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

本申请是对NIAID RFA-AI-21-075的响应，标题为 《精选原生动物病原体的持续机制》，明确了对间日疟原虫的研究 催眠药作为研究对象。间日疟在全球范围内给人们造成了负担，而且 致病性比之前所认识到的要高。寄生虫的流行病学和临床影响是由潜伏的 肝脏阶段称为催产素，这是复发的血液期感染的来源。分子调控 直到最近，肝细胞中催眠素阶段的形成、持续和激活仍未得到研究。 主要是由于缺乏针对这种寄生虫的实验室工具。这一点在过去十年中发生了变化， 我们健壮的人肝嵌合小鼠模型(FRG huHep小鼠)的开发使详细的 催眠素的形成和持久性以及复发和体外原发的健壮性分析 感染的肝细胞模型。此外，我们最近与合作伙伴一起开发了基因定义的P. 用于催产素研究的间日疟原虫Chesson菌株，取代复杂的野外分离的衍生子孢子 感染。使用这些工具，此应用程序的目标是识别寄生虫的分子驱动因素 影响催产素形成和持续的催产素和宿主肝细胞因素 坚持不懈。我们将通过三个独立但相辅相成的目标来实现这些目标。在目标1中，我们将 生成和询问催产素基因表达数据，重点关注已知的调节因素 唾液腺子孢子中的类持久性。候选因子，如AP2转录因子， EIF2a/ik2/UIS2翻译抑制系统及SAP1和PUF应激颗粒基因存储系统 将在间日疟原虫催眠虫中接受全面检查。可能推动和维持的隐含因素 然后，将使用转基因表达和基因敲除来从功能上询问催眠体的持久性 在啮齿动物疟疾模型中过度表达。在目标2中，我们将建立一个间日疟原虫转基因系统，使用 间日疟原虫感染模型的建立及其报告基因表达的研究 寄生虫系，包括我们最近发现的表达催眠虫特异性标记GRP94的一株。我们 还将在功能上分析间日疟原虫在目标1至直接中表现出强持久性表型的因素 询问它们在催眠体形成和持久性中的作用。在目标3中，我们将识别和审问主机 在催产素感染的肝细胞中强调宿主防御和代谢的因素。主控因素 与催产素的形成和持久性有关，将使用siRNA介导法进行功能分析 体外催产素感染模型中的基因敲除和过表达技术。总而言之，此应用程序 将对寄生虫对肝脏阶段潜伏期的内在分子调控以及 可以进一步塑造潜伏期表型的寄主因素。这些发现可能会导致识别新的 以寄生虫为靶标和以宿主为靶标的治疗方法，防止疟疾感染复发。

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.