The Intracellular Pathogen Response Triggers Defense Against Co-evolved Pathogens

项目摘要 传染病对人类健康造成重大负担，上皮细胞处于最前线 可以在这些细胞内入侵和复制的致病微生物的攻击。除了……之外 病原体引起的腹泻和肺炎等疾病，防御通路激活不当 在上皮细胞中会导致炎症性疾病。因此，了解更多关于上皮防御的知识是至关重要的 对抗细胞内病原体。特别是，对微孢子虫门的防御知之甚少。 与真菌相关的胞内寄生虫，其中14种可感染人类并导致疾病，最常见的是 感染肠道上皮细胞。我们开发了一种方便的全动物模型来研究防御 通过表征线虫的自然肠道感染来对抗微孢子虫。我们的 长期目标是剖析上皮细胞防御共同进化的细胞内的机制。 像微孢子虫这样的病原体。弥合这一认识上的差距将为治疗 传染病和炎症性疾病。我们的中心假设是来自共同进化的病原体的攻击 导致宿主基因的扩展和多样化成为“物种特有的”，尽管这些基因可能 控制保守的免疫途径。这里的目的是描述物种特有的pals基因。 在线虫中，它扩展到39个PALS基因，而人类只有一个PALS基因。 几乎对PALS在任何系统中的蛋白质结构或生化功能一无所知，尽管它们 通过对线虫的序列分析和遗传学研究，发现该基因与泛素连接酶有关。 我们发现PALS-22和PALS-25是天然转录反应的关键调节者。 线虫体内的微孢子虫和病毒感染，我们称之为细胞内病原体反应或IPR。这个 IPR似乎定义了一个完整的生理程序，我们的正向遗传筛查发现PALS-22是 负调节器和PALS-25作为知识产权的正调节器。失去PALS-22导致增强 对细胞内病原体的免疫力，增加的RNA干扰，以及健康后果，如 由于发育迟缓，所有这些都依赖PALS-25。在具体目标1中，我们将描述 PALS-22和PALS-25调控的基因表达与免疫应答的关系 它们活动的组织，并定义它们所针对的微孢子虫的阶段。在具体目标2中，我们将 重组表达PALS-22和PALS-25蛋白以表征它们之间的相互作用以及它们的 用X射线结晶学和低温电子显微镜对其结构进行了分析。在具体目标3中，我们将分析其他伙伴的角色 蛋白质、RNAi机制和确定IPR的新调节者。这种方法是创新的，因为它专注于 调节一种新的上皮性免疫的未知蛋白质。这项拟议的研究意义重大， 因为它可能导致传染病和炎症性疾病的新疗法。

Project Summary Infectious diseases impose a significant burden on human health, with epithelial cells on the front lines of attack by disease-causing microbes that can invade and replicate inside of these cells. In addition to diseases such as diarrhea and pneumonia caused by pathogens, inappropriate activation of defense pathways in epithelial cells can lead to inflammatory disease. Therefore, it is critical learn more about epithelial defense against intracellular pathogens. In particular, little is known about defense against the Microsporidia phylum of fungal-related intracellular parasites, 14 of which can infect and cause disease in humans, most commonly infecting intestinal epithelial cells. We have developed a convenient whole-animal model for studying defense against microsporidia through characterization of natural intestinal infection in the nematode C. elegans. Our long-term goal is to dissect the mechanisms by which epithelial cells defend against co-evolved intracellular pathogens like microsporidia. Closing this gap in our understanding will provide new insights for treating infectious disease and inflammatory disorders. Our central hypothesis is that attack from co-evolved pathogens causes expansion and diversification of host genes to become ‘species-specific’ although these genes may control conserved immune pathways. The objective here is to characterize the species-specific pals gene family, which expanded to 39 pals genes in C. elegans, whereas there is only one pals gene in humans. Virtually nothing is known about PALS protein structure or biochemical function in any system, although they have been connected to ubiquitin ligases through sequence analysis and genetic studies in C. elegans. We found PALS-22 and PALS-25 to be key regulators of a common transcriptional response to natural microsporidia and viral infections in C. elegans that we call the Intracellular Pathogen Response or IPR. The IPR appears to define an entire physiological program and our forward genetic screens identified PALS-22 as a negative regulator and PALS-25 as a positive regulator of the IPR. Loss of PALS-22 leads to enhanced immunity against intracellular pathogens, increased RNA interference, as well as fitness consequences such as delayed development, all of which depend on PALS-25. In Specific Aim 1 we will characterize the relationship between gene expression and immune responses regulated by PALS-22 and PALS-25, identify the tissues where they act, and define the stage of microsporidia they target. In Specific Aim 2 we will recombinantly express PALS-22 and PALS-25 proteins to characterize their interaction, as well as their structure using X-ray crystallography and cryo EM. In Specific Aim 3 we will analyze the role of other PALS proteins, RNAi machinery and identify new regulators of the IPR. The approach is innovative as it focuses on uncharacterized proteins that regulate a novel form of epithelial immunity. The proposed research is significant, because it could lead to new treatments for infectious diseases and inflammatory disorders.