Novel activities and the T. gondii vacuolar membrane

描述(申请人提供)：原虫寄生虫弓形虫是一种重要的机会性寄生虫，在子宫内感染时会导致危及生命的免疫损害和严重的出生缺陷。寄生虫生长在一个特殊的隔间里，即受感染宿主细胞内的寄生液泡(PV)。定义这一生态位的边界是光伏膜(PVM)，它充当寄生虫和宿主之间的界面。尽管它很重要，但对PVM的调查一直是有限的。我们最近完成了对弓形虫PVM的蛋白质组分析，结果鉴定出123种蛋白质，其中70种似乎是新发现的。在这些新的蛋白质中，26个被注释为“假设的开放阅读框架”，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主题，表明它们确实是新的。虽然有几个似乎是弓形虫独有的，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。暗示着保守的功能。作为潜在定位于PVM的新蛋白质，PVM是一种仅在寄生虫感染背景下发现的细胞器，这些假想的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，我们利用最近开发的技术来表位、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。总而言之，这些研究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，它非常适合于RO_3供资机制。通过这项建议，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。 公共卫生相关性：弓形虫会在免疫受损的个体中引起严重疾病，是几种实验上不太容易驯化的寄生虫的模式生物。这项研究的目的是研究被鉴定为寄生虫液泡膜成分的真正新的蛋白质，寄生虫液泡膜是一种独特的和必需的寄生虫细胞器。我们预计，随着这项工作的完成，将开辟寄生虫生物学研究的新领域。

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.