Role of cruzain in T. cruzi and macrophage interactions

描述(申请人提供)：克氏锥虫的免疫逃避，恰加斯病的病原体可能是多因素的。然而，与免疫逃避有关的一个关键的寄生虫衍生因子是Cruzain(或CruziPain)，它是这种有机体的主要蛋白酶。我们的实验室已经成功地用半胱氨酸蛋白酶抑制剂靶向了这种蛋白酶。我们可以阻断用这种抑制剂治疗的实验动物的寄生虫生命周期。我们还通过药物筛选分离到了一个克隆菌素缺乏的寄生虫克隆。该克隆Cai-KR在野生型小鼠中无致病性，但可在RAG1-/-小鼠中引起急性疾病。我们的初步结果表明，克鲁扎因可能通过降解感染细胞胞浆中的核因子-kB而起到阻止巨噬细胞激活的作用。在这一应用中，我们将使用分子、生化和细胞方法来确定克氏锥虫是否干扰核因子-kB信号并描述其机制。我们还将比较感染克氏毛滴虫的巨噬细胞与经典激活和交替激活的巨噬细胞以及感染细胞内细菌的巨噬细胞的表达谱。最后，我们建议制作一只过量表达克鲁萨因的转基因小鼠，以确定其在体内对免疫系统的影响。

DESCRIPTION (provided by applicant): Immune evasion by Trypanosoma cruzi, the causative agent of Chagas disease is likely multifactorial. However, one key parasite derived factor implicated in immune evasion is cruzain (or cruzipain), the major protease of this organism. Our laboratory has successfully targeted this protease with a cysteine protease inhibitor. We can block the parasite life cycle in experimental animals treated with this inhibitor. We have also isolated a cruzain deficient parasite clone via drug selection. This clone, Cai-KR is non-pathogenic in wildtype mice, but can cause acute disease in RAG1-/- mice. Our preliminary results suggest that cruzain could play a role in preventing macrophage activation by degrading NF-kB in the cytoplasm of infected cells. In this application, we will use molecular, biochemical and cellular approaches to determine if T. cruzi interferes with NF-kB signaling and describe its mechanism. We will also compare the expression profile of T. cruzi infected macrophages with macrophages classically and alternatively activated with cytokines, as well as infected with intracellular bacteria. Finally, we propose to make a transgenic mouse over expressing cruzain to determine its effects on the immune system in vivo.