SERINE PROTEINASES IN APICOMPLEXAN PARASITES

描述(改编自申请者摘要)：显然需要 弓形虫及其相关疾病的替代化疗策略 顶端复合体寄生虫。这些寄生虫的入侵在形态上是相似的 并伴随着对蛋白质的降解过程 微丝细胞器的内容物。丝氨酸蛋白酶抑制剂已经被 最近被证明能阻止弓形虫的宿主细胞入侵。蛋白酶抑制剂 在各种致病条件下都取得了很高的成功。弓形虫 将被用作评估类枯草杆菌菌素适合性的模型系统 丝氨酸蛋白酶(枯草杆菌酶)作为尖端复合体化疗靶点的研究 寄生虫。弓形虫的两种枯草杆菌酶已被克隆，并且至少有一种 这些可能局限于微线粒。初步研究表明， 丝氨酸蛋白酶抑制剂DFP阻止微体蛋白H4和H4的加工 MIC2.推测弓形虫枯草杆菌(S)介导寄主入侵 细胞和抑制枯草杆菌酶将被证明是有用的治疗 感染其他重要的Apicomplexan寄生虫。这项提议将 弓形虫枯草杆菌酶基因的克隆与鉴定 贡地。核糖核酸的合成、加工和亚细胞定位模式 枯草杆菌酶将用抗蛋白酶的抗体和 有表位标记的蛋白质。具有酶活性的重组枯草杆菌酶将是 表达。速殖子细胞组分和重组蛋白酶将被测试 用于人工底物的蛋白质水解性切割。这些底物将是 用来表征T. 弓形虫枯草杆菌。枯草杆菌酶的功能将由基因决定。 颠覆。敲除寄生虫入侵宿主细胞的能力，过程 微线体蛋白，并在动物身上引起疾病将进行测试。最后，它 将确定微线体蛋白H4和MIC2是否具有蛋白水解性 在入侵前或入侵期间被弓形虫枯草杆菌裂解。这些研究将 进一步了解蜜蜂复合体寄生虫的入侵并将导致 针对这些生物的新的化疗策略。

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.