CRISPR/Cas systems in bacterial gene regulation and virulence

描述（由申请人提供）：CRISPR/Cas系统最近被描述为介导细菌防御来自噬菌体或质粒的入侵外来核酸，它们以降解为目标（1,2）。这些系统以前没有被证明靶向mRNA或控制内源性基因表达。我们证明了CRISPR/Cas蛋白Cas9靶向内源性mRNA，揭示了一种新的细菌RNA沉默机制和遗传调控范式(3)。Cas9与2个小rna一起作用，抑制细胞内病原体新弗朗西斯菌的内源性细菌脂蛋白（BLP）(3)。由于BLP通过toll样受体2 （TLR2）触发促炎先天免疫反应（4,5），旨在对抗病原体，因此CRISPR/ cas介导的BLP抑制对于新发假丝酵母抑制宿主反应至关重要，并且对体内毒力至关重要3,6)。有趣的是，我们的数据显示，当细菌处于巨噬细胞吞噬体中时，Cas9调控系统被激活，其中含有TLR2（3,6）。在那里，细菌面临许多诱导膜损伤的抗菌剂(7)，我们假设这是诱导Cas9系统的触发因素，导致TLR2的逃避。Cas9被至少63种细菌病原体（以及至少22种共栖细菌）编码（3,8），我们已经证明了它在脑膜炎奈瑟菌的毒力特性中起作用(3)，而最近其他研究表明它在空肠弯曲杆菌中起作用(9)。这些数据表明，Cas9系统广泛用于介导细菌病原体以及许多共生体与真核宿主的相互作用。由于Cas9靶向F. novicida中的BLP mRNA是目前已知的唯一CRISPR/Cas介导内源性基因调控的例子，我们将利用该模型来回答有关该系统的作用机制以及控制其诱导的参数的基本问题。这将导致对Cas9和CRISPR/Cas生物学基本原理的阐明。我们将研究脑膜炎奈瑟菌的Cas9调控系统，利用我们从新乳杆菌实验中获得的见解作为指导，可能有助于加强我们对两种系统中存在的核心成分的了解，以及识别潜在的差异。这项拟议的研究将对我们对Cas9、CRISPR/Cas系统、RNA沉默、遗传调控机制、细菌毒力和先天免疫逃避的理解产生持续而强大的影响，并为更广泛地了解不同细菌病原体如何引起疾病奠定框架。

DESCRIPTION (provided by applicant): CRISPR/Cas systems have recently been described to mediate bacterial defense against invading foreign nucleic acid derived from bacteriophages or plasmids, which they target for degradation (1, 2). These systems have not previously been shown to target mRNA or control endogenous gene expression. We demonstrated that the CRISPR/Cas protein Cas9 targets an endogenous mRNA, revealing a novel bacterial RNA silencing machinery and genetic regulatory paradigm (3). Cas9 functions with 2 small RNAs to repress an endogenous bacterial lipoprotein (BLP) in the intracellular pathogen Francisella novicida (3). Since BLPs trigger a proinflammatory innate immune response through Toll-like Receptor 2 (TLR2)(4, 5), aimed at combating pathogens, CRISPR/Cas-mediated repression of BLP is critical for F. novicida to dampen this host response and is critical for virulence in vivo 3, 6). Interestingly, our data show that the Cas9 regulatory system is activated when the bacteria are in the macrophage phagosome, which contains TLR2 (3, 6). There, the bacteria are confronted with numerous antimicrobials that induce membrane damage (7), which we hypothesize is the trigger for the induction of the Cas9 system, leading to evasion of TLR2. Cas9 is encoded by at least 63 bacterial pathogens (as well as at least 22 commensals)(3, 8), and we have demonstrated its role in virulence traits of Neisseria meningitidis (3), while others have recently shown a role in Campylobacter jejuni (9). These data suggest that the Cas9 system is broadly used to mediate the interaction of bacterial pathogens, as well as numerous commensals, with eukaryotic hosts. Since Cas9 targeting of a BLP mRNA in F. novicida is the only currently known example of CRISPR/Cas- mediated endogenous gene regulation, we will employ this model to answer fundamental questions about the mechanism of action of this system, as well as the parameters controlling its induction. This will lead to the elucidation of basic foundational principles governing Cas9 and CRISPR/Cas biology. We will study the Cas9 regulatory system in N. meningitidis, using the insights we gain from experiments with F. novicida as a guide, likely serving to both strengthen our knowledge of core components present in both systems, as well as identifying potential differences. The proposed research will have a sustained and powerful impact on our understanding of Cas9, CRISPR/Cas systems, RNA silencing, genetic regulatory mechanisms, bacterial virulence, and innate immune evasion, and lay the framework for a much broader knowledge of how diverse bacterial pathogens cause disease.