CRISPR/Cas systems in bacterial gene regulation and virulence

描述（由申请人提供）：最近已经描述了CRISPR/Cas系统介导细菌防御来自噬菌体或质粒的入侵外源核酸，它们靶向这些外源核酸以进行降解（1，2）。这些系统以前没有显示出靶向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模式（3）。Cas9与2种小RNA一起发挥作用，以抑制细胞内病原体新弗朗西斯菌中的内源性细菌脂蛋白（BLP）（3）。由于BLP通过Toll样受体2（TLR 2）（4，5）触发促炎性先天免疫应答，旨在对抗病原体，因此CRISPR/Cas介导的BLP抑制对于F. novicida抑制这种宿主反应，并且对体内毒力至关重要3，6）。有趣的是，我们的数据显示，当细菌处于含有TLR 2的巨噬细胞吞噬体中时，Cas9调节系统被激活（3，6）。在那里，细菌面临着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TLR 2的逃避。Cas9由至少63种细菌病原体（以及至少22种细菌）编码（3，8），并且我们已经证明了其在脑膜炎奈瑟氏球菌的毒力性状中的作用（3），而其他人最近已经显示出在空肠弯曲杆菌中的作用（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细菌与真核宿主的相互作用。由于Cas9靶向F. novicida是CRISPR/Cas介导的内源性基因调控的唯一目前已知的例子，我们将使用该模型来回答关于该系统的作用机制以及控制其诱导的参数的基本问题。这将有助于阐明Cas9和CRISPR/Cas生物学的基本原理。我们将研究N.脑膜炎，利用我们从F. novicida作为指南，可能有助于加强我们对两个系统中存在的核心组件的了解，并确定潜在的差异。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的细菌病原体如何导致疾病奠定框架。

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.