Proteomic analysis of the T. gondii vacuolar membrane

描述(由申请人提供)：专性细胞内弓形虫的成功寄生关键依赖于有效的营养清除和对多种宿主功能的颠覆。寄主细胞质内营养物质和细胞功能的获得受到寄生液泡膜(PVM)的限制，PVM是一种寄生虫修饰的结构，作为定义复制允许生态位的边界。PVM的活动在营养获取、结构完整性和宿主信号级联的操纵中至关重要，包括潜在的对寄生虫的免疫反应。虽然PVM涉及感染的许多方面，但这些活动的分子基础仍然难以捉摸。由于无法提纯PVM，因此无法对这些活性进行详细的生化分析。在这项建议中，我们的目标是用质谱学方法结合新的多克隆抗血清作为亲和试剂来表征弓形虫PVM的蛋白质组。除了传统的质谱学技术外，我们还将应用多维蛋白质鉴定技术(MUDIT)，它可以分析复杂的蛋白质混合物。最近在蛋白质的质谱学鉴定、开放获取弓形虫基因组数据库和分析数据的生物信息学算法方面的进展使这些确定PVM蛋白质组的方法成为可能。这种方法的最终目的是通过定义PVM的蛋白质组成，为表征PVM中的活性奠定基础。这些方法将可能导致识别潜在的新的弓形虫和其他顶体复合体的化疗靶点，包括微小隐孢子虫和肝脏阶段的疟原虫。

DESCRIPTION (provided by the applicant): Successful parasitism by the obligate intracellular Toxoplasma gondii is critically dependent on efficient nutrient scavenging and subversion of diverse host functions. Access to nutrients and cellular functions within the host cytoplasm is restricted by the parasitophorous vacuole membrane (PVM), a parasite modified structure that serves as the boundary defining the replication permissive niche. Activities at the PVM are critical in nutrient acquisition, structural integrity and the manipulation of host signaling cascades, including potentially the immune response to the parasite. While the PVM is involved in many facets of infection the molecular basis for these activities remains elusive. The inability to purify the PVM has prevented detailed biochemical analyses of these activities. In this proposal we aim to characterize the proteome of the T. gondii PVM using mass spectroscopic approaches coupled with novel polyclonal antisera as affinity reagents. In addition to conventional mass spectroscopic techniques we will apply multi-dimensional protein identification technology (MudPit) which permits the analysis of complex protein mixtures. These approaches to define the PVM proteome are made feasible by recent advances in the mass spectrometric identification of proteins, open access to the T. gondii genome databases and bioinformatic algorithms to analyze the data. The culmination of this approach is to establish the foundation for the characterization of activities in the PVM by defining its protein composition. These approaches will likely lead to the identification of potentially novel chemotherapeutic targets for Toxoplasma and other apicomplexan including Cryptosporidium parvum and hepatic stages of Plasmodia.