Mechanisms of Giardia Lamblia Cyst Wall Formation

描述：（由申请人提供）：蓝氏贾第鞭毛虫的囊壁是 这是它成功寄生的关键这种原始的细胞外基质 负责环境中的心血管存活， 感染新的宿主基于我们对心包膜的广泛研究，我们 已经制定了一个四阶段模型的囊壁蛋白（CWP）和囊肿 成熟本修订提案的目标是验证和扩展 模型，以便更全面地了解这种基本差异。 我们将深入关注煤矸石的加工、运输和组装。我们的中央 假设是囊壁的组装需要协同活动 在几个细胞内室，但最终的成熟发生在 细胞表面具体目标1是评价新型心血管 溶酶体/内体外周囊泡（PV）区室在CWP加工中的作用。 我们将检验通过PV是CWP所必需的假设 成熟或者，CWP可以通过PV以内吞的方式运输。 恢复已提前释放的CWP的途径。我们将使用 四环素调节表位标记的CWP和细胞表面的表达 标记以追踪CWP。这些研究将阐明CWP交通的作用 通过PV实现成熟的囊壁结构和功能。在 具体目标2，我们将测试假设，CWP经历连续阶段 S-S键的形成和重排由不寻常的心血管蛋白催化 二硫键异构酶，导致在细胞上形成抗性原纤维 面具体目标3是检验异肽键 形成是分化成包囊和鉴定蛋白质所必需的 交联，使用特定的标记和抑制剂。 这一修订后的提案将揭示形成，功能， 进化的一个古老的真核细胞外基质，使用整合 生物化学、细胞生物学和分子方法。由于囊性阶段 是许多寄生虫传播的关键，囊壁形成是 生命周期的中断。

DESCRIPTION: (provided by the applicant): The cyst wall of Giardia lamblia is critical to its success as a parasite. This primitive extracellular matrix is responsible for giardial survival in the environment and for its ability to infect new hosts. Based on our extensive studies of giardial encystation, we have formulated a four-stage model of cyst wall protein (CWP) and cyst maturation. The goal of this revised proposal is to validate and expand that model for a more comprehensive understanding of this essential differentiation. We will focus in depth on CWP processing, transport, and assembly. Our central hypothesis is that assembly of the cyst wall requires co-coordinated activities within several intracellular compartments, but final maturation occurs on the cell surface. Specific Aim 1 is to evaluate the role of the novel giardial lysosomal/endosomal peripheral vesicle (PV) compartment in processing of CWPs. We will test the hypothesis that passage through the PV is necessary for CWP maturation. Alternatively, CWP may traffic through the PV in an endocytic pathway to recover CWP that has been released prematurely. We will use tetracycline-regulated expression of epitope-tagged CWPs and cell surface labeling to trace CWP. These studies will elucidate the role of CWP traffic through the PV in achieving mature cyst wall architecture and function. In Specific Aim 2, we will test the hypothesis that CWPs undergo sequential stages of S-S bond formation and re-arrangement catalyzed by unusual giardial protein disulfide isomerases, leading to formation of resistant fibrils on the cell surface. Specific Aim 3 is to test the hypothesis that iso-peptide bond formation is needed for differentiation into cysts and to identify the proteins cross linked, using specific labels and inhibitors. This revised proposal will reveal basic insights into formation, function, and evolution of an ancient eukaryotic extracellular matrix, using integrated biochemical, and cell biological, and molecular approaches. Since cystic stages are key to transmission of many parasites, cyst wall formation is a target for interruption of the life cycle.