Serine Proteinases in Apicomplexan Parasites

描述（由申请人提供）：刚地弓形虫和相关的顶复合体寄生虫，包括隐孢子虫，引起重大的人类疾病，特别是艾滋病患者。弓形虫和小弓形虫也是B类生物恐怖主义制剂，对免疫功能低下者构成特别威胁。顶复合体因其独特的顶分泌细胞器而得名，这些分泌细胞器包括微粒、细粒和致密颗粒。分泌细胞器内容物的蛋白水解过程发生在整个顶复合体中。我们从刚地弓形虫中鉴定了两种枯草杆菌样丝氨酸蛋白酶TgSUB1和TgSUB2。TgSUB1定位于微组，而TgSUB2是一种看似是一种虫体蛋白成熟酶的虫体蛋白。TgSUB2不能被破坏，并且TgSUB2的反义缺失导致寄生虫分裂不良，具有很少的异构体，并且具有巨大的异常细胞质囊泡。相比之下，TgSUB1破坏者是可行的。我们推测枯草弓形虫酶在生物发生或微单位和异体的功能中起重要作用。我们计划使用遗传学方法测试TgSUB2的重要性，并表征TgSUB2的生物底物。由于蛋白酶抑制剂通常模拟底物，鉴定TgSUB2的天然底物将加速特异性抑制剂的合成，并阐明TgSUB2在寄生虫存活中的作用。我们将使用重组TgSUB2开发TgSUB2活性的体外测定方法，该方法可用于鉴定TgSUB2的底物和独特的抑制剂。最后，我们将研究弓形虫基因组中其他枯草酶基因的表达。我们假设TgSUB1不是必需的，因为它的功能与其他枯草酶家族成员是冗余的。我们将确定其他枯草酶在弓形虫速殖子中的表达模式和定位，并利用遗传技术确定它们是否与TgSUB1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酶是否是治疗人类顶复体寄生虫感染的可行化疗靶点。

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.