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

项目摘要 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，以推动许多抗噬菌体免疫途径的进化，如限制- 修改和CRISPR-CAS系统。最近发现的新的抗噬菌体免疫系统激增 揭示了原核生物中令人难以置信的多样性的防御机制。令人惊讶的是，其中许多 这些系统似乎与哺乳动物的抗病毒免疫途径同源，这表明一些人类 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 该建议最早出现在细菌中，现在在哺乳动物中被广泛研究为cGAS-刺痛。在哺乳动物中，cGAs 酶直接与胞质双链DNA(DsDNA)结合，触发环状GMP-2的产生。 AMP(CGAMP)分子与蛋白质结合，最终刺激干扰素基因。CGAS是 通常通过一系列抑制性的翻译后修饰保持在关闭状态，直接蛋白质 相互作用、核小体拴系和相分离。然而，抑制的监管机制 或者激活细菌中的同源系统，称为CBass，目前尚不清楚。CBASS(循环 基于寡核苷酸的抗噬菌体信号系统)目前被认为是导致流产感染的原因 结果，其中许多潜在的环状寡核苷酸(c-寡核苷酸)的产生激活了一种共同的- 编码有毒效应蛋白并诱导细胞死亡。考虑到激活的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 在噬菌体感染期间打开CBASS。生化分析表明，cGAS样酶在 细菌，称为CD-NTase，在体外结构性地产生c-寡糖，结构研究表明CD-NTase 处于激活状态，催化位置永久地能够与底物核苷酸结合。 矛盾的是，在没有噬菌体的情况下，CD-NTase和效应器在细菌中的过度表达是无毒的， 确认该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 我们这群人。我们将首先进行无偏见和有针对性的基因筛查，以确定内源性CBASS抑制剂 或抑制物，允许维持和防止CBASS的自我毒性。同时，我们将确定 噬菌体成分(S)，通过分离获得使CBASS逃逸的突变的噬菌体来触发CBASS。 遗传学和生化实验将被用来验证触发因素。令人惊讶的是，初步实验 用这种方法揭示了第一个编码抗CBass蛋白的噬菌体，这将是机械的 在这项研究中表现出的特点。总之，这些实验将提供一种机械性的理解，即 CBASS免疫系统与噬菌体复制相互作用，并抑制其复制。

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.