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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（由申请方提供）：专性细胞内弓形虫的成功寄生主要依赖于有效的营养清除和多种宿主功能的破坏。进入宿主细胞质内的营养物质和细胞功能受到寄生虫空泡膜（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的活动对于营养获取、结构完整性和宿主信号级联的操纵（可能包括对寄生虫的免疫反应）至关重要。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物化学分析。在这个提议中，我们的目标是表征T。弓形虫PVM使用质谱方法与新的多克隆抗血清作为亲和试剂偶联。除了传统的质谱技术，我们将应用多维蛋白质鉴定技术（MudPit），它允许分析复杂的蛋白质混合物。蛋白质质谱鉴定的最新进展、T.弓形虫基因组数据库和生物信息学算法来分析数据。这种方法的最终结果是通过定义PVM的蛋白质组成，为PVM中的活性表征奠定基础。这些方法将可能导致识别弓形虫和其他顶复门的潜在新的化疗靶点，包括隐孢子虫和疟原虫的肝阶段。