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Proteomic analysis of the T. gondii vacuolar membrane

弓形虫液泡膜的蛋白质组学分析

基本信息

批准号：
7140472
负责人：
ANTHONY P. SINAI
金额：
$ 17.59万
依托单位：
UNIVERSITY OF KENTUCKY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-15 至 2007-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7140472
关键词：
Toxoplasma gondii bioinformatics biotechnology cell membrane host organism interaction immunoprecipitation intracellular parasitism mass spectrometry membrane activity molecular cloning parasite infection mechanism protein protein interaction protein quantitation /detection proteomics vesicle /vacuole

项目摘要

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.

描述（由申请人提供）：专性细胞内刚地弓形虫的成功寄生严重依赖于有效的营养清除和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取受到寄主液泡膜（PVM）的限制，PVM是一种寄生虫修饰的结构，作为定义复制许可生态位的边界。PVM的活性在营养获取、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参与感染的许多方面，但这些活动的分子基础仍然难以捉摸。由于无法纯化PVM，因此无法对这些活性进行详细的生化分析。在这个提议中，我们的目的是利用质谱方法结合新型多克隆抗血清作为亲和试剂来表征弓形虫PVM的蛋白质组。除了传统的质谱技术外，我们还将应用多维蛋白质鉴定技术（MudPit），该技术允许分析复杂的蛋白质混合物。这些定义PVM蛋白质组的方法是可行的，因为最近在蛋白质质谱鉴定、开放访问弓形虫基因组数据库和生物信息学算法方面取得了进展。该方法的最终成果是通过定义PVM的蛋白质组成，为PVM活性的表征奠定了基础。这些方法可能会导致鉴定潜在的新的化疗靶点弓形虫和其他顶端复合体，包括小隐孢子虫和肝期疟原虫。