Plasmodium cynomolgi as a model for P. vivax.

食蟹猴疟原虫作为间日疟原虫的模型。

• 批准号: 8177389
• 负责人: MARY R GALINSKI
• 金额: $ 30.8万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8177389
• 关键词: Address; Animal Model; Anopheles Genus; Applied Genetics; Area; Basic Science; Biochemical; Biochemistry; Biological; Biological Models; Biology; Blood; Cataloging; Catalogs; Caveolae; Cebidae; Cell membrane; Cells; Cellular biology; Cleaved cell; Color; Complex; Culicidae; Cytoplasm; Dependence; Development; Disease; Erythrocytes; Face; Future; Genetic; Genetic Transformation; Goals; Human; Immunology; In Vitro; Infection; Intervention; Investigation; Knowledge; Laboratories; Life Cycle Stages; Liver; Location; Macaca mulatta; Malaria; Membrane; Microscopy; Modality; Modeling; Morphology; Parasitemia; Parasites; Pathway interactions; Performance; Phylogenetic Analysis; Pilot Projects; Plasmodium cynomolgi; Plasmodium falciparum; Plasmodium vivax; Procedures; Proteins; Proteomics; Regulation; Research; Resources; Reticulocytes; Rodent; Sporozoites; Staging; Surface; System; Technology; Tertiary Protein Structure; Therapeutic; Time; Transfection; Vacuole; Vesicle; biological research; experience; feeding; in vivo; in vivo Model; interest; model development; novel; pathogen; programs; protein transport; research study; success; tool; trafficking; vaccine development

DESCRIPTION (provided by applicant): This R21 proposal focuses on developing in vivo and in vitro Plasmodium cynomolgi model systems to advance cell biological and biochemical research on P. vivax, a major widespread human malaria pathogen. In particular, the goal is to enable the routine application of transfection technologies that will address hypothesis-driven questions regarding the unique biology held in common between P. vivax and P. cynomolgi, which is not found in the other major human malaria, P. falciparum or any of the popular rodent malaria models. This biology includes, among many other differences, the development and reactivation of dormant liver-stage forms known as hypnozoites, and the synthesis and functioning of numerous caveolae vesicle complexes (CVCs) and other membrane structures in infected red blood cells (iRBCs). While P. vivax cannot be cultured continuously in vitro, to facilitate such experimentation, P. cynomolgi blood-stage parasites can be easily manipulated genetically in vivo and ex vivo from rhesus monkey infections where large quantities of parasites can be obtained as well as be adequately propagated and experimentally manipulated by in vitro culture for several days to weeks. The phylogenetic close kinship and nearly identical basic morphology and biology shared by these two species strongly support the proposed utility of P. cynomolgi model systems. No rodent malaria model or P. falciparum culture system offers any biology that mimics the unique biology of these species, and thus they cannot serve as model systems to forward this research. In Aim 1 we will focus on attaining transformed P. cynomolgi parasites that constitutively throughout the life-cycle or at specific developmental time points express single or dual-color fluorescent tags, as the first step for future studies aiming to identify, purify and investigate specific life cycle stages, with a primary interest at this time on the development and activation of the elusive hypnozoite stage. In Aim 2, transformed parasites will be developed to study the trafficking machinery, pathways and unique membrane structures produced by P. vivax and P. cynomolgi blood-stage parasites, which include the CVCs, extensive networks of cleft membranes and novel proteins catalogued by proteomics, immunochemical studies and microscopy. One specific question to be addressed is how and when the PHIST81-95 protein traffics from the parasite beyond the parasitophorous vacuole membrane to its RBC cytosolic location on the cytoplasmic face of the CVCs. For this, the Conditional Aggregation Domain (CAD) protein regulation system will be applied. Critically, P. cynomolgi parasites provide a superior model for the proposed studies, and development of this model will enable research on initial hypothesis-driven questions posed here and ongoing research to understand and define biological and biochemical targets of intervention for P. vivax. PUBLIC HEALTH RELEVANCE: The performance of research directly on the human malaria parasite Plasmodium vivax comes with major impediments. However, the closely related simian malaria species, P. cynomolgi has proven to be a valuable and faithful stand in as a model organism for P. vivax. This proposal aims to develop and apply genetic transformation technologies using P. cynomolgi ex vivo and in vivo model systems to capitalize on the potential of this species to reveal basic biological and biochemical knowledge relevant for identifying targets of intervention against P. vivax.

描述（由申请人提供）：该 R21 提案侧重于开发体内和体外食蟹猴疟原虫模型系统，以推进对间日疟原虫（一种主要广泛传播的人类疟疾病原体）的细胞生物学和生化研究。具体而言，目标是实现转染技术的常规应用，以解决有关间日疟原虫和食蟹猴疟原虫之间共同的独特生物学的假设驱动的问题，而这种生物学在其他主要人类疟疾、恶性疟原虫或任何流行的啮齿动物疟疾模型中都没有发现。这种生物学差异包括许多其他差异，包括被称为休眠子的休眠肝脏阶段形式的发育和重新激活，以及受感染红细胞 (iRBC) 中众多小窝囊泡复合物 (CVC) 和其他膜结构的合成和功能。虽然间日疟原虫不能在体外连续培养，但为了便于此类实验，食蟹猴血期寄生虫可以很容易地在体内和体外从恒河猴感染中进行遗传操作，在恒河猴感染中可以获得大量寄生虫，并通过体外培养数天至数周进行充分繁殖和实验操作。这两个物种所共有的系统发育密切的亲缘关系和几乎相同的基本形态和生物学强烈支持食蟹猴模型系统的拟议用途。没有啮齿动物疟疾模型或恶性疟原虫培养系统提供任何模仿这些物种独特生物学的生物学，因此它们不能作为模型系统来推进这项研究。在目标 1 中，我们将重点关注获得在整个生命周期或在特定发育时间点组成性表达单色或双色荧光标签的转化食蟹猴寄生虫，作为未来研究的第一步，旨在识别、纯化和研究特定生命周期阶段，目前主要关注难以捉摸的催眠阶段的发育和激活。在目标 2 中，将开发转化的寄生虫来研究间日疟原虫和食蟹猴血期寄生虫产生的运输机制、途径和独特的膜结构，其中包括 CVC、广泛的裂膜网络和通过蛋白质组学、免疫化学研究和显微镜分类的新型蛋白质。需要解决的一个具体问题是 PHIST81-95 蛋白如何以及何时从寄生虫穿过寄生液泡膜到达 CVC 细胞质表面上的红细胞胞质位置。为此，将应用条件聚集域（CAD）蛋白质调节系统。至关重要的是，食蟹猴寄生虫为拟议的研究提供了一个优越的模型，该模型的开发将使我们能够研究此处提出的最初假设驱动的问题，以及正在进行的研究，以了解和定义间日疟原虫干预的生物和生化目标。 公共卫生相关性：直接针对人类疟原虫间日疟原虫进行研究存在重大障碍。然而，与猿猴疟疾密切相关的物种食蟹猴疟原虫已被证明是间日疟原虫的有价值且忠实的模式生物。该提案旨在开发和应用食蟹猴离体和体内模型系统的遗传转化技术，以利用该物种的潜力来揭示与确定间日疟原虫干预目标相关的基本生物学和生化知识。