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。在病毒学、分子生物学和计算方法方面的训练帮助我回答了各种各样的科学问题。这里提出的项目是为了让我从博士后过渡到学术环境中的独立研究者，继续我的追求，用创造性的方法进行高影响力的、冒险的研究，以了解宿主对致病性感染的反应。研究：先天免疫是指人体在接触入侵生物时抑制感染的初始反应。虽然检测病原体相关分子是一种古老的宿主防御形式，但如果功能失调，它可能导致自身免疫性疾病，影响超过2000万美国人。先天免疫反应是微生物感染的第一道防线，它是通过激活受体来识别病原感染的特征保守分子而启动的。近年来，人们发现了一种胞内DNA传感器STING （STimulator of INterferon Genes）。在病毒和细菌感染期间，STING对先天免疫反应至关重要，然而在出生时表现出STING过度激活的动物表现为炎症性自身免疫性疾病。虽然对STING激活后发生的下游信号事件已经很清楚，但对STING激活的机制知之甚少。为了解决这个问题，我提出了一种正交方法，利用果蝇来研究STING功能和刺激它的分子。这种方法将补充正在进行的哺乳动物系统中STING的研究。在Aim 1中，我们将确定人类STING和果蝇STING （dSTING）的相似之处，为dSTING是人类STING的真正祖先提供证据。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Aim 2中，我们将创建一个dSTING敲除蝇来研究dSTING在感染先天免疫应答中的作用。基因表达分析将用于从功能上确定与基因组救援蝇相比，dSTING的缺失如何导致先天免疫反应受损。Aim 3将利用下一代测序技术鉴定微生物感染过程中与STING结合的核酸，从而深入了解STING配体的特异性，这是一个潜在的治疗干预位点。总之，这些使用遗传可延展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以进一步实验，提高我们对STING功能的理解。本研究获得的信息将对先天免疫和自身免疫的发展产生广泛的影响，以治疗微生物感染和自身免疫性疾病。环境：这个项目是对我的导师格伦·巴伯博士正在进行的先天免疫研究的补充。这项工作还将与我的共同导师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，为我提供必要的培训，使我能够独立。迈阿密大学拥有一流的设施、设备和优秀的教师，他们通过使用令人兴奋的技术和动物模型来研究微生物学和免疫学。

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.