Functional genomic characterization of diverse bat innate immune mechanisms

不同蝙蝠先天免疫机制的功能基因组特征

• 批准号: 10589378
• 负责人: Anna Bruchez
• 金额: $ 24.15万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-11-07 至 2024-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10589378
• 关键词: 2019-nCoV; Address; Amino Acids; Animals; Biological; Biotechnology; COVID-19 pandemic; Cell Line; Cells; Cessation of life; Characteristics; Chimera organism; Chiroptera; Complement; Cytoplasm; DNA biosynthesis; Development; Ebola virus; Exposure to; Future; Genes; Genetic; Genetic Determinism; Glycoproteins; Goals; Health; Homeostasis; Homologous Protein; Human; Immune; Immune system; Immunity; Immunologics; Immunology; Individual; Infection; Interferons; Investigation; Knowledge; Learning; Lemurs; Libraries; Link; Mammals; Metabolic; Methodology; Methods; Modernization; Molecular; Mutagenesis; Mutagens; Natural Immunity; Nipah Virus; Nucleic Acids; Orthologous Gene; Pathogenicity; Pathway interactions; Phosphorylation; Proteins; Proxy; RNA; RNA Virus Infections; RNA Viruses; Rabies virus; Recombinant DNA; Regulation; Resistance; Rodent; Role; Scientist; Signal Transduction; Site; Societies; Technology; Temperature; Testing; Vesicular stomatitis Indiana virus; Viral Physiology; Virus; Virus Replication; Work; Zoonoses; base; causal variant; cellular engineering; functional genomics; future pandemic; genome-wide; improved; innate immune mechanisms; innate immune pathways; insight; mutation screening; new technology; prevent; resistance factors; resistance gene; technology/technique; tool; trait; whole genome; zoonotic spillover

PROJECT SUMMARY Human health can be inextricably linked to our understanding of bats, as bats are known or predicted reservoirs for the precursors of many zoonotic viruses. Unfortunately, we still know little about bat immunology, and we do not know what characteristics allow them to survive with the same viruses that can readily kill other hosts such as humans. This problem is exacerbated by the fact that bats are an incredibly diverse order of mammals, so what we learn about the immunological mechanisms of one bat species may not be true for another. We need new techniques and technologies to efficiently explore the vast unknown universe of immunological mechanisms that exist across diverse bats. In this application, we propose to develop key tools needed to functionally characterize the genetic underpinnings of bat immunology. In the first aim, we propose to pair modern advances in recombinant DNA synthesis and cell engineering to more comprehensively investigate the function of orthologous genes already proposed to be important in bat immune homeostasis. We will focus our investigations on STING, a key node in the cytoplasmic nucleic acid sensing pathway. We will characterize a dozen diverse STING orthologs across Chiroptera to determine whether the same regulatory mechanisms exist across the order, or if multiple distinct regulatory mechanisms exist. We will narrow down the genetic determinants underlying these mechanisms by developing and applying “chimeric mutational scanning”, where we will create libraries of human-bat STING chimeras to identify the key residues that confer bat-specific regulation to signaling. In the second aim, we will shift our focus to identifying previously unappreciated immune genes and pathways present in bats. We will perform this with an unbiased transposon mutagenesis approach, which will be applied to cell lines from diverse bat species. Selection and sequencing of mutagenized bat cells resistant to the cytopathic effects of Vesicular Stomatitis Virus (VSV) replication, a proxy for rabies virus, will reveal innate immune genes that allow bats to survive infection. The mutagenized cells will also be exposed to VSV-EboGP (VSV pseudotyped with the Ebola virus glycoprotein), mimicking the mechanism used by Ebola virus to enter cells. Thus, with little extra effort, the same library will be used to identify bat innate immune genes that confer resistance to two zoonotic viruses thought to use bats as an animal reservoir. This work will both create the technologies needed to explore and understand the unknown aspects of bat immunity, and generate biological findings directly improving our understanding of bat innate immune mechanisms. These complementary approaches span a wide field of scope, from the functional interrogation of the importances of individual amino acids within known innate immune genes, to the whole genome level to highlight additional genes that should be the subject of focused investigation in the future.

项目概要 人类健康与我们对蝙蝠的了解有着千丝万缕的联系，因为蝙蝠是已知的或预测的 不幸的是，我们对蝙蝠免疫学仍然知之甚少。 我们不知道什么特征使它们能够与可以轻易杀死其他病毒的相同病毒一起生存 蝙蝠是一个极其多样化的目，这一事实加剧了这个问题。 哺乳动物，因此我们对一种蝙蝠物种的免疫机制的了解可能并不适用于 另一方面，我们需要新的技术和技术来有效地探索广阔的未知宇宙。 不同蝙蝠之间存在的免疫机制在此应用中，我们建议开发关键工具。 在第一个目标中，我们提出需要从功能上表征蝙蝠免疫学的遗传基础。 将重组 DNA 合成和细胞工程的现代进步与更全面的 研究已经被认为对蝙蝠免疫稳态很重要的直系同源基因的功能。 我们将重点研究 STING，这是细胞质核酸传感途径的关键节点。 将表征翼手目中十几个不同的 STING 直向同源物，以确定是否相同 整个秩序都存在监管机制，或者如果存在多种不同的监管机制，我们也会这样做。 通过开发和应用“嵌合体”缩小这些机制背后的遗传决定因素 突变扫描”，我们将创建人-蝙蝠 STING 嵌合体文库来识别关键残基 赋予蝙蝠特定的信号调节能力在第二个目标中，我们将把重点转向识别。 我们将公正地进行这项研究。 转座子诱变方法，将应用于来自不同蝙蝠物种的细胞系。 对水泡性口炎病毒 (VSV) 细胞病变作用具有抵抗力的诱变蝙蝠细胞进行测序 复制是狂犬病病毒的代表，它将揭示使蝙蝠能够在感染中存活下来的先天免疫基因。 诱变细胞也将暴露于 VSV-EboGP（用埃博拉病毒糖蛋白假型化的 VSV）， 模仿埃博拉病毒进入细胞的机制，因此，只需很少的额外努力，相同的库就可以实现。 用于识别蝙蝠先天免疫基因，这些基因能够抵抗两种被认为利用蝙蝠的人畜共患病毒 作为动物储存库，这项工作将创造探索和理解所需的技术。 蝙蝠免疫的未知方面，并产生生物学发现，直接提高我们对蝙蝠的了解 这些互补的方法涵盖了广泛的领域，从功能性免疫机制到免疫机制。 询问已知先天免疫基因中单个氨基酸对整体的重要性 基因组水平以突出显示应成为未来重点研究主题的其他基因。