Role of cruzain in T. cruzi and macrophage interactions

描述（由申请方提供）：克氏锥虫（恰加斯病的病原体）的免疫逃避可能是多因素的。然而，涉及免疫逃避的一个关键寄生虫衍生因子是cruzain（或cruzipain），其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的寄生虫生命周期。我们还通过药物选择分离了cruzain缺陷型寄生虫克隆。该克隆Cai-KR在野生型小鼠中是非致病性的，但可在RAG 1-/-小鼠中引起急性疾病。我们的初步结果表明，cruzain可以发挥作用，防止巨噬细胞活化，通过降解感染细胞的细胞质中的NF-κ B。在本申请中，我们将使用分子，生物化学和细胞的方法来确定T。cruzi干扰NF-kB信号传导并描述其机制。我们还将比较T. Cruzi感染巨噬细胞，其中巨噬细胞经典地和交替地被细胞因子激活，以及被细胞内细菌感染。最后，我们提出了一个转基因小鼠过表达cruzain，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.