议的长期目标是在分子和细胞水平上阐明阴道毛滴虫的病理生物学机制。我们建立了一个人阴道上皮细胞的体外培养系统，以研究寄生虫与宿主细胞的相互作用。阴道毛滴虫体外感染宿主细胞可导致细胞破坏。寄生虫分泌/释放的可溶性部分(SF)以种属特有的方式诱导血管内皮细胞的凋亡。阴道毛滴虫SF的部分纯化活性成分(S)含有一种半胱氨酸蛋白酶(S)，该酶似乎与诱导血管内皮细胞凋亡有关。其具体目标是：1)纯化和鉴定诱导细胞凋亡的蛋白酶(S)；包括克隆、测序、表达重组蛋白和表达活性分子(S)的突变体；2)研究寄生虫分子与阴道毛滴虫对凋亡蛋白酶(S)的生物合成、分泌和激活有关的生理/细胞生物学；以及3)鉴定由寄生虫分子(S)包括细胞表面死亡受体在内的导致血管内皮细胞凋亡的初始事件。活性毒剂将通过一系列的层析程序进行纯化。每一步都将使用细胞毒性和蛋白水解酶分析进行监测。纯化的材料将经过蛋白水解酶的消化，并对分离的多肽进行测序。序列信息将用于克隆该cdna。然后，该基因将被用于生产重组蛋白，该重组蛋白将在功能分析中进行测试，以证明克隆的蛋白诱导血管内皮细胞凋亡。将产生抗体(抗体)活性蛋白酶(S)，以研究细胞毒剂在各种条件下的最佳释放，并表明它是以可溶性形式合成或与膜结合。针对死亡受体的拮抗剂将与纯化试剂一起使用，以阐明哪些受体参与触发凋亡途径。Caspase-8、-9和-3的激活将通过使用特定抗体的Western印迹/流式细胞术分析进行监测，并使用特定的多肽底物进行定量。对寄生虫引起的宿主细胞病理的分子理解的知识将揭示滴虫病的新策略/有效治疗。

DESCRIPTION (provided by applicant): Trichomonas vaginalis, a parasite, causes one of the most common non-viral sexually transmitted disease in humans, trichomoniasis. Trichomoniasis is a serious disease that causes vaginitis in women and urethritis in men. It has been linked to infertility, preterm delivery, low birth weight infants and cervical cancer. T. vaginalis may be emerging as one of the most important cofactors in amplifying HIV transmission, especially in African-American communities of the United States. The long-term goal of this proposal is to elucidate the mechanisms of T. vaginalis pathobiology at the molecular and cellular levels. We have developed an in vitro culture system of human vaginal epithelial cells (HVECs) to study parasite-host cell interaction. T. vaginalis infection of host cells in vitro induces cell destruction. The soluble fraction (SF) elaborated (secreted/released) by the parasites induces apoptosis in the HVECs in a species-specific manner. The partially purified active agent(s) from T. vaginalis-SF contains a cysteine protease(s) that appears to be responsible for inducing apoptosis in HVECs. The specific aims are: 1) to purify and characterize the protease(s) that induce apoptosis; including, cloning, sequencing, expression of recombinant protein and express mutants of the active molecule(s); 2) study the physiology/cell biology of the parasite molecules with respect to biosynthesis, secretion, and activation of the apoptotic protease(s) by T. vaginalis; and 3) identify the initial events leading to HVEC apoptosis triggered by the parasite molecule (s), including cell surface death receptors. The active toxic agents will be purified by a series of chromatographic procedures. Each step will be monitored using both cytotoxicity and protease assays. The purified material will be subjected to proteolytic digestion and isolated peptides will be sequenced. Sequence information will be used to clone the cDNA. The cDNA will then be used to produce recombinant protein, which will be tested in functional assays to demonstrate that the cloned protein induces HVEC apoptosis. Antibodies (Abs) will be generated against active protease(s) to study the optimal release of cytotoxic agent under a variety of conditions and to show if it is synthesized in a soluble form or associated with membranes. Antagonist Abs against death receptors will be utilized with the purified agent to elucidate which of the receptors is involved in triggering apoptotic pathways. Activation of caspases-8, -9, and -3 will be monitored by Western blot/flow cytometry analyses using specific Abs and quantified using specific peptide substrates. Knowledge of the molecular understanding of host cell pathology induced by parasite will reveal new strategies/effective treatment for trichomoniasis.