Conserved Immune Response to Sensing Cytosolic DNA

描述（由申请人提供）：在我的科学生涯中，我一直参与宿主-病原体相互作用的研究，重点是生物工程，微生物学，免疫学和疫苗开发。病毒学、分子生物学和计算方法的训练帮助我回答了各种各样的科学问题。这里提出的项目旨在弥合我从博士后到学术环境中的独立调查员的过渡，继续我的追求，使用创造性的方法来高影响力，冒险的研究，以了解宿主对病原体感染的反应。研究：先天免疫是指身体在接触入侵生物体时抑制感染的最初反应。虽然病原体相关分子的检测是一种古老的宿主防御形式，但如果功能失调，它可能会导致自身免疫性疾病，影响超过2000万美国人。先天性免疫应答是微生物感染的第一道防线，并且其通过识别作为病原性感染的标志的保守分子的受体的激活而启动。近年来，人们发现了一种细胞内DNA传感器STING（STimulator of Interferon Genes）。STING对病毒和细菌感染期间的先天免疫应答至关重要，然而在出生时表现出STING过度活化的动物显示炎性自身免疫性疾病。虽然STING激活后发生的下游信号传导事件已被很好地理解，但对负责STING激活的机制知之甚少。为了解决这个问题，我提出了一个正交的方法，利用果蝇研究STING功能和刺激它的分子。这种方法将补充正在进行的研究STING在哺乳动物系统。在目标1中，我们将确定人类STING和果蝇STING（dSTING）是如何相似的，提供证据证明dSTING是人类STING的真正祖先。我们将评估dSTING结合核酸的能力，并表征dSTING的结构域，以确定哪些结构域对核酸结合和细胞内定位至关重要。在目标2中，我们将创建dSTING敲除果蝇以研究dSTING在感染的先天免疫应答中的作用。基因表达分析将用于在功能上确定与基因组拯救果蝇相比，dSTING的缺失如何导致先天免疫应答受损。目标3将利用下一代测序来鉴定在微生物感染期间结合tdSTING的核酸，从而提供对STING配体的特异性的了解，STING是潜在治疗干预的位点。总之，这些使用遗传可塑性果蝇模型系统的研究将通过将结果外推到哺乳动物系统中进行进一步实验来提高我们对STING功能的理解。本研究获得的信息将对先天性和自身免疫性疾病产生广泛的影响，有助于开发治疗微生物感染和自身免疫性疾病的疗法。环境：这个项目补充了我的导师Glen Barber博士正在进行的先天免疫研究。这项工作也将与我的共同导师Grace Zhai博士建立新的合作关系。两位导师将在整个K99阶段提供宝贵的指导，为我提供独立所需的培训。迈阿密大学拥有一流的设施，设备和优秀的教师谁研究微生物学和免疫学，通过使用令人兴奋的技术和动物模型。

DESCRIPTION (provided by applicant): Throughout my scientific career I have been involved in the study of host-pathogen interactions, with an emphasis on bioengineering, microbiology, immunology, and vaccine development. Training in virology, molecular biology, and computational methods has helped me answer a wide variety of scientific questions. The project proposed here is designed to bridge my transition from a postdoctoral fellow to an independent investigator in an academic setting to continue my pursuit of using creative approaches to high-impact, adventurous research to understand the host response to pathogenic infection. Research: Innate immunity refers to the body's initial response to curb infection upon exposure to invading organisms. While the detection of pathogen-associated molecules is an ancient form of host defense, if dysfunctional, it can cause autoimmune disease, which affects over 20 million Americans. The innate immune response is the first line of defense to microbial infection, and it is initiated through the activation of receptors recognizing conserved molecules that are signature of pathogenic infection. Recently, STING (STimulator of INterferon Genes), an intracellular sensor of cytosolic DNA was discovered. STING is critical to the innate immune response during viral and bacterial infection, yet animals exhibiting STING hyperactivation at birth display inflammatory autoimmune disease. While the downstream signaling events occurring after STING activation are well understood, little is known about the mechanisms responsible for STING activation. To address this question, I propose an orthogonal approach utilizing Drosophila melanogaster to investigate STING function and the molecules that stimulate it. This approach will complement the ongoing studies of STING in a mammalian system. In Aim 1, we will determine how human STING and Drosophila STING (dSTING) are similar, providing evidence that dSTING is the bone fide ancestor of human STING. We will assess the capacity of dSTING to bind nucleic acids and characterize the domains of dSTING to determine which are critical for nucleic acid binding and intracellular localization. In Aim 2, we will create a dSTING knockout fly to study the role of dSTING in the innate immune response to infection. Gene expression analyses will be used to functionally determine how the loss of dSTING contributes to an impaired innate immune response, as compared to that of a genomic rescue fly. Aim 3 will utilize next-generation sequencing to identify the nucleic acids that bind t dSTING during microbial infection, providing insight to the specificity of the ligand for STING, a site for potential therapeutic intervention. Together, these studies using the genetically malleable Drosophila model system will improve our understanding of STING function through the extrapolation of the results into the mammalian system for further experimentation. The information gained in this study will have broad-ranging impacts in innate and autoimmunity towards to the development of therapeutics to treat microbial infection and autoimmune disorders. Environment: This project complements the ongoing research on innate immunity of my proposed mentor, Dr. Glen Barber. The work will also setup new collaborations with my co-mentor, Dr. Grace Zhai. Both mentors will provide invaluable mentorship throughout the K99 phase of the award, giving me the training necessary for the rise to independence. The University of Miami has superb facilities, equipment, and outstanding faculty who study microbiology and immunology, through the use of exciting techniques and animal models.