Role of Proteases in Cryptosporidium-Host Cell Interactions

顶端复合体寄生虫隐孢子虫是全球腹泻疾病的重要原因， 特别是在免疫功能受损的宿主中，如艾滋病患者。这个项目的总体目标是 细小弧菌类枯草杆菌丝氨酸蛋白酶在宿主寄生虫中的作用 互动。编码可能的枯草杆菌酶的两个基因(分别命名为CpSUB1和CpSUB2)已经被 在微小隐孢子虫基因组中鉴定，但尚未被调查。初步研究表明， 这两个基因在体外感染微小弧菌期间都有表达，这表明它们编码的蛋白质 很可能在宿主与寄生虫的相互作用中起重要作用。假设CpSUB1和/或CpSUB2 处理表面和根尖复合蛋白，如gp40/15，并在体外介导微小弧菌感染。 在第一个特定目标中，我们将量化CpSUB1和2的mRNA表达，鉴定前体， 在微小隐孢子虫中表达的CpSUB1和2蛋白的加工和成熟形式，研究它们的 翻译后处理，并确定它们的亚细胞定位。此外，我们还将表示 具有酶活性的重组CpSUB1和CpSUB2，并对其酶活性进行了表征。在……里面 第二个具体目标是，我们将确定这两种枯草杆菌酶是否处理重组gp40/15。 此外，我们将确定前肽抑制剂或CpSubs抗体是否能抑制微小弧菌 肠上皮细胞的体外感染。长期目标是合理的、基于结构的设计 这些酶的抑制剂，作为预防或治疗隐孢子虫病的药物。项目说明/相关性 肠道寄生虫隐孢子虫是腹泻疾病的重要原因 在世界范围内，特别是在免疫功能受损的宿主，如艾滋病患者。在这 我们将研究在宿主细胞感染中起重要作用的酶。 寄生虫，长期目标是开发这些酶的抑制剂作为治疗 隐孢子虫病。因为目前还没有一贯有效的治疗方法。 这些研究对于新药的开发具有重要意义。

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. Project Narrative/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.