Role of Proteases in Cryptosporidium-Host Cell Interactions

描述（由申请人提供）：顶复合体寄生虫隐孢子虫是世界范围内腹泻疾病的重要原因，特别是在免疫功能低下的宿主，如艾滋病患者中。本项目的总体目标是研究枯草杆菌样丝氨酸蛋白酶（枯草酶）在宿主-寄生虫相互作用中的作用。两个编码假定枯草酶的基因（被命名为CpSUB1和CpSUB2）已经在小孢子虫基因组中被鉴定出来，但尚未被研究。初步研究表明，这两个基因在体外感染小孢子虫过程中都有表达，这表明它们编码的蛋白质可能在宿主-寄生虫相互作用中起重要作用。假设CpSUB1和/或CpSUB2加工表面和根尖复合体蛋白，如gp40/15，并介导体外小孢子虫感染。在第一个具体目标中，我们将量化CpSUB1和2的mRNA表达，鉴定CpSUB1和2蛋白在小孢子虫中表达的前体、加工和成熟形式，研究它们的翻译后加工并确定它们的亚细胞定位。此外，我们将表达具有酶活性的重组CpSUB1和CpSUB2，并对其酶活性进行表征。在第二个具体目标中，我们将确定是否有一种枯草酶处理重组gp40/15。此外，我们将确定CpSUBs的前肽抑制剂或抗体是否能在体外抑制细小梭菌对肠上皮细胞的感染。长期目标是合理的、基于结构的设计这些酶的抑制剂作为药物来预防或治疗隐孢子虫病。公共卫生相关性：肠道寄生虫隐孢子虫是世界范围内腹泻疾病的重要原因，特别是在免疫功能低下的宿主（如艾滋病患者）中。在这个项目中，我们将研究寄生虫感染宿主细胞的重要酶，长期目标是开发这些酶的抑制剂作为隐孢子虫病的药物。由于这种疾病没有持续有效的治疗方法，这些研究对开发新药很重要。

DESCRIPTION (provided by applicant): The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide, particularly in immunocompromised hosts such as AIDS patients. The overall goal of this project is to investigate the role of subtilisin-like serine proteases (subtilases) of C. parvum in host-parasite interactions. Two genes encoding putative subtilases (designated CpSUB1 and CpSUB2) have been identified in the C. parvum genome but have not yet been investigated. Preliminary studies show that both genes are expressed during C. parvum infection in vitro suggesting that the proteins they encode are likely to be important in host-parasite interactions. The hypothesis is that CpSUB1 and/or CpSUB2 process surface and apical complex proteins such as gp40/15 and mediate C. parvum infection in vitro. In the first specific aim we will quantify mRNA expression of CpSUB1 and 2, identify the precursor, processed and mature forms of the CpSUB1 and 2 proteins expressed in C. parvum, investigate their post-translational processing and determine their subcellular localization. In addition, we will express enzymatically active recombinant CpSUB1 and CpSUB2 and characterize their enzymatic activity. In the second specific aim we will determine whether either subtilase processes recombinant gp40/15. In addition we will determine whether propeptide inhibitors or antibodies to the CpSUBs inhibit C. parvum infection of intestinal epithelial cells in vitro. The long term goal is the rational, structure-based design of inhibitors of these enzymes as drugs to prevent or treat cryptosporidiosis. PUBLIC HEALTH RELEVANCE: The intestinal parasite Cryptosporidium is a significant cause of diarrheal disease worldwide, particularly in immunocompromised hosts such as AIDS patients. In this project we will study enzymes that are important for infection of host cells by the parasite, with the long term goal of developing inhibitors of these enzymes as drugs for cryptosporidiosis. Since there is no consistently effective treatment available for this disease, these studies are important for development of new drugs.