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.

本申请是对NIAID RFA-AI-21-075的响应，其标题为“ 选择原生动物病原体的持久性机制”，其中明确说明了间日疟原虫的研究 催眠虫作为研究对象。间日疟原虫疟疾在全球范围内给人们带来负担， 比以前认识到的致病性。寄生虫的流行病学和临床影响是由潜在的 肝脏阶段称为催眠虫，这是复发性血液阶段感染的来源。的分子调控 直到最近，肝细胞中催眠子阶段的形成、持续和激活仍未被研究， 主要是因为缺乏针对这种寄生虫的实验室工具。这在过去十年中发生了变化， 我们强大的人肝嵌合小鼠模型（FRG huHep小鼠）的开发， 催眠子形成和持久性以及复发和稳健的体外原发性的发生的分析 肝细胞感染模型。此外，我们最近与合作伙伴一起开发了基因定义的P。 间日疟原虫Chesson株用于催眠子研究，代替复杂的野外分离衍生的子孢子， 感染有了这些工具，本申请的目标是鉴定寄生虫的分子驱动因素， 催眠子形成和持久性以及影响催眠子形成宿主肝细胞因子， 坚持不懈我们将通过三个独立但互补的目标来实现这些目标。在目标1中， 生成和询问催眠子基因表达数据，重点是已知的调节因子 唾液腺子孢子中的准持久性。候选因子如AP 2转录因子， eIF 2a/IK 2/UIS 2翻译抑制系统和SAP 1和PUF应激颗粒mRNA储存系统 将在间日疟原虫催眠体中进行全面检查。可能驱动和维持 然后，还将使用转基因表达和敲除功能性地询问催眠子持久性 在啮齿动物疟疾模型中的过度表达。目的二：建立间日疟原虫转基因系统， 子孢子转染和Saimiri猴感染模型建立间日疟原虫报告基因表达 寄生虫系，包括我们最近鉴定的表达催眠虫特异性标记GRP 94的系。我们 还将对间日疟原虫中在目的1中表现出强持久性表型的因子进行功能分析， 询问它们在催眠体形成和持续中作用。在aim 3中，我们将识别并询问宿主 在催眠虫感染的肝细胞中强调宿主防御和代谢的因素。宿主因素， 与催眠子的形成和持久性相关，将使用siRNA介导的 体外催眠虫感染模型中的敲减和过表达技术。总之，这个应用程序 将获得前所未有的见解寄生虫内在的分子调节肝脏阶段的潜伏期，以及 可以进一步塑造潜伏期表型的宿主因素。这些发现可能会导致识别新的 寄生虫靶向和宿主靶向治疗，预防复发性疟疾感染。