Role of cruzain in T. cruzi and macrophage interactions

描述（由申请人提供）：恰加斯病的病原体克氏锥虫的免疫逃避可能是多因素的。然而，一个关键的寄生虫衍生因子涉及免疫逃避是cruzain（或cruzipain），这是该生物体的主要蛋白酶。我们的实验室已经成功地用半胱氨酸蛋白酶抑制剂靶向这种蛋白酶。我们可以用这种抑制剂阻断实验动物体内寄生虫的生命周期。我们还通过药物选择分离出一种缺乏cruzain的寄生虫克隆。该克隆Cai-KR在野生型小鼠中无致病性，但在RAG1-/-小鼠中可引起急性疾病。我们的初步结果表明，cruzain可能通过降解感染细胞细胞质中的NF-kB来阻止巨噬细胞活化。在这个应用中，我们将使用分子、生化和细胞方法来确定T. cruzi是否干扰NF-kB信号并描述其机制。我们还将比较克氏锥虫感染的巨噬细胞与经典、细胞因子交替激活的巨噬细胞以及细胞内细菌感染的巨噬细胞的表达谱。最后，我们建议构建过表达cruzain的转基因小鼠，以确定其对体内免疫系统的影响。

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.