EAGER: Prokaryotic Adaptive Immunity and the CRISPR Associated Complex for Antiviral Defense (CASCADE)

哺乳动物和其他真核生物的获得性免疫系统已经知道很多年了，但单细胞细菌和古生菌也具有获得性免疫系统的事实直到最近才得到证实。这种被称为CRISPR/CA的原核生物适应性免疫系统所依据的分子机制与真核生物所使用的机制有根本的不同。该项目旨在通过研究这些蛋白质如何识别入侵病毒并终止病毒感染，来研究构成CRISPR/Cas系统的基因和蛋白质机制的功能。这种方法包括一种称为X射线结晶学的结构技术，在这种技术中，CRISPR/Cas蛋白质被分析以确定其三维结构。然后研究这些结构，以寻找这些蛋白质如何向病毒传递免疫力的线索，否则这些病毒可能会感染并杀死具有CRISPR/Cas系统的细菌和古生菌。由于几个原因，硫叶藻被选为这些研究的模式生物。首先，这种生物是一种嗜热的古生物，生活在接近200华氏度的地方，比如黄石国家公园的温泉。它在高温环境中存活的能力是由于其非常稳定的蛋白质在这些极端温度下不会失活。这种耐热蛋白质更容易研究，而且已经证明，这个系统与大肠杆菌等细菌的系统有很大的相似之处。因此，在研究热稳定性CRISPR/CAS系统的同时，也有可能了解其他生物体中的CRISPR/CAS。其次，解冻沙门氏菌也令人感兴趣，因为它是嗜热病毒的宿主，研究这些病毒将有助于回答病毒学中的基本问题。这些问题包括：什么是病毒？它们已经存在多久了？几十亿年？他们是从哪里来的？随着时间的推移，病毒是如何进化的？病毒对包括高等生物在内的宿主的进化有何贡献？了解CRISPR/CAS与了解这些嗜热病毒直接相关。这项拟议的工作将对我们理解单细胞生物体中的病毒和病毒与宿主的相互作用做出开创性的贡献，这一研究领域有望产生许多工业和生物技术应用。此外，劳伦斯实验室是蒙大拿州立大学热生物学研究所(TBI)的成员。因此，我们正在进行的工作的结果通过TBI外展计划向公众传达。这些面向K-12、部落学院、其他本科院校和普通公众的外展工作将继续下去。此外，TBI还与国家公园管理局/黄石国家公园合作，向公众宣传，活动包括准备和审查供公园内公开展示的材料。公园系统和普通公众一样，越来越意识到居住在这些世界著名的热特征中的独特微生物，并努力增加公园内描述这些微生物的教育标志。还将拟议的研究与教育目标紧密结合起来。高中、本科生、博士和博士后级别的学生和教师将接受生物化学、结构生物学和热生物学方面的重要培训。积极鼓励代表性不足的群体，特别是妇女和美洲土著人的参与。

Adaptive immune systems in mammals and other eukaryotes have been known for many years, but the fact that single celled bacteria and archaea also have adaptive immune systems has only recently been established. The molecular machinery that underlies this prokaryotic adaptive immune system, known as CRISPR/Cas, is fundamentally different than that employed by eukaryotes. This project seeks to investigate the functions of the genes and protein machinery that constitute the CRISPR/Cas system, by studying how these proteins recognize invading viruses and terminate the viral infection. The approach includes a structural technique known as X-ray crystallography, in which the CRISPR/Cas proteins are analyzed to determine their three dimensional structure. These structures are then studied for clues about how these proteins impart immunity to viruses that might otherwise infect and kill the bacteria and archaea that have the CRISPR/Cas system. Sulfolobus solfataricus has been chosen as the model organism for these studies for several reasons. First, this organism is a thermophilic archaeal organism that lives at temperatures near 200 degrees Fahrenheit in places like the hot springs of Yellowstone National Park. Its ability to survive in a high temperature environment is due to its very stable proteins that are not deactivated at these extreme temperatures. Such thermostable proteins can be easier to study, and it has already been shown that this system has significant similarities to that of bacteria like E. coli. Thus, while studying a heat stable CRISPR/Cas system, it is possible to learn about CRISPR/Cas in other organisms as well. Second, S. solfataricus is also of interest because it is a host for hyperthermophilic viruses, and studying these viruses will help answer fundamental questions in virology. These include questions such as: What is a virus? How long have they been around; billions of years? Where did they come from? How have viruses evolved over time? How have viruses contributed to the evolution of their hosts, including higher organisms? Understanding CRISPR/Cas is directly relevant to understanding these hyperthermophilic viruses.Broader Impacts. The proposed work will make seminal contributions to our understanding of viruses and virus-host interactions in single celled organisms, a research area that is expected to give rise to a number of industrial and biotechnology applications. In addition, the Lawrence laboratory is a member of the Montana State University Thermal Biology Institute (TBI). As such, the results of our ongoing work is communicated to the general public by TBI outreach programs. These outreach efforts to K-12, tribal colleges, other undergraduate institutions, and the general public will continue. In addition, TBI also partners with the National Park Service /Yellowstone National Park in outreach to the general public, with activities that include preparation and review of materials for public presentation within the Park. The Park System is increasingly aware, as is the general public, of the unique microbial organisms inhabiting these world famous thermal features, and has worked to increase the educational signage within the park that describes these organisms. There is also strong integration of the proposed research with educational goals. Students and teachers at the high school, undergraduate, doctoral and post-doctoral levels will receive significant training in biochemistry, structural biology and thermal biology. The involvement of under represented groups, particularly women and Native Americans, is actively encouraged.