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

基本信息

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
项目类别：
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

项目摘要

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表征细菌中数十种抗噬菌体和抗质粒防御系统。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在先前针对广泛且多样化的感染的工作中，协调他们的活动以协同对抗感染。 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 CapW 和 CapH+CapP 共同与 DNA 损伤反应的表达相关，约 10% 是相关的。 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包括 Pycsar、DISARM 和 BREX 在内的系统这些发现表明 CapW 和 CapH+CapP 可能。介导抗病毒防御的激活，以响应细胞应激的普遍信号，即 DNA 损伤。首先确定在 DNA 损伤时结合并激活 CapW 的小分子或核酸配体。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。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，我们追求 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）来强化防御反应，我将通过感染编码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，以确定是否另外，这些系统的组合会产生协同抗病毒免疫力，我将测试DNA是否如此。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 消除了 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。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因此，这项工作不是孤立地观察细菌防御系统。确定它们如何合作组成全面的细菌“免疫系统”。