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

项目摘要 噬菌体（Bacteriophage，简写为BPHs）是一种能够感染并杀死细菌的病毒。这些病毒捕食者提供了 选择性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多样性令人惊讶的是，其中许多 系统似乎是同源的哺乳动物抗病毒免疫途径，这表明，一些人类 先天免疫应答可能起源于抗噬菌体系统。免疫系统的利益在这方面 该提议首先出现在细菌中，现在作为cGAS-STING在哺乳动物中被广泛研究。在哺乳动物中，cGAS 酶直接结合到细胞质双链DNA（dsDNA），并触发环GMP的生产- AMP（cGAMP）分子，其结合蛋白STING以最终刺激干扰素基因。CGAS是 通常通过广泛的抑制性翻译后修饰保持在关闭状态，直接蛋白质 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 目前认为，基于抗噬菌体肽的抗噬菌体信号传导系统（anti-phage signaling systems 结果，其中许多潜在的环状寡核苷酸（c-寡核苷酸）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。鉴于激活的CBASS的细胞死亡结果，我们 假设严格的监管机制必须阻止它，这些机制必须迅速逆转 生物化学分析表明，在噬菌体感染过程中， 细菌，称为CD-NTases，在体外组成性产生c-寡核苷酸，结构研究表明，CD-NTases 处于活化状态，催化位点永久地胜任底物核苷酸结合。 巧合的是，细菌中CD-NT酶和效应子的过表达在没有噬菌体的情况下是无毒的， 证实所述细胞具有至少一种抑制或抑制功能的机制。为了我们的研究，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， 我们的团队我们将首先进行无偏见和有针对性的基因筛选，以确定内源性CBASS抑制剂 或允许维持和防止CBASS自身毒性的阻遏物。同时，我们将确定 噬菌体组分，其通过分离获得能够使CBASS逃逸的突变的噬菌体来触发CBASS。 遗传学和生化实验将用于验证触发器。令人惊讶的是，初步实验 用这种方法揭示了第一个噬菌体编码的抗CBASS蛋白，这将是机械的 在这项研究中，总之，这些实验将提供一个机械的理解， CBASS免疫系统与噬菌体复制相互作用并抑制噬菌体复制。

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.