Defining the role of cyclic trinucleotide signaling in bacteriophage immunity through the activation and regulation of HORMA-associated cGAS/DncV-related nucleotidyltransferases

通过激活和调节 HORMA 相关 cGAS/DncV 相关核苷酸转移酶来定义环三核苷酸信号在噬菌体免疫中的作用

• 批准号: 10198852
• 负责人: Rebecca Kavaler Lau
• 金额: $ 3.84万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198852
• 关键词: ATP phosphohydrolase; Adopted; Affinity; Bacteria; Bacterial Proteins; Bacteriophages; Behavior; Binding; Binding Proteins; Biochemical; Biological; Cells; Cellular Assay; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Conflict (Psychology); Cytology; DNA; DNA Methylation; DNA-Binding Proteins; Data; Deinococcus; Deoxyribonuclease I; Detection; Diffuse; Dinucleoside Phosphates; Environment; Enzymes; Escherichia coli; Eukaryota; Feedback; Fluorescence Microscopy; Genetic Transcription; Genome; Goals; Growth; Health; Helix-Turn-Helix Motifs; Homologous Gene; Human; Immunity; Infection; Ions; Lead; Mass Spectrum Analysis; Mediating; Metalloproteases; Microbial Biofilms; Modification; Molecular; Molecular Conformation; Names; Nucleotides; Oligonucleotides; Operon; Orthologous Gene; Pathogenicity; Pathway interactions; Patients; Peptide Hydrolases; Peptide Signal Sequences; Peptides; Periodicity; Pharmaceutical Preparations; Play; Production; Proteins; Pseudomonas aeruginosa; Regulation; Role; Second Messenger Systems; Signal Pathway; Signal Transduction; Signaling Molecule; Structure; System; Tertiary Protein Structure; Testing; Therapeutic; Transcriptional Regulation; Work; bacterial resistance; base; genome integrity; human pathogen; interdisciplinary approach; interest; microbial community; novel; nuclease; nucleotidyltransferase; response; screening; sensor; small molecule; training opportunity

PROJECT SUMMARY Defining the role of cyclic trinucleotide signaling in bacteriophage immunity through the activation and regulation of HORMA-associated cGAS/DncV-related nucleotidyltransferases In their natural environment, bacterial cells are in a constant conflict with the surrounding cells and bacteriophages. Therefore, they have developed a variety of signaling pathways which are responsible for detecting and adapting to changes both their internal and external environment. These signals can be propagated through the synthesis of small molecules, also called second messengers, which amplify the signal and can bind to downstream effectors. It has recently been discovered that one class of enzymes, cGAS/DncV- related nucleotidyltransferases, are responsible for synthesizing a wide variety of cyclic di- and tri-nucleotides, which have been shown to act as second messengers. In bacteria, dinucleotides regulate a plethora of behaviors ranging from biofilm formation, to intracellular ion concentration, to defense against bacteriophage infection. With the rise in bacterial resistance to small molecule drugs and increased interest in alternative approaches, including phage therapy, it is imperative that we understand the responses to bacteriophage infection. Furthermore, there is growing evidence that cyclic dinucleotides are sensed by hosts upon infection, leading to high interest in bacterial cyclic oligonucleotide signaling. In this study, we propose to study the molecular mechanisms and biological roles of a novel bacteriophage defense pathway recently discovered in a diverse set of environmental and pathogenic bacterial strains. This pathway, whose components are contained in an operon, has an enzyme, CdnC, which synthesizes cyclic tri-AMP (cAAA) and co-exists with the first-identified bacterial proteins containing the HORMA domain, a peptide-binding domain found in many critical signaling pathways in eukaryotes. The operons also encode an ortholog of the AAA+ ATPase Pch2, an important regulator of eukaryotic HORMA domain proteins. Our extensive preliminary data shows that CdnC is inactive on its own, but synthesizes cAAA when bound to the HORMA in a closed conformation as well as DNA. cAAA in turn binds and activates a DNA endonuclease, NucC, also found in the operon. I have found that this pathway confers immunity to bacteriophage , and that immunity requires the CdnC, HORMA, and NucC proteins. In this project, I will identify the bacteriophage protein(s) recognized by HORMA to initiate signaling, and define the spectrum of bacteriophage immunity imparted by the operon. I will further define the activities of NucC and determine whether it specifically targets phage DNA, or rather degrades the bacterium’s own genome. Finally, I will determine how the CdnC operon is regulated transcriptionally, through the activity of a DNA binding protein/metallopeptidase pair with homology to transcriptional control proteins in Deinococcus. Overall, this work will determine the regulation of a novel trinucleotide signaling pathway in bacteria, and further the understanding of the mechanisms through which it provides bacteriophage immunity.

项目概要 通过激活和定义环三核苷酸信号在噬菌体免疫中的作用 HORMA 相关 cGAS/DncV 相关核苷酸转移酶的调节 在自然环境中，细菌细胞与周围细胞不断发生冲突， 因此，它们开发了多种负责的信号通路。 检测并适应这些信号的内部和外部环境的变化。 通过小分子的合成传播，也称为第二信使，放大信号 并且可以与下游效应子结合，最近发现一类酶，cGAS/DncV-。 相关的核苷酸转移酶，负责合成多种环状二核苷酸和三核苷酸， 已被证明可以作为细菌中的第二信使，调节大量的行为。 从生物膜形成到细胞内离子浓度，再到防御噬菌体感染。 细菌对小分子药物耐药性的增加以及对替代方法的兴趣增加，包括 在噬菌体治疗中，我们必须了解对噬菌体感染的反应。 越来越多的证据表明，宿主在感染时能够感知到环状二核苷酸，这引起了人们的高度兴趣 在这项研究中，我们建议研究细菌环状寡核苷酸信号传导的分子机制和 最近在多种环境中发现的新型噬菌体防御途径的生物学作用 该途径的成分包含在操纵子中，具有酶， CdnC，合成环状三磷酸腺苷 (cAAA)，并与首次鉴定的细菌蛋白共存，其中含有 HORMA 结构域，一种在真核生物许多关键信号通路中发现的肽结合结构域。 操纵子还编码 AAA+ ATPase Pch2 的直系同源物，它是真核 HORMA 的重要调节因子 我们广泛的初步数据表明，CdnC 本身不活跃，但合成 cAAA。 当以闭合构象与 HORMA 结合时，cAAA 反过来结合并激活 DNA。 核酸内切酶 NucC 也存在于操纵子中，我发现该途径赋予噬菌体免疫力。 ，而免疫需要 CdnC、HORMA 和 NucC 蛋白。在这个项目中，我将识别这些蛋白。 HORMA 识别的噬菌体蛋白可启动信号传导，并定义噬菌体谱 我将进一步定义NucC的活性并确定它是否具有特异性。 靶向噬菌体 DNA，或者更确切地说，降解细菌自身的基因组。最后，我将确定 CdnC 是如何发挥作用的。 操纵子通过 DNA 结合蛋白/金属肽酶对的活性进行转录调节 总体而言，这项工作将确定与奇异球菌中转录控制蛋白的同源性。 细菌中新型三核苷酸信号通路，并进一步了解其机制 它提供噬菌体免疫力。