Serine Proteinases in Apicomplexan Parasites

描述(由申请人提供)：弓形虫和相关的复合体寄生虫，包括隐孢子虫，会导致严重的人类疾病，特别是在艾滋病患者中。弓形虫和微小弓形虫也是B类生物恐怖分子，对免疫受损的人构成了特别的威胁。顶端复合体因其独特的顶端分泌细胞器--微丝、棒状体和致密颗粒而得名。分泌细胞器内容物的蛋白水解性处理发生在整个顶丛中。我们鉴定了弓形虫的两种类似枯草杆菌蛋白的丝氨酸蛋白酶，TgSUB1和TgSUB2。TgSUB1定位于微线体，而TgSUB2是一种棒状蛋白，似乎是一种棒状蛋白成熟酶。TgSUB2不能被破坏，而TgSUB2的反义缺失会导致寄生虫分裂不良，胞质小泡数目少，并有大量异常的细胞质小泡。相比之下，TgSUB1破坏剂是可行的。我们推测，弓形虫枯草杆菌酶在微线虫和棒状体的生物发生或功能中起着重要作用。我们计划使用遗传方法测试TgSUB2的重要性，并对TgSUB2的生物底物进行表征。由于蛋白酶抑制剂通常模拟底物，因此确定TgSUB2的天然底物将加速特定抑制剂的合成，并阐明TgSUB2在寄生虫生存中的作用。我们将使用重组TgSUB2来建立一种TgSUB2活性的体外检测方法，该方法可用于鉴定TgSUB2的底物和唯一的抑制剂。最后，我们将研究弓形虫基因组中存在的其他枯草杆菌酶基因的表达。我们假设TgSUB1不是必需的，因为它的功能与其他枯草杆菌酶家族成员是多余的。我们将确定其他枯草杆菌酶在弓形虫速殖子中的表达模式和定位，并使用基因技术来确定它们是否与TgSUB1具有重叠功能。除了阐明弓形虫分泌细胞器的功能和生物学外，这些研究还将确定枯草杆菌酶是否为治疗人类感染Apicomplexan寄生虫的可行化疗靶点。

DESCRIPTION (provided by applicant): Toxoplasma gondii and related Apicomplexan parasites including Cryptosporidium cause significant human disease particularly in AIDS patients. T. gondii and C. parvum are also category B bioterrorism agents that pose a particular threat to the immunocompromised. The Apicomplexa are named for their unique apical secretory organelles-micronemes, rhoptries and dense granules. Proteolytic processing of secretory organelle contents occurs throughout the Apicomplexa. We have characterized two subtilisin-like serine proteinases (subtilases) from T. gondii, TgSUB1 and TgSUB2. TgSUB1 localizes to the micronemes, whereas TgSUB2 is a rhoptry protein that appears to be a rhoptry protein maturase. TgSUB2 cannot be disrupted, and antisense depletion of TgSUB2 results in parasites that divide poorly, have few rhoptries, and have large aberrant cytoplasmic vesicles. In contrast, TgSUB1 disruptants are viable. We hypothesize that Toxoplasma subtilases play an essential role in the biogenesis or function of micronemes and rhoptries. We plan to test the importance of TgSUB2 using genetic methods and to characterize TgSUB2 biological substrates. Because proteinase inhibitors typically mimic substrates, identification of a natural substrate for TgSUB2 would accelerate synthesis of specific inhibitors and elucidate the role of TgSUB2 in parasite survival. We will use recombinant TgSUB2 to develop an in vitro assay for TgSUB2 activity that can be used to identify substrates and unique inhibitors of TgSUB2. Finally, we will investigate the expression of other subtilase genes present in the T. gondii genome. We hypothesize that TgSUB1 is not essential because its function is redundant with other subtilase family members. We will determine the expression pattern and localization of other subtilases in T. gondii tachyzoites and use genetic techniques to determine if they have overlapping function with TgSUB1. In addition to illuminating the function and biology of T. gondii secretory organelles, these studies will determine whether subtilases are a viable chemotherapeutic target for treatment of human infections with Apicomplexan parasites.