Proteolytic modulation of toxoplasma invasion proteins

描述（由申请方提供）：专性细胞内原生动物刚地弓形虫是一般人群后葡萄膜炎、免疫缺陷个体脑炎和先天性感染儿童认知障碍的主要原因。我们试图了解弓形虫如何准备和实现细胞入侵。虽然它已被公认为许多弓形虫入侵蛋白进行蛋白水解成熟的途中顶端分泌细胞器，很少有人知道有关的蛋白酶或成熟发生在哪里和为什么。我们假设T.基于初步的免疫定位、基因敲除和抑制剂研究，以及沿着蛋白酶特异性分析和推定底物的体外处理，弓形虫使用组织蛋白酶蛋白酶来蛋白水解激活和稳定新型多泡内体（MVE）内的侵入蛋白。为了验证这一假设，我们将：（1）描述CPL敲除寄生虫的缺陷和补偿机制;（2）通过遗传和抑制剂研究证实组织蛋白酶在proMIC成熟中的重叠作用;（3）测试蛋白水解成熟稳定proMIC蛋白复合物的“锁定和加载”假设。这项工作将阐明分泌性蛋白质运输和蛋白水解调节蛋白质功能的基本机制，可能与相关的apicomplexan寄生虫和其他利用调节分泌的真核细胞共享。公共卫生相关性：我们的目标是找到新的方法来干扰弓形虫对人体细胞的入侵，作为控制这种寄生虫引起的感染的策略。弓形虫蛋白酶可能是新的抗寄生虫药物的良好靶点;这项工作旨在测试两个这样的蛋白酶的功能和意义。

DESCRIPTION (provided by applicant): The obligate intracellular protozoan Toxoplasma gondii is a leading cause of posterior uveitis in the general population, encephalitis in immunodeficient individuals, and cognitive impairment in congenitally infected children. We seek to understand how Toxoplasma prepares for and achieves cell invasion. Although it has been well recognized that many Toxoplasma invasion proteins undergo proteolytic maturation en route to apical secretory organelles, little is known about the proteases involved or where and why maturation occurs. We hypothesize that T. gondii uses a cathepsin proteases to proteolytically activate and stabilize invasion proteins within a novel multivesicular endosome (MVE), based on preliminary immunolocalization, gene knockout, and inhibitor studies, along with protease specificity analysis and in vitro processing of a putative substrate. To test this hypothesis we will: (1) Characterize defects and compensatory mechanisms in CPL knockout parasites; (2) Confirm the overlapping roles of cathepsins in proMIC maturation by genetic and inhibitor studies; and (3) Test the "lock and load" hypothesis that proteolytic maturation stabilizes proMIC protein complexes. This work will illuminate basic mechanisms of secretory protein trafficking and proteolytic modulation of protein function that may be shared with related apicomplexan parasites and other eukaryotic cells that utilize regulated secretion. PUBLIC HEALTH RELEVANCE: Our goal is to find new ways of interfering with Toxoplasma invasion of human cells as a strategy of controlling infections caused by this parasite. Toxoplasma proteases may be good targets for new anti-parasitic drugs; this work is designed to test the function and significance of two such proteases.