Novel activities and the T. gondii vacuolar membrane

描述（由申请方提供）：原生动物寄生虫弓形虫是一种重要的机会性寄生虫，在子宫内获得时，可导致免疫受损和严重出生缺陷的危及生命的感染。寄生虫生长在一个专门的隔间，寄生虫空泡（PV）内感染的宿主细胞。定义这个生态位的边界是PV膜（PVM），作为寄生虫和宿主之间的界面。尽管它的重要性，PVM的调查一直是有限的。我们最近完成了T.弓形虫PVM鉴定了123个蛋白质，其中70个是新的。在这些新的蛋白质中，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，表明它们确实是新的。虽然有几种似乎是弓形虫特有的，但其他人在其他不太容易处理的顶复门中也有类似的注释同源物，包括疟原虫和隐孢子虫属。提示功能保守。作为可能定位于PVM（仅在寄生虫感染的背景下发现的细胞器）的新型蛋白质，这些假设的ORF代表了可能用作药物靶标的潜在独特活性。在这项研究中，我们利用最近开发的技术，表位标签和定位的假设ORF的克隆验证的基因，并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供资机制。有了这个建议，我们的目标是执行这些孤儿基因的初步表征，以建立它们在寄生虫生物学中的功能。 公共卫生相关性：刚地弓形虫会导致免疫功能低下的个体严重的疾病，并且是几种不太容易实验处理的寄生虫的模式生物。本研究的目的是研究真正的新蛋白质，被确定为寄生虫空泡膜，一个独特的和必要的寄生虫细胞器的组成部分。我们期望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.