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

项目摘要 我的研究目标是调查细菌CRISPR-CAS免疫系统是如何调节的，以及如何 这一调节有助于细菌免疫和毒力。CRISPR-CAS系统保护细菌免受 噬菌体和其他可移动的遗传元件，从而防止细胞裂解，但也限制了 水平基因转移，是抗生素耐药基因传播的主要途径。尽管如此， 从机械论的角度了解CRISPR-CAS生物学，还有很多事情需要了解 这些系统在它们的天然细菌宿主中发挥作用。尤其是，我们缺乏对通过 哪些细菌调节CRISPR-Cas的表达以最大限度地提高免疫力，同时降低自身免疫力 构成系统表达的代谢负担。此外，Cas9本身在许多情况下都是一个毒力因素 人类病原体，包括化脓性链球菌、无乳链球菌、新城疫杆菌、脑膜炎奈瑟氏菌和空肠弯曲杆菌。 目前尚不清楚Cas9在发病机制中的作用。出于这些原因，关键是要了解是否和 CRISPR-Cas的表达如何为即将到来的噬菌体感染或在 一个人类的主人。 为了解决这一知识差距，我们进行了一次筛查，以确定CRISPR-CAS的调节因子 豁免权。有趣的是，我们发现化脓性链球菌CRISPR-Cas基因座中的非编码RNA Trl是 能够折叠成自然的单引导RNA，指导Cas9转录沉默Cas操纵子 推动者(Workman等人，2020年)。而Trl缺失可使Cas基因的表达增加约50倍 刺激CRISPR-Cas免疫3000倍，目前尚不清楚Trl是如何在 生理条件。在这项提案中，我将确定介导Trl de-De的条件和遗传途径 抑制并评估这一调节对细菌免疫和毒力的影响。我们已经获得了 初步数据表明，CRISPR RNAs(CrRNAs)和生长阶段特定信号调节Cas9 然而，Cas9诱导的机制和结果仍有待测试。在目标1中，我们 验证crRNAs和trl形成控制CRISPR-CAS的完整遗传回路的假设 表达，提供了一种新的机制，通过这种机制，间隔区，感染的分子“记忆”， 不同地影响免疫力。在目标2中，我们调查了生理线索和遗传网络 Cas9在稳定期晚期蓄积，我们探讨了这一调节是否影响免疫和 化脓性链球菌的致病力。拟议的研究将帮助我们了解病原菌是如何调节 CRISPR-Cas的表达是为了在恶劣的环境中生存。最后，我们的工作将告知 开发可调节的Cas9工具以及针对人类病原体的新治疗靶点和策略。

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.