Molecular mechanisms of bacterial immune signaling through DNA damage

通过 DNA 损伤产生细菌免疫信号的分子机制

• 批准号: 10677417
• 负责人: Chelsea Lee Blankenchip
• 金额: $ 4.03万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677417
• 关键词: Address; Antiviral Response; Bacteria; Bacterial Antibiotic Resistance; Bacterial Genome; Bacterial Infections; Bacteriophages; Binding; Biochemical; Bioinformatics; Biological Assay; Cells; Cellular Stress; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Restriction-Modification Enzymes; Escherichia coli; Gene Expression; Generations; Genetic Transcription; Genome; Immune Targeting; Immune response; Immune signaling; Immune system; Individual; Infection; Island; Ligand Binding; Ligands; Mainstreaming; Mediating; Modeling; Modification; Molecular; Molecular Conformation; Mutation; Nucleic Acids; Oligonucleotides; Oranges; Periodicity; Plasmids; Population; Prophages; Proteins; Regulation; Resolution; Role; Signal Transduction; Structure; System; Testing; Transcriptional Regulation; Viral; Virus; Work; X-Ray Crystallography; antiviral immunity; bioinformatics tool; experimental study; fighting; pathogen; pressure; rational design; response; sensor; small molecule; synergism; transcription factor

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”.

项目概要 通过 DNA 损伤产生细菌免疫信号的分子机制 数万个细菌基因组序列的可用性，加上新的生物信息学工具和 对细菌基因组组织的新认识，使得发现和实验成为可能 细菌中数十种抗噬菌体和抗质粒防御系统的表征。由于一个典型的 细菌基因组编码 3-6 个不同的防御系统，一个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。在之前关于广泛且多样化的工作中 CBASS（基于环状寡核苷酸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 – 增强 CBASS 基因 DNA 损伤时的表达。 CapW 和 CapH+CapP 一起与约 10% 的 CBASS 系统，并且还被发现与广泛的已知和预测的细菌防御系统相邻 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 损伤的普遍信号。在这里，我将 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 将 CapW 与其靶 DNA 及其配体结合的生化测定与 X 射线晶体学相结合 表征配体所施加的构象变化，以控制 CapW-DNA 结合。这项工作将 建立 CapW（一种广泛存在的细菌转录因子）的机制。接下来，我将测试这个想法 CapW 和 CapH+CapP 通过感知 DNA 损伤来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，如限制性修饰和 CRISPR-Cas 产生由 CapW 或 CapH+CapP 感知的 DNA 损伤，以激活二级防御系统 (CBASS 或其他）来加强防御反应。我将通过感染编码的细胞来系统地测试这个模型 限制修改系统和 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力。另外，我会测试DNA是否 损伤感知在防御系统协同作用中发挥着重要作用，使用基于结构的突变来检测 CapW 或 CapP 消除 DNA 损伤传感。这些实验将共同揭示分子机制 CapW 的作用，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。这 研究结果有可能建立一个新的范式，其中 DNA 靶向防御系统构成了 抗病毒防御的第一道防线，DNA损伤激活系统构成第二道防线 正交机制。因此，这项工作不是孤立地观察细菌防御系统，而是将 确定它们如何合作组成一个全面的细菌“免疫系统”。