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.

项目总结