PARASITE CYSTEINE PROTEASES AND HOST CELL APOPTOSIS

描述（由申请方提供）：阴道毛滴虫是一种寄生虫，可引起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，导致女性阴道炎和男性尿道炎。它与不孕症、早产、低出生体重婴儿和宫颈癌有关。T.流浪汉可能正在成为扩大艾滋病毒传播的最重要的辅助因素之一，特别是在美国的非洲裔美国人社区。本研究的长期目标是阐明T.迷走神经病理生物学在分子和细胞水平。我们建立了一个体外培养人阴道上皮细胞（HVECs）的系统，以研究寄生虫-宿主细胞的相互作用。T.体外宿主细胞的迷走神经感染诱导细胞破坏。由寄生虫精心制作（分泌/释放）的可溶性组分（SF）以物种特异性方式诱导HVECs的凋亡。将部分纯化的T. vaginalis-SF含有半胱氨酸蛋白酶，其似乎负责诱导HVEC中的细胞凋亡。具体目标是：1）纯化和表征诱导细胞凋亡的蛋白酶;包括克隆、测序、表达重组蛋白和表达活性分子的突变体; 2）研究寄生虫分子的生理学/细胞生物学，涉及T.迷走神经;和3）鉴定导致由寄生虫分子（包括细胞表面死亡受体）触发的HVEC凋亡的初始事件。将通过一系列色谱程序纯化活性毒剂。将使用细胞毒性和蛋白酶试验监测每个步骤。将对纯化的材料进行蛋白水解消化，并对分离的肽进行测序。序列信息将用于克隆cDNA。然后将cDNA用于产生重组蛋白，将在功能测定中测试该重组蛋白以证明克隆的蛋白诱导HVEC凋亡。将针对活性蛋白酶生成抗体（Ab），以研究细胞毒性剂在各种条件下的最佳释放，并显示其是否以可溶形式合成或与膜缔合。针对死亡受体的拮抗剂Ab将与纯化的试剂一起使用，以阐明哪种受体参与触发细胞凋亡途径。使用特异性Ab通过Western印迹/流式细胞术分析监测半胱天冬酶-8、-9和-3的活化，并使用特异性肽底物定量。了解寄生虫引起的宿主细胞病理学的分子机制，将为毛滴虫病的治疗提供新的策略和有效的治疗方法。

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.