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”.

项目摘要 通过DNA损伤，细菌免疫信号传导的分子机制 数以万计的细菌基因组序列的可用性，以及新的生物信息学工具和 对细菌基因组组织的新理解已使发现和实验性 细菌中数十种抗细菌噬菌体和抗质粒防御系统的表征。由于典型 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可以 协调他们的活动以协同作用。在宽度和潜水员的先前工作中 CBASS（基于循环寡核苷酸的抗流量信号系统）防御系统，我们确定了两个 转录调节器 - CAPW和两蛋白CAPH+CAPP系统 - 增强CBASS基因 响应DNA损伤的表达。 CAPW和CAPH+CAPP一起与〜10％ CBASS系统，也发现与广泛的已知和预测细菌防御相邻 包括Pycsar，disman和Bret在内的系统。这些发现表明CAPW和CAPH+CAPP可能 响应细胞应激的通用信号，DNA损伤，介导抗病毒防御的激活。在这里，我会的 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 将生化测定与X射线晶体学结合与其靶DNA及其配体的CAPW结合 表征配体对控制CAPW-DNA结合的构象变化。这项工作将 建立CAPW的机制，CAPW是一种宽度细菌转录因子。接下来，我将测试 CAPW和CAPH+CAPP通过感测DNA损伤来调解抗病毒防御系统之间的合作。 具体而言，我们假设靶向DNA靶向免疫系统，例如限制 - 修饰和CRISPR-CAS 创建CAPW或CAPH+CAPP感知的DNA损伤以激活二级防御系统（CBASS 或其他）加强防御反应。我将通过感染的单元编码系统地测试此模型 限制性修改系统和CAPW-或CAPH+CAPP相关的CBASS系统都可以确定是否 这些系统的组合产生协同的抗病毒免疫学。另外，我将测试是否DNA 损害敏感性在防御系统协同作用中起作用，使用基于结构的突变来capw或 消除DNA损伤敏感性的CAPP。这些实验将共同揭示分子机制 CAPW，以及DNA损伤传感器在细菌防御系统中介导协同作用中的作用。这 调查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 抗病毒防御的第一线和DNA损伤激活的系统构成了第二道防线 正交机制。这项工作将不是孤立地观看细菌防御系统，而是 确定它们如何协调以组成综合细菌“免疫系统”。