Plasmodium cynomolgi as a model for P. vivax.

描述(申请人提供)：这份R21提案侧重于开发体内和体外食蟹猴疟原虫模型系统，以推进间日疟原虫的细胞生物学和生化研究，间日疟原虫是人类主要的流行疟疾病原体。特别是，目标是使转基因技术的常规应用能够解决假说驱动的问题，这些问题涉及间日疟原虫和食蟹猴之间共有的独特生物学，这种独特的生物学在其他主要的人类疟疾、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。这种生物学包括许多差异，包括休眠的肝期形式的发展和重新激活称为催眠体，以及受感染的红细胞(IRBC)中大量小窝囊泡复合体(CVCs)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，但为了便于进行这样的实验，食蟹猴血液期寄生虫可以很容易地在体内和体外通过遗传操作从恒河猴感染中获得大量寄生虫，并通过体外培养进行充分繁殖和实验操作数天至数周。这两个物种在系统发育上的密切亲缘关系以及几乎相同的基本形态和生物学特征有力地支持了食蟹猴模型系统的提出的实用性。没有任何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，因此它们不能作为推动这项研究的模型系统。在目标1中，我们将专注于获得在整个生命周期或在特定发育时间点结构性地表达单色或双色荧光标记的食蟹猴寄生虫，作为未来旨在识别、纯化和调查特定生命周期阶段的研究的第一步，目前主要关注难以捉摸的催眠体阶段的发育和激活。在目标2中，将开发转化寄生虫，以研究间日疟原虫和食蟹猴血液期寄生虫产生的转运机制、途径和独特的膜结构，包括CVCs、广泛的裂膜网络和蛋白质组学、免疫化学研究和显微镜分类的新蛋白。一个需要解决的具体问题是PHIST81-95蛋白是如何以及何时从寄生虫通过寄生虫的液泡膜运输到它在CVCs细胞质表面的红细胞胞浆位置。为此，将应用条件聚集域(CAD)蛋白质调控系统。重要的是，食蟹猴寄生虫为拟议的研究提供了一个更好的模型，该模型的发展将使对本文提出的初始假设驱动的问题的研究和正在进行的研究能够理解和定义干预间日疟原虫的生物学和生化目标。 公共卫生相关性：直接对人类疟疾寄生虫间日疟原虫进行研究会遇到重大障碍。然而，密切相关的猿猴疟疾物种食蟹猴已被证明是间日疟原虫的有价值和忠实的模式生物。这项建议旨在开发和应用利用食蟹猴体内和体外模型系统的遗传转化技术，以充分利用该物种的潜力，揭示与识别干预间日疟原虫靶标相关的基本生物学和生化知识。

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.