SERINE PROTEINASES IN APICOMPLEXAN PARASITES

描述（改编自申请人的摘要）：显然需要 弓形虫及相关疾病的替代化疗策略 顶复寄生虫这些寄生虫的入侵在形态上是相似的 在整个门，并伴随着蛋白水解加工的 微线细胞器的内容物。丝氨酸蛋白酶抑制剂已经被 最近显示阻断T.刚地。蛋白酶抑制剂 在各种致病性疾病中取得了非常成功。T.弓形虫 将被用作模型系统，以评估枯草杆菌蛋白酶样 丝氨酸蛋白酶（枯草杆菌酶）作为顶复生中的化疗靶点 寄生虫从T.已经克隆了弓形虫， 这些可以定位于微线。初步研究表明， 丝氨酸蛋白酶抑制剂DFP阻断微线体蛋白H4的加工， MIC 2。推测T.弓形虫枯草杆菌酶介导宿主入侵 细胞，并且枯草杆菌酶的抑制将证明可用于治疗 感染其他重要的顶复门寄生虫。这项建议会 完成弓形虫枯草杆菌酶基因的克隆和鉴定 刚地。蛋白质的合成、加工和亚细胞定位模式 使用蛋白酶的抗体澄清枯草杆菌酶， 表位标记的蛋白质。酶活性重组枯草杆菌酶将是 表达。将检测速殖子细胞组分和重组蛋白酶 用于人工底物的蛋白水解裂解。这些基板将是 用于表征T. 弓形虫枯草杆菌酶枯草杆菌酶的功能将使用基因工程来确定。 破坏基因敲除寄生虫侵入宿主细胞的能力， 微线蛋白，并导致动物疾病将进行测试。最后 如果微线体蛋白H4和MIC2是蛋白水解的， 被T.弓形虫枯草杆菌酶在入侵之前或期间。这些研究将 进一步了解顶复门寄生虫的入侵，并将导致 针对这些生物体的新的化疗策略。

DESCRIPTION (Adapted from Applicant's Abstract): There is a clear need for alternative chemotherapeutic strategies for Toxoplasma gondii and related apicomplexan parasites. Invasion by these parasites is morphologically similar throughout the phylum and is accompanied by proteolytic processing of the contents of the microneme organelle. Serine proteinase inhibitors have been recently shown to block host cell invasion by T. gondii. Proteinase inhibitors have been highly successful in a variety of pathogenic conditions. T. gondii will be used as a model system to evaluate the suitability of subtilisin-like serine proteinases (subtilases) as chemotherapeutic targets in the Apicomplexan parasites. Two subtilases from T. gondii have been cloned and at least one of these may localize to the micronemes. Preliminary studies indicate that the serine proteinase inhibitor DFP blocks processing of microneme proteins H4 and MIC2. It is hypothesized that T. gondii subtilase(s) mediate invasion of host cells and that inhibition of subtilases will prove useful for treatment of infections with other important Apicomplexan parasites. This proposal will complete the cloning and characterization of subtilase genes from Toxoplasma gondii. The pattern of synthesis, processing and subcellular localization of the subtilases will be clarified using antibody to proteinases and epitope-tagged proteins. Enzymatically active recombinant subtilases will be expressed. Tachyzoite cell fractions and recombinant proteinases will be tested for proteolytic cleavage of artificial substrates. These substrates will be used to characterize the inhibitor profile and substrate specificity of T. gondii subtilases. The function of the subtilases will be determined using gene disruption. The ability of knockout parasites to invade host cells, process microneme proteins, and cause disease in animals will be tested. Finally, it will be determined if the microneme proteins H4 and MIC2 are proteolytically cleaved by T. gondii subtilases prior to or during invasion. These studies will further understanding of invasion by Apicomplexan parasites and will lead to novel chemotherapeutic strategies for these organisms.