Identifying the mechanism of bacteriophage detection by cyclic-oligonucleotide signaling systems

通过环状寡核苷酸信号系统识别噬菌体检测机制

基本信息

批准号：
10550270
负责人：
Joseph Bondy-Denomy
金额：
$ 20.19万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-14 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10550270
关键词：
Adaptor Signaling Protein Amino Acids Back Bacteria Bacterial Proteins Bacteriophages Binding Biochemical Biological Assay Biological Models Candidate Disease Gene Catalytic Domain Cell Death Cell Death Induction Cells Clustered Regularly Interspaced Short Palindromic Repeats Cyclic GMP Cytoplasm DNA Binding Data Detection Effector Cell Engineering Enzyme Interaction Enzymes Escherichia coli Eukaryota Evolution Future Gene Deletion Genes Genetic Genetic Screening Genome Genomic DNA Homologous Protein Human Immune Immune system Immunity In Vitro Infection Innate Immune Response Interferon Type I Interferons Length Libraries Life Maintenance Mammalian Cell Mammals Measurement Modification Mutagenesis Mutation Natural Resistance Nucleic Acids Nucleosomes Nucleotides Oligonucleotides Operon Outcome Pathway interactions Periodicity Phase Phospholipase Post-Translational Protein Processing Production Prokaryotic Cells Proteins Pseudomonas aeruginosa Recording of previous events Repression Resistance Resolution Role Signal Transduction Signaling Molecule Stimulator of Interferon Genes System Terminator Codon Toxic effect Viral Virus Work Zinc antiviral immunity cell killing ds-DNA experimental study genome-wide human pathogen immune function in vivo inhibitor interest mutant nucleotidyltransferase opportunistic pathogen overexpression pressure prevent reconstitution response tool viral DNA viral detection

项目摘要

Project Summary Bacteriophages (phages) are viruses that infect and kill bacteria. These viral predators have provided the selective pressure to drive the evolution of numerous anti-phage immune pathways such as restriction- modification and CRISPR-Cas systems. A recent surge in the discovery of new anti-phage immune systems has uncovered an incredible diversity of defensive mechanisms across prokaryotes. Strikingly, many of these systems appear to be homologous to mammalian anti-viral immune pathways, suggesting that some human innate immune responses may have originated as anti-phage systems. The immune system of interest in this proposal first arose in bacteria and is now widely studied in mammals as cGAS-STING. In mammals, the cGAS enzyme directly binds to cytoplasmic double-stranded DNA (dsDNA) and triggers the production of cyclic GMP- AMP (cGAMP) molecules that bind to the protein STING to ultimately stimulate interferon genes. cGAS is normally held in the off state through a wide array of inhibitory post-translational modifications, direct protein interactions, nucleosome tethering, and phase separation. However, the regulatory mechanisms that inhibit or activate the homologous system in bacteria, called CBASS, remain unknown. CBASS (cyclic oligonucleotide-based anti-phage signaling systems) systems are currently thought to drive an abortive infection outcome, in which the production of one of many potential cyclic oligonucleotides (c-oligos) activates a co- encoded toxic effector protein and induces cell death. Given this cell death outcome of activated CBASS, we hypothesize that tight regulatory mechanisms must keep it off, and these mechanisms must be rapidly reversed during phage infection to turn CBASS on. Biochemical assays have shown that the cGAS-like enzymes in bacteria, called CD-NTases, constitutively produce c-oligos in vitro and structural work shows that CD-NTases are in an activated state with the catalytic site permanently competent for substrate nucleotide binding. Paradoxically, the overexpression of the CD-NTase and effector in bacteria is not toxic in the absence of phage, confirming that the cell has at least one mechanism to repress or inhibit function. For our studies, we will use the first described native model system for CBASS anti-phage function, established in Pseudomonas aeruginosa, in our group. We will first conduct unbiased and targeted genetic screens to identify endogenous CBASS inhibitors or repressors that allow maintenance and prevent self-toxicity by CBASS. In conjunction, we will identify the phage component(s) that triggers CBASS by isolating phages that acquire mutations that enable CBASS escape. Genetics and biochemical experiments will be used to validate the trigger. Surprisingly, preliminary experiments with this approach revealed the first phage encoded anti-CBASS protein, which will be mechanistically characterized during this study. Together, these experiments will provide a mechanistic understanding for how CBASS immune systems interface with, and inhibit, phage replication.

项目概要噬菌体（噬菌体）是感染并杀死细菌的病毒。这些病毒掠食者提供了选择压力驱动许多抗噬菌体免疫途径的进化，例如限制性- 修饰和 CRISPR-Cas 系统。最近新的抗噬菌体免疫系统的发现激增发现了原核生物防御机制的令人难以置信的多样性。引人注目的是，其中许多系统似乎与哺乳动物的抗病毒免疫途径同源，这表明某些人类先天免疫反应可能起源于抗噬菌体系统。对此感兴趣的免疫系统该提议首先在细菌中提出，现在在哺乳动物中作为 cGAS-STING 进行了广泛研究。在哺乳动物中，cGAS 酶直接与细胞质双链 DNA (dsDNA) 结合并触发环状 GMP- 的产生 AMP (cGAMP) 分子与蛋白质 STING 结合，最终刺激干扰素基因。 cGAS 是通常通过一系列抑制性翻译后修饰、直接蛋白质保持在关闭状态相互作用、核小体束缚和相分离。然而，抑制的调节机制或激活细菌中的同源系统（称为 CBASS）仍然未知。 CBASS（循环基于寡核苷酸的抗噬菌体信号系统）系统目前被认为可驱动流产感染结果，其中许多潜在的环状寡核苷酸（c-oligos）之一的产生激活了一种共- 编码毒性效应蛋白并诱导细胞死亡。鉴于激活 CBASS 的细胞死亡结果，我们假设严格的监管机制必须阻止它，并且这些机制必须迅速逆转在噬菌体感染期间打开 CBASS。生化分析表明，cGAS 样酶称为 CD-NTase 的细菌在体外组成型产生 c-oligos，结构研究表明 CD-NTase 处于激活状态，催化位点永久能够与底物核苷酸结合。矛盾的是，在没有噬菌体的情况下，细菌中 CD-NTase 和效应子的过度表达并没有毒性，确认细胞具有至少一种抑制或抑制功能的机制。对于我们的研究，我们将使用首次描述了在铜绿假单胞菌中建立的 CBASS 抗噬菌体功能的天然模型系统，我们组。我们将首先进行公正和有针对性的基因筛选，以鉴定内源性CBASS抑制剂或阻遏物，允许维持并防止 CBASS 的自毒性。结合起来，我们将确定通过分离获得使 CBASS 逃脱的突变的噬菌体来触发 CBASS 的噬菌体成分。遗传学和生化实验将用于验证触发因素。令人惊讶的是，初步实验通过这种方法揭示了第一个噬菌体编码的抗 CBASS 蛋白，该蛋白将在机制上本研究期间进行了表征。总之，这些实验将为如何 CBASS 免疫系统与噬菌体复制相互作用并抑制噬菌体复制。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Core defense hotspots within Pseudomonas aeruginosa are a consistent and rich source of anti-phage defense systems.

铜绿假单胞菌内的核心防御热点是抗噬菌体防御系统的一致且丰富的来源。

DOI：
发表时间：
2023-06-09
期刊：
影响因子：
14.9
作者：
Johnson, Matthew C;Laderman, Eric;Huiting, Erin;Zhang, Chi;Davidson, Alan;Bondy
通讯作者：
Bondy

Single phage proteins sequester TIR- and cGAS-generated signaling molecules.

单噬菌体蛋白隔离 TIR 和 cGAS 生成的信号分子。

DOI：
发表时间：
2023-11-16
期刊：
影响因子：
0
作者：
Li, Dong;Xiao, Yu;Xiong, Weijia;Fedorova, Iana;Wang, Yu;Liu, Xi;Huiting, Erin;Ren, Jie;Gao, Zirui;Zhao, Xingyu;Cao, Xueli;Zhang, Yi;Bondy;Feng, Yue
通讯作者：
Feng, Yue

Defining the expanding mechanisms of phage-mediated activation of bacterial immunity.

定义噬菌体介导的细菌免疫激活的扩展机制。

DOI：
发表时间：
2023-08
期刊：
影响因子：
5.4
作者：
Huiting, Erin;Bondy
通讯作者：
Bondy

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

噬菌体抗 CBASS 蛋白同时隔离环状三核苷酸和二核苷酸。

DOI：
发表时间：
2023-06-01
期刊：
影响因子：
0
作者：
Cao, Xueli;Xiao, Yu;Huiting, Erin;Cao, Xujun;Li, Dong;Ren, Jie;Guan, Linlin;Wang, Yu;Li, Lingyin;Bondy;Feng, Yue
通讯作者：
Feng, Yue

Phage anti-CBASS protein simultaneously sequesters cyclic trinucleotides and dinucleotides.

噬菌体抗 CBASS 蛋白同时隔离环状三核苷酸和二核苷酸。

DOI：
发表时间：
2024-01-18
期刊：
影响因子：
16
作者：
Cao, Xueli;Xiao, Yu;Huiting, Erin;Cao, Xujun;Li, Dong;Ren, Jie;Fedorova, Iana;Wang, Hao;Guan, Linlin;Wang, Yu;Li, Lingyin;Bondy;Feng, Yue
通讯作者：
Feng, Yue