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.

项目摘要噬菌体（噬菌体）是感染和杀死细菌的病毒。这些病毒捕食者提供了选择性压力以驱动众多抗流量免疫途径的演变，例如限制 - 修改和CRISPR-CAS系统。最新发现新的抗流量免疫系统的激增已有揭示了跨原核生物的防御机制令人难以置信的多样性。令人惊讶的是，其中许多系统似乎与哺乳动物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流量系统。在此中的免疫系统感兴趣的系统提案首先是在细菌中提出的，现在在哺乳动物中被广泛研究为CGAS刺。在哺乳动物中，CGA 酶直接与细胞质双链DNA（DSDNA）结合，并触发环状GMP-的产生与蛋白质sting结合以最终刺激干扰素基因的AMP（CGAMP）分子。 CGA是通常通过多种抑制性翻译后修饰，直接蛋白质保持在离子状态相互作用，核小体链接和相位分离。但是，抑制的调节机制或激活细菌中的同源系统（称为CBAS）仍然未知。 cbass（循环目前认为基于寡核苷酸的抗流量信号系统）系统会驱动流产感染结果，其中许多潜在环状寡核苷酸（C-Oligos）的产生激活了共同编码有毒效应蛋白并诱导细胞死亡。鉴于激活CBAS的细胞死亡结果，我们假设严格的监管机制必须保持关闭，并且这些机制必须迅速逆转在噬菌体感染期间，打开CBAS。生化测定表明，CGAS样酶细菌，称为CD-NTase，在体外和结构工作中产生C-Oligos，表明CD-NTases 处于激活状态，具有催化位点具有永久胜任底物核苷酸结合的活化态。矛盾的是，在没有噬菌体的情况下，细菌中CD-NTase和效应子的过表达无毒确认细胞至少具有一种抑制或抑制功能的机制。对于我们的学习，我们将使用首先描述了在铜绿假单胞菌中建立的CBASS抗流量功能的天然模型系统，我们的小组。我们将首先进行无偏和靶向遗传筛选以鉴定内源性CBASS抑制剂或允许维护并防止CBASS自我激素的阻遏物。结合使用，我们将确定噬菌体成分（S）通过隔离噬菌体而触发CBAS，从而获得使CBASS逃脱的突变。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-bass蛋白，这将是机械上的在这项研究中的特征。这些实验将共同为如何提供机械理解 CBASS免疫系统与噬菌体复制并抑制。

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）

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：
10.1101/2023.06.01.543220
发表时间：
2023
期刊：
bioRxiv : the preprint server for biology
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-Denomy,Joseph;Feng,Yue
通讯作者：
Feng,Yue

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：
10.1101/2023.11.15.567273
发表时间：
2023
期刊：
bioRxiv : the preprint server for biology
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-Denomy,Joseph;Feng,Yue
通讯作者：
Feng,Yue

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

DOI：
10.1093/nar/gkad317
发表时间：
2023-06-09
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Johnson, Matthew C.;Laderman, Eric;Huiting, Erin;Zhang, Chi;Davidson, Alan;Bondy-Denomy, Joseph
通讯作者：
Bondy-Denomy, Joseph