Molecular mechanisms of bacterial immune signaling through DNA damage

项目摘要 DNA损伤介导的细菌免疫信号分子机制 数以万计的细菌基因组序列的可用性，加上新的生物信息学工具， 对细菌基因组组织的新认识，使发现和实验 鉴定了细菌中的几十种抗噬菌体和抗质粒防御系统。由于典型 细菌基因组编码3-6种不同的防御系统，关键问题是这些系统是否以及如何能够 协调他们的活动以协同对抗感染。在以前关于广泛而多样的 CBASS（基于环磷脂的抗噬菌体信号系统）防御系统，我们确定了两个 转录调节因子- CapW和双蛋白CapH+CapP系统-促进CBASS基因 表达对DNA损伤的反应。总的来说，CapW和CapH+CapP与约10%的 CBASS系统，并且还发现邻近广泛的已知和预测的细菌防御系统。 包括Pycsar、DISARM和BREX。这些发现表明，CapW和CapH+CapP可能 介导抗病毒防御的激活，以响应细胞应激、DNA损伤的通用信号。来，我来 首先鉴定在DNA损伤时结合并激活CapW的小分子或核酸配体。我会 用X射线晶体学对CapW结合其靶DNA及其配体的联合收割机进行生物化学测定 表征由配体施加的构象变化以控制CapW-DNA结合。这项工作将 建立一种广泛存在的细菌转录因子CapW的机制。接下来，我将测试这个想法， CapW和CapH+CapP通过感知DNA损伤介导抗病毒防御系统之间的合作。 具体来说，我们假设DNA靶向免疫系统如限制修饰和CRISPR-Cas 产生DNA损伤，由CapW或CapH+CapP感知，以激活二级防御系统（CBASS 或其他）以加强防御反应。我将系统地测试这个模型， 限制修饰系统和CapW-或CapH+ CapP-相关的CBASS系统，以确定是否 这些系统的组合产生协同抗病毒免疫。另外，我会检测DNA 损伤感知在防御系统协同中发挥作用，使用基于结构的突变， 消除DNA损伤传感的CapP。这些实验将揭示 以及DNA损伤传感器在介导细菌防御系统协同作用中的作用。的 这些发现有可能建立一个新的范式，其中DNA靶向防御系统构成了一个新的防御系统。 抗病毒的第一道防线，DNA损伤激活系统构成了第二道防线， 正交机制因此，这项工作将不会孤立地看待细菌防御系统， 确定它们如何合作组成一个全面的细菌“免疫系统”。

PROJECT SUMMARY Molecular mechanisms of bacterial immune signaling through DNA damage The availability of tens of thousands of bacterial genome sequences, plus new bioinformatics tools and new understanding of bacterial genome organization, has enabled the discovery and experimental characterization of dozens of anti-bacteriophage and anti-plasmid defense systems in bacteria. Since a typical bacterial genome encodes 3-6 distinct defense systems, a key question is whether and how these systems can coordinate their activities to synergistically fight an infection. In prior work on the widespread and diverse CBASS (Cyclic oligonucleotide-Based Anti-phage Signaling System) defense systems, we identified two transcriptional regulators – CapW and the two-protein CapH+CapP system – that boost CBASS gene expression in response to DNA damage. Together, CapW and CapH+CapP are associated with ~10% of CBASS systems, and are also found adjacent to a broad range of known and predicted bacterial defense systems including Pycsar, DISARM, and BREX. These findings suggest that CapW and CapH+CapP may mediate activation of antiviral defense in response to a universal signal of cell stress, DNA damage. Here, I will first identify the small-molecule or nucleic acid ligand that binds and activates CapW upon DNA damage. I will combine biochemical assays for CapW binding to both its target DNA and its ligand with x-ray crystallography to characterize the conformational changes imposed by the ligand to control CapW-DNA binding. This work will establish a mechanism for CapW, a widespread bacterial transcription factor. Next, I will test the idea that CapW and CapH+CapP mediate cooperation between antiviral defense systems by sensing DNA damage. Specifically, we hypothesize that DNA-targeting immune systems like restriction-modification and CRISPR-Cas create DNA damage that is sensed by CapW or CapH+CapP to activate a secondary defense system (CBASS or others) to reinforce the defensive response. I will systematically test this model by infecting cells encoding both a restriction-modification system and a CapW- or CapH+CapP-associated CBASS system to determine if the combination of these systems yields synergistic antiviral immunity. Additionally, I will test whether DNA damage sensing plays a role in defense-system synergy, using structure-based mutations to either CapW or CapP that eliminate DNA damage sensing. Together, these experiments will reveal the molecular mechanism of CapW, and the role of DNA damage sensors in mediating synergy in bacterial defense systems. The findings have the potential to establish a new paradigm in which DNA targeting defense systems constitute a first line of antiviral defense, and DNA damage-activated systems constitute a second line of defense with orthogonal mechanisms. Thus, instead of viewing bacterial defense systems in isolation, this work will establish how they cooperate to compose a comprehensive bacterial “immune system”.