Mechanistic Characterization of CRISPR-Cas Complexes that Mediate Pathogenicity in the Bacterium Francisella tularensis novicida (Rajan)

介导土拉弗朗西斯菌 (Rajan) 致病性的 CRISPR-Cas 复合物的机制表征

• 批准号: 9360238
• 负责人: Rakhi Rajan
• 金额: $ 21.48万
• 依托单位: UNIVERSITY OF OKLAHOMA
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9360238
• 关键词: Antimicrobial Resistance; Archaea; BCAR1 gene; Bacteria; Bacterial Drug Resistance; Bacterial Infections; Bacterial Physiology; Bacteriophages; Behavior; Biochemical; Biological Assay; Biology; Campylobacter jejuni; Cell physiology; Cell surface; Cells; Centers of Research Excellence; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complex; Crystallization; DNA; DNA Probes; Data; Development; Dimensions; Enzymes; Exhibits; Foundations; Francisella tularensis; Future; Genes; Genetic; Genome; Goals; Health; Horizontal Gene Transfer; Human; Immune response; Immune system; In Vitro; Infection; Inherited; Intervention; Ions; Knowledge; Life Cycle Stages; Lipoproteins; Mediating; Memory; Messenger RNA; Microbial Antibiotic Resistance; Molecular Conformation; Multi-Drug Resistance; Neisseria meningitidis; Nucleic Acids; Oklahoma; Organism; Orthologous Gene; Pathogenicity; Physiological; Predatory Behavior; Production; Proteins; RNA; Recruitment Activity; Research; Resistance development; Resistance to infection; Roentgen Rays; Role; Staphylococcus aureus; Structure; System; Therapeutic; Transcript; Virulence; Work; X-Ray Crystallography; antimicrobial; bacterial resistance; base; combat; drug resistant bacteria; ds-DNA; immunogenic; in vivo; innovation; mRNA Transcript Degradation; novel; novel therapeutics; nuclease; pathogenic bacteria; plasmid DNA; protein complex; ribonuclease E; scaffold; structural biology; tool

Project Summary (Rajan Project) CRISPR-Cas systems are comprised of RNA-protein complexes that inactivate foreign DNA or RNA entering a bacterial or archaeal cell by producing sequence specific cleavage in the intruding DNA or RNA. CRISPR was given the status of a “bacterial/archaeal immune system” because of its ability to protect against recurring phage infections, based on genetic memory retained from the past infections. The RNA-based DNA targeting function of CRISPR-Cas systems is widely used for gene editing and has promise for the treatment of inherited and infectious diseases. One of the less characterized aspects of CRISPR-Cas systems is their role in alternate functions in bacteria other than phage predation. For example, CRISPR-Cas systems have been shown to enhance bacterial pathogenicity in Francisella tularensis (Ft) novicida, Neisseria meningitidis, and Campylobacter jejuni. Paradoxically, in highly drug resistant bacteria such as Staphylococcus aureus and Francisella tularensis, components of the CRISPR-Cas systems are inactivated possibly to capitalize on the benefits of horizontal gene transfer. It is critical that we understand how bacteria turn on and off the benefits of CRISPR-Cas systems. The proposed work aims to characterize the biochemical, structural, and functional mechanisms by which the CRISPR-Cas complexes of Ft. novicida enhance bacterial pathogenicity by down- regulating a bacterial lipoprotein (BLP) essential for host recognition and immune response. A non-canonical, RNA-independent, Mn2+-specific DNA cleavage activity for Cas9 and Cpf1 (two Ft. novicida Cas proteins) was observed recently in the PL's lab. The structural and conformational basis by which Cas9 and Cpf1 differentiate RNA-DNA, RNA-RNA, and DNA-Mn2+ as their substrates will be further characterized using a combination of in vitro and in vivo assays, X-ray crystallography, and Small Angle X-ray Scattering. The results from the proposed studies will identify the nuclease involved in blp mRNA degradation and the mechanistic aspects of how Cas9 associates with other cellular components for regulating bacterial pathogenicity. The basis of substrate recognition by Cas9 and Cpf1 will provide mechanistic details of a previously unknown activity of these proteins and how these enzymes perform RNA-independent DNA cleavage. The results will provide the basis of future studies on characterizing CRISPR-Cas mediated pathogenicity in other bacteria and the physiological relevance of RNA-independent, Mn2+-dependent DNA cleavage in bacterial physiology. This information can be used to develop innovative strategies for combating microbial antibiotic resistance and development of anti-microbials.

项目概要（Rajan 项目） CRISPR-Cas系统由RNA-蛋白质复合物组成，可灭活外来DNA或RNA进入 通过在侵入的 DNA 或 RNA 中产生序列特异性切割来攻击细菌或古细菌细胞。 考虑到“细菌/古菌免疫系统”的地位，因为它能够防止复发 噬菌体感染，基于过去感染中保留的遗传记忆。 CRISPR-Cas系统的功能广泛用于基因编辑，并有望用于遗传治疗 CRISPR-Cas 系统较少被表征的方面之一是它们在疾病和传染病中的作用。 细菌中除了噬菌体捕食之外还有其他功能，例如 CRISPR-Cas 系统。 显示可增强土拉弗朗西斯菌 (Ft) 新杀者、脑膜炎奈瑟菌和 矛盾的是，在金黄色葡萄球菌等高度耐药的细菌中。 土拉弗朗西斯菌，CRISPR-Cas 系统的组件被灭活，可能是为了利用 水平基因转移的好处至关重要，我们了解细菌如何开启和关闭水平基因转移的好处。 拟议的工作旨在表征 CRISPR-Cas 系统的生化、结构和功能。 Ft novicida 的 CRISPR-Cas 复合物通过下调增强细菌致病性的机制。 调节宿主识别和免疫反应所必需的细菌脂蛋白（BLP）。 Cas9 和 Cpf1（两种 Ft. novicida Cas 蛋白）的不依赖于 RNA 的 Mn2+ 特异性 DNA 切割活性为 最近在 PL 实验室观察到 Cas9 和 Cpf1 的结构和构象基础。 区分 RNA-DNA、RNA-RNA 和 DNA-Mn2+，因为它们的底物将使用 结合体外和体内测定、X 射线晶体学和小角 X 射线散射结果。 从拟议的研究中将确定参与 blp mRNA 降解的核酸酶及其机制 Cas9 如何与其他细胞成分结合来调节细菌致病性。 Cas9 和 Cpf1 底物识别的基础将提供以前未知的机制细节 这些蛋白质的活性以及这些酶如何进行不依赖于 RNA 的 DNA 切割。 为未来研究其他细菌中 CRISPR-Cas 介导的致病性特征提供基础 细菌生理学中不依赖 RNA、依赖 Mn2+ 的 DNA 切割的生理相关性。 信息可用于制定对抗微生物抗生素耐药性的创新策略， 抗微生物药物的开发。