Role of Autophagy in Malaria Sporozoite Differentiation

 描述(由申请人提供)：疟原虫在其脊椎动物宿主和蚊子媒介之间循环时会遇到各种情况。为了适应这些不同的环境，寄生虫需要彻底改变其形态和新陈代谢。寄生虫在肝脏细胞内成功发育的一个关键是将细长的子孢子转化为圆形滋养体，这一过程为寄生虫在肝细胞中的复制做好了准备。人们对这种表型转化所涉及的生物系统知之甚少。我们发表的工作概述了转化子孢子会排出环境中的细胞器，如微线虫，这些细胞器是宿主细胞入侵所需的，但对寄生虫的复制无用。我们发现，寄生虫自噬机制的ATG8结合系统在子孢子分化过程中上调，表明在转换过程中激活了一个类似自噬的过程。我们的初步数据显示，寄生虫将微线虫隔离到由寄生虫ATG8的存在定义的自噬体内。我们提供的形态证据表明，PbATG8标记的结构与高尔基体蛋白PbGRASP结合，并与内体多囊体结合形成PbVPS4和PbGRASP阳性的自噬间隔，或双体。在真核细胞中，GRAP促进双体与质膜的融合，从而在细胞外释放双体内容物。这项提案的总体目标是提供一幅详细的图景，说明子孢子转化为肝脏形式期间与自噬相关的途径的潜在机制。我们的假设模型强调了肝内疟原虫内、外和自噬系统之间的合作，以促进微线体的胞吐。具体目标1将获得自噬小体成熟的实时视图和细胞外微核处理的路线。特定目的2将分析疟原虫ATG8条件敲除寄生虫的表型特征，以评估寄生虫ATG8结合系统对微线体胞吐的重要性。具体目标3将通过产生缺乏GRAP表达的寄生虫来研究疟原虫GRAP在分泌型自噬过程中的作用。虽然针对肝脏阶段的新疗法的开发有可能阻止疟疾感染的发生，但必须做出广泛的努力 部署以更好地了解在受感染肝脏中发生的事件。拟议的研究结果可能确定细胞器重塑途径的独特和基本成分，这些成分可以作为药物治疗的有效靶点。

 DESCRIPTION (provided by applicant): Plasmodium parasites encounter diverse conditions as they cycle between their vertebrate host and the mosquito vector. Adaptation to these distinct environments requires the parasite to drastically change its morphology and metabolism. A key to the parasite's successful intracellular development in the liver is the conversion of the elongated sporozoite to the round trophozoite, a process that prepares the parasite for the replication in hepatocytes. Very little is known about the biological systems involved in this phenotypic transformation. Our published work has outlined that converting sporozoites expel into their environment organelles such as micronemes that are needed for host cell invasion but useless for parasite replication. We found that the ATG8-conjugation system of the parasite's autophagy machinery is upregulated during sporozoite differentiation, suggesting that an autophagy-like process is activated during conversion. Our preliminary data reveal that the parasites sequester micronemes into autophagosomal compartments defined by the presence of parasite ATG8. We provide morphological evidence that PbATG8-labeled structures associate with the Golgi protein PbGRASP and combine with endosomal multivesicular bodies to form PbVPS4- and PbGRASP-positive autophagic compartments, or amphisomes. In eukaryotic cells, GRASP promotes the fusion of amphisomes with the plasma membrane to release the amphisomal content extracellularly. The overall goal of this proposal is to provide a detailed picture of the mechanisms underlying autophagy-related pathways in sporozoites during their conversion into liver forms. Our hypothetical model highlights a cooperation between the endocytic-exocytic and autophagic systems in intrahepatic Plasmodium to promote microneme exocytosis. Specific Aim 1 will obtain a real-time view of autophagosome maturation and itinerary for extracellular microneme disposal. Specific Aim 2 will analyze the phenotypic traits of Plasmodium Atg8 conditional knockout parasites to evaluate the importance of the parasite ATG8-conjugation system for microneme exocytosis. Specific Aim 3 will investigate the contribution of Plasmodium GRASP to the process of secretory autophagy by generating parasites lacking GRASP expression. While the development of new therapeutics targeting the liver stage has the potential to arrest the onset of a malaria infection, extensive efforts must be deployed to better understand the events that take place in the infected liver. The results of the proposed studies are likely to identify unique and essential components of the organelle remodeling pathways that can serve as potent targets for pharmacologic therapy.