Mechanisms and functions of CRISPR-Casautoregulation in bacterial immunity and pathogenesis

CRISPR-Casautoregulation在细菌免疫和发病机制中的机制和功能

基本信息

批准号：
10624292
负责人：
Rachael Workman
金额：
$ 3.17万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-05-15 至 2023-11-10
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10624292
关键词：
Adaptive Immune System Address Affect Antibiotic Resistance Autoimmunity Bacteria Bacteriophages Binding Biochemical Biological Biological Assay Biology Campylobacter jejuni Cells Clustered Regularly Interspaced Short Palindromic Repeats Cues Cytolysis DNA Data Development Environment Exclusion Gene Expression Gene Silencing Genes Genetic Genetic Transcription Genome engineering Goals Growth Guide RNA Homeostasis Horizontal Gene Transfer Human Immune system Immunity Infection Knowledge Laboratories Mediating Memory Metabolic Microbial Biofilms Mobile Genetic Elements Molecular Molecular Genetics Neisseria meningitidis Operon Pathogenesis Pathway interactions Phase Physiological Plasmids Proteins RNA Regulation Repression Reproducibility Research Route Stimulus Streptococcus Group B Streptococcus pyogenes System Testing Untranslated RNA Variant Virulence Virulence Factors Work derepression environmental change experimental study gene repression human pathogen improved in vivo insight mortality new therapeutic target novel nuclease pathogenic bacteria pathogenic microbe prevent promoter resistance gene scaffold tool virulence gene

项目摘要

Project Summary The goal of my research is to investigate how bacterial CRISPR-Cas immune systems are regulated and how this regulation contributes to bacterial immunity and virulence. CRISPR-Cas systems protect bacteria from bacteriophages and other mobile genetic elements and thereby prevent cell lysis but also limit the potential for horizontal gene transfer, a major route for the dissemination of antibiotic-resistance genes. Despite all that is known about CRISPR-Cas biology from a mechanistic standpoint, much remains to be understood about how these systems function in their native bacterial hosts. In particular, we lack an understanding of the ways by which bacteria regulate CRISPR-Cas expression to maximize immunity while mitigating autoimmunity and the metabolic burden of constitutive system expression. Furthermore, Cas9 is itself a virulence factor in many human pathogens, including S. pyogenes, S. agalactiae, F. novicida, N. meningitidis, and C. jejuni although it is unclear how Cas9 contributes to pathogenesis. For these reasons, it is critical to understand whether and how CRISPR-Cas expression is regulated to prepare bacteria for an impending phage infection or for growth in a human host. To address this gap in knowledge, we performed a screen to identify regulators of CRISPR-Cas immunity. Interestingly, we discovered that trL, a noncoding RNA within the S. pyogenes CRISPR-Cas locus, is capable of folding into a natural single-guide RNA that directs Cas9 to transcriptionally silence the Cas operon promoter (Workman et al., 2020). While a trL deletion enhances Cas gene expression by ~50-fold and stimulates CRISPR-Cas immunity by 3000-fold, it remains unknown how trL de-repression occurs under physiological conditions. In this proposal, I will identify the conditions and genetic pathways that mediate trL de- repression and assess the impact of this regulation on bacterial immunity and virulence. We have obtained preliminary data demonstrating that CRISPR RNAs (crRNAs) and growth-phase specific cues modulate Cas9 expression; however, the mechanisms and consequences of Cas9 induction remain to be tested. In Aim 1, we test the hypothesis that crRNAs and trL form an integrated genetic circuit that controls CRISPR-Cas expression, providing a novel mechanism through which spacers, the molecular “memories” of infection, differentially affect immunity. In Aim 2, we investigate the physiological cues and genetic networks that cause Cas9 to accumulate in late stationary phase, and we probe whether this regulation affects immunity and virulence in S. pyogenes. The proposed studies will help us understand how pathogenic bacteria regulate CRISPR-Cas expression in order to survive in hostile environments. Finally, our work will inform the development of regulatable Cas9 tools and new therapeutic targets and strategies for human pathogens.

项目概要我的研究目标是研究细菌 CRISPR-Cas 免疫系统是如何被调节的以及如何这种调节有助于细菌免疫和毒力，从而保护细菌免受感染。噬菌体和其他可移动遗传元件，从而防止细胞裂解，但也限制了水平基因转移是抗生素抗性基因传播的主要途径。虽然从机制的角度了解了 CRISPR-Cas 生物学，但关于其如何发挥作用还有很多需要了解的地方。特别是，我们对这些系统在其天然细菌宿主中的运作方式缺乏了解。哪些细菌调节 CRISPR-Cas 表达以最大限度地提高免疫力，同时减轻自身免疫和此外，Cas9 本身就是许多毒力因子。人类病原体，包括化脓性链球菌、无乳链球菌、新凶杆菌、脑膜炎奈瑟菌和空肠弯曲菌，尽管它目前还不清楚 Cas9 如何促进发病机制，因此了解是否以及如何参与发病机制至关重要。如何调节 CRISPR-Cas 表达来为即将到来的噬菌体感染或在细菌中生长做好准备人类宿主。为了弥补这一知识空白，我们进行了筛选来鉴定 CRISPR-Cas 的调控因子隐含地，我们发现化脓性链球菌 CRISPR-Cas 位点内的非编码 RNA trL 是能够折叠成天然的单向导RNA，指导Cas9在转录上沉默Cas操纵子启动子（Workman et al., 2020），而 trL 缺失可将 Cas 基因表达增强约 50 倍。刺激 CRISPR-Cas 免疫力达 3000 倍，但 trL 去抑制如何发生仍不清楚在本提案中，我将确定介导 trL de- 的条件和遗传途径。我们已经获得了抑制并评估了这种调节对细菌免疫和毒力的影响。初步数据表明 CRISPR RNA (crRNA) 和生长期特异性信号可调节 Cas9 然而，Cas9 诱导的机制和后果仍有待测试。检验 crRNA 和 trL 形成控制 CRISPR-Cas 的集成遗传电路的假设表达，提供了一种新的机制，通过间隔区，感染的分子“记忆”，在目标 2 中，我们研究了导致免疫力的生理线索和遗传网络。 Cas9 在稳定期后期积累，我们探讨这种调节是否会影响免疫和拟议的研究将帮助我们了解致病菌如何调节。最后，我们的工作将为在恶劣环境中生存提供信息。开发可调节的 Cas9 工具以及人类病原体的新治疗靶点和策略。