Proteomics of the Plasmodium Ookinete

描述(申请人提供)：该项目将应用蛋白质组学中的先进和新兴技术，以发现阻止疟疾传播的新分子靶点。虽然这一建议主要是探索性的，但也基于两个假设：1)疟原虫受精卵(蚊子中肠配子融合/受精的产物)表达可能是阻断疟疾传播的免疫学靶标的新的、可能较低丰度但迄今尚未确定的蛋白质；以及2)卵形疟原虫(接替受精卵并积极入侵中肠的运动发育阶段)表达新的分泌和细胞表面相关分子，这些分子专门参与蚊子中肠上皮的寄生虫入侵，是阻断疟疾传播的潜在靶标。有两个具体的目标：1)利用多维蛋白质鉴定技术(MUDPIT)、定量同位素编码的亲和标记质谱仪和定量2D凝胶电泳的互补技术来描绘鸡疟原虫受精卵和动子的蛋白质组，并使用生物信息学工具来比较鉴定现有的疟原虫基因组数据库中的受精卵和动子阶段的基因，特别是在致命的人类疟疾寄生虫恶性疟原虫中；以及2)通过在传播阻断免疫的动物模型中测试选定的一些新鉴定的分子来验证受精卵/动子表达的蛋白质。数据将放入PlamoDB公共数据库。由于本申请的重点是使用蛋白质组学来发现和验证新的阻断疟疾传播的分子靶点，因此本申请与母公司R01授予的AI45999“阻断疟疾传播的分子靶点”直接相关。这一建议也非常适合由母公司R01赠款支持的强大的现有基础设施，以测试新发现的寄生虫分子作为阻断疟疾传播的目标。这一应用与R21机制的探索/开发性质是一致的，因为它建议应用新的和新兴的技术来发现未知但潜在重要和有趣的疟原虫分子。

DESCRIPTION (provided by applicant): This project will apply advanced and emerging technologies in proteomics to the discovery of novel molecular targets of blocking malaria transmission. While primarily exploratory, this proposal is also based on two hypotheses: 1) Plasmodium zygotes (the products of gamete fusion/fertilization in the mosquito midgut) express novel, perhaps low abundance, but hitherto unidentified proteins that may be immunological targets of blocking malaria transmission; and 2) Plasmodium ookinetes (the motile developmental stage that succeeds the zygote and actively invades the midgut) express novel secreted and cell surface-associated molecules that are specifically involved in parasite invasion of the mosquito midgut epithelium and are potential targets of blocking malaria transmission. There are two Specific Aims: 1) To delineate the proteome of Plasmodium gallinaceum zygotes and ookinetes using complementary techniques of multidimensional protein identification technology (MUDPIT), quantitative isotope-coded affinity tag mass spectrometry, and quantitative 2D gel electrophoresis, and use bioinformatics tools to comparatively identify zygote and ookinete stage genes in the extant Plasmodium genome databases, particular in the lethal human malaria parasite P. falciparum; and 2) To initiate the validation of zygote/ookinete-expressed proteins by testing a select number of newly identified molecules in an animal model of transmission-blocking immunity, both with a new DNA vaccine technology and traditional recombinant protein expression/vaccination. Data will be placed into the PlasmoDB public database. This application directly relates to the parent R01 grant AI45999, "Molecular Targets of Blocking Malaria Transmission," since the focus of the present application is on using proteomics to discover and validate new molecular targets of blocking malaria transmission. This proposal also fits well into the robust extant infrastructure supported by the parent R01 grant to test newly identified parasite molecules as targets of blocking malaria transmission. This application is consistent with the exploratory/development nature of the R21 mechanism since it proposes to apply new and emerging technologies to discovering unknown but potentially important and interesting Plasmodium molecules.