Parasite and host cell factors involved in the formation and persistence of Plasmodium vivax hypnozoites

寄生虫和宿主细胞因子参与间日疟原虫休眠子的形成和持续存在

• 批准号: 10564073
• 负责人: Stefan HI Kappe
• 金额: $ 82.34万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-09 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10564073
• 关键词: Antimalarials; Biological; Biology; Blood; Cells; Cellular biology; Clinical; Complex; Culicidae; Data; Development; Environment; Epidemiology; Erythrocytes; Event; Feeds; Frequencies; GRP94; Gene Deletion; Gene Expression; Gene Expression Profiling; Gene Transfer Techniques; Genes; Genome; Goals; Growth; Hepatocyte; Host Defense; Human; In Vitro; Infection; Integration Host Factors; Knock-out; Knowledge; Laboratories; Liver; Malaria; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Monkeys; Mus; National Institute of Allergy and Infectious Disease; Parasites; Pathogenicity; Persons; Phase; Phenotype; Plasmodium; Plasmodium vivax; Primary Infection; Process; Recombinants; Regulation; Relapse; Reporter Genes; Research; Rodent; Role; Saimiri; Salivary Glands; Shapes; Small Interfering RNA; Source; Sporozoites; System; Techniques; Transfection; Transgenic Organisms; Translational Repression; Vivax Malaria; Work; enhancer-binding protein AP-2; humanized mouse; insight; knock-down; liver infection; malaria infection; mouse model; overexpression; pathogen; relapse prevention; response; stress granule; symptom treatment; targeted treatment; tool; transcription factor; transgene expression; vector control

This application is in response to the NIAID RFA-AI-21-075 entitled “Identification and Characterization of Persistence Mechanisms of Select Protozoan Pathogens”, which explicitly states the study of P. vivax hypnozoites as a research objective. P. vivax malaria burdens people within a wide global range and is more pathogenic than previously appreciated. The parasite’s epidemiology and clinical impact is governed by latent liver stages called hypnozoites, which are the source of relapsing blood stage infection. The molecular regulation of hypnozoite stage formation, persistence and activation in hepatocytes has until recently remained unstudied, mainly due to the lack of laboratory tools for this parasite. This has changed of the past ten years with the development of our robust human liver-chimeric mouse model (FRG huHep mouse) that enables the detailed analysis of hypnozoite formation and persistence as well the occurrence of relapses and robust in vitro primary hepatocyte models of infection. In addition, with partners we have recently developed the genetically defined P. vivax Chesson strain for use in hypnozoite studies, replacing complex field-isolate derived sporozoites for infection. With these tools, the goals of this application are the identification of the parasite molecular drivers of hypnozoite formation and persistence and host hepatocyte factors that impact hypnozoite formation and persistence. We will achieve these goals through three independent but complementary aims. In Aim 1 we will generate and interrogate hypnozoite gene expression data with emphasis on factors that are known to regulate quasi-persistence in salivary gland sporozoites. Candidate factors such as AP2 transcription factors, the eIF2a/IK2/UIS2 translational repression system and the, SAP1 and PUF stress granule mRNA storage system will undergo comprehensive examination in P. vivax hypnozoites. Implicated factors that might drive and maintain hypnozoite persistence will then be functionally interrogated using transgene expression and knockout as well as overexpression in a rodent malaria model. In aim 2, we will establish a P. vivax transgenesis system using sporozoite transfection and the Saimiri monkey infection model to establish P. vivax reporter gene-expressing parasite lines, including a line that expresses a hypnozoite-specific marker GRP94, recently identified by us. We will also functionally analyze factors in P. vivax that show strong persistence phenotypes in aim 1 to directly interrogate their role in hypnozoite formation and persistence. In aim 3, we will identify and interrogate host factors with emphasis on host defenses and metabolism in hypnozoite-infected hepatocytes. Host factors that are associated with hypnozoite formation and persistence will be functionally analyzed using siRNA-mediated knockdown and overexpression techniques in an in vitro hypnozoite infection model. Together, this application will gain unprecedented insights into the parasite-intrinsic molecular regulation of liver stage latency as well as the host factors that can further shape the latency phenotype. The findings might lead to the identification of new parasite-targeted and host-targeted therapeutics that prevent relapsing malaria infection.

该应用是对NIAID RFA-AI-21-075的响应，标题为“识别和表征 精选原生动物病原体的持久性机制”，该病原体明确指出了对体内疟原虫的研究 催眠症作为研究目标。 P. Vivax Malaria Burnens在全球范围内的人，更多 致病性比以前所欣赏的。寄生虫的流行病学和临床影响受潜在的约束 肝脏阶段称为催眠症，这是复发性血期感染的来源。分子调节 直到最近才被研究 主要是由于该寄生虫缺乏实验室工具。这已经改变了过去十年 开发我们强大的人肝chimeric小鼠模型（FRG Huhep小鼠），该模型可以详细介绍 分析催眠岩的形成和持久性以及复发的发生和强大的体外原发性 感染的肝细胞模型。此外，与合作伙伴一起，我们最近开发了普遍定义的P。 可用于催眠症研究的Vivax Chesson菌株，取代复杂的场分离衍生的孢子岩作为 感染。使用这些工具，此应用的目标是识别寄生虫分子驱动因素 催眠岩形成和持久性和宿主肝细胞因子，影响催眠症状形成和 持久性。我们将通过三个独立但完全的目标来实现这些目标。在目标1中，我们将 生成和审问催眠基因表达数据，重点是已知的因素 唾液腺子孢子中的准持久性。候选因素，例如AP2转录因子， EIF2A/IK2/UIS2翻译表达系统以及SAP1和PUF应力颗粒mRNA存储系统 将在维瓦克斯催眠症状疟原虫中进行全面检查。暗示可能驱动和维护的因素 然后，使用变换表达式和基因敲除，催眠症持久性将在功能上进行询问 作为啮齿动物疟疾模型中的过表达。在AIM 2中，我们将使用 Sporozoite转化和Saimiri猴子感染模型，以建立P. Vivax报告基因表达基因 寄生虫线，包括表达催眠岩特异性标记GRP94的线，该线最近被我们确定。我们 还将在疟原虫中的功能分析因素，这些因素在AIM 1中显示出强的持久性表型直接 询问他们在催眠症状形成和持久性中的作用。在AIM 3中，我们将识别和询问主机 强调催眠症肝细胞中宿主防御和代谢的因素。主机因素 与催眠岩形成和持久性有关，将使用siRNA介导的功能分析 体外催眠症感染模型中的敲低和过表达技术。一起，这个应用程序 将获得对肝脏潜伏期的寄生虫 - 内部分子调节的前所未有的见解 可以进一步塑造潜伏表型的宿主因素。这些发现可能导致识别新的 以寄生虫为目标和宿主靶向的疗法可防止疟疾感染。