Functional genomic characterization of diverse bat innate immune mechanisms

项目总结 人类的健康与我们对蝙蝠的了解密不可分，因为蝙蝠是已知的或预测的 许多人畜共患病病毒前体源的蓄水池。不幸的是，我们仍然对蝙蝠免疫学知之甚少， 我们不知道是什么特征使它们能够在相同的病毒下存活，而这些病毒很容易杀死其他 像人类这样的宿主。蝙蝠是一个极其多样化的目，这一事实加剧了这个问题 因此，我们所了解的一种蝙蝠物种的免疫机制可能并不适用于 又一个。我们需要新的技术和技术来有效地探索浩瀚的未知宇宙 存在于不同蝙蝠身上的免疫机制。在此应用程序中，我们建议开发关键工具 需要从功能上描述蝙蝠免疫学的遗传基础。在第一个目标中，我们建议 将重组DNA合成和细胞工程的现代进展更全面地结合起来 研究已被认为在蝙蝠免疫动态平衡中起重要作用的同源基因的功能。 我们将重点研究胞质核酸传感途径中的关键节点--STING。我们 将对翼手目中十几种不同的刺直系物进行鉴定，以确定是否相同 整个订单存在监管机制，或者如果存在多个不同的监管机制。我们会 通过开发和应用“嵌合体”来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-蝙蝠叮咬嵌合体的文库来识别关键残基 这赋予了对信号进行蝙蝠特有的监管。在第二个目标中，我们将把重点转移到识别 蝙蝠体内存在着以前未被重视的免疫基因和免疫途径。我们将不偏不倚地执行这项工作 转座子诱变方法，这将应用于不同蝙蝠物种的细胞系。选择和 抗水疱性口炎病毒致细胞病变BAT细胞的序列测定 复制是狂犬病病毒的代用品，它将揭示使蝙蝠在感染后存活的先天免疫基因。这个 诱变细胞还将暴露于VSV-EboGP(VSV假型与埃博拉病毒糖蛋白)， 模仿埃博拉病毒进入细胞的机制。因此，只需很少的额外工作，相同的库就可以 被用来识别蝙蝠的先天免疫基因，这些基因对两种被认为利用蝙蝠的人畜共患病病毒具有抵抗力 作为动物的储藏室。这项工作将创造探索和理解 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理解 先天免疫机制。这些互补的方法跨越了广泛的领域，从功能 已知的先天免疫基因中个别氨基酸对整体重要性的询问 基因组水平，突出其他基因，这些基因应该是未来重点研究的主题。

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.