Novel activities and the T. gondii vacuolar membrane

描述（由申请人提供）：弓形虫是一种重要的机会性寄生虫，在子宫内获得的弓形虫会导致免疫功能低下和严重出生缺陷的人感染危及生命。寄生虫在被感染宿主细胞内的寄生液泡（PV）内生长。定义这个生态位的边界是PV膜（PVM），它是寄生虫和宿主之间的界面。尽管它很重要，但对PVM的调查却很有限。我们最近完成了对弓形虫PVM的蛋白质组学分析，鉴定出123个蛋白质，其中70个似乎是新的。在这些新蛋白质中，有26个被注释为“假设的ORF”，排除了任何功能分配。作为假设的ORF，这些基因座缺乏任何可识别的基序，表明它们确实是新颖的。虽然有几种似乎是弓形虫所特有的，但其他一些在其他不太容易处理的顶复合体中也有类似的注释同源物，包括疟原虫和隐孢子虫，这表明它们具有保守功能。作为可能定位于PVM（一种仅在寄生虫感染背景下发现的细胞器）的新蛋白，这些假设的ORF代表了可能作为药物靶点的潜在独特活性。在这项研究中，我们利用最近发展的技术来标记和定位假设的ORF，作为克隆验证基因和尝试基因敲除的前奏。这些研究共同代表了对现有数据集的二次分析，作为一个有限的短期项目，具有揭示新活动的高潜力。因此，它非常适合RO3融资机制。有了这个建议，我们的目标是对这些孤儿基因进行初步表征，以确定它们在寄生虫生物学中的功能。

DESCRIPTION (provided by applicant): The protozoan parasite Toxoplasma gondii is an important opportunistic parasite causing life threatening infections in the immune compromised and severe birth defects when acquired in utero. The parasite grows within a specialized compartment, the parasitophorous vacuole (PV) within the infected host cell. The boundary defining this niche is the PV membrane (PVM) that serves as the interface between the parasite and host. Despite its importance, investigation of the PVM has been limited. We have recently completed a proteomic analysis of the T. gondii PVM resulting in the identification of 123 proteins of which 70 appear to be novel. Of these novel proteins, 26 are annotated as "hypothetical ORF's" precluding any assignment of function. As hypothetical ORF's, these loci lack any identifiable motif indicating they are truly novel. While several appear unique to Toxoplasma, others have similarly annotated homologs in other less tractable Apicomplexa including Plasmodium and Cryptosporidium spp. suggesting conserved function. As novel proteins that are potentially localized to the PVM, an organelle found only in the context of parasite infection, these hypothetical ORF's represent potentially unique activities that may serve as drug targets. In this study we exploit recently developed technologies to epitope tag and localize the hypothetical ORF's as a prelude to cloning the validated genes and attempting the gene knockouts. Together these studies represent a secondary analysis of an existing data set as a limited short term project with a high potential of revealing novel activities. As such it is ideally suited to the RO3 funding mechanism. With this proposal we aim to perform the initial characterization of these orphan genes toward establishing their functions in parasite biology. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii causes serious illness in immune compromised individuals and is a model organism for several less experimentally tractable parasites. This study aims to investigate truly novel proteins that were identified as being components of the parasitophorous vacuole membrane, a unique and essential parasite organelle. We expect with the completion of this work to open new areas of investigation in parasite biology.