Defining the role of cyclic trinucleotide signaling in bacteriophage immunity through the activation and regulation of HORMA-associated cGAS/DncV-related nucleotidyltransferases

项目总结 确定环三核苷酸信号在噬菌体免疫中的作用 荷尔蒙相关cGAS/DncV相关核苷酸转移酶的调控 在它们的自然环境中，细菌细胞与周围的细胞不断发生冲突， 噬菌体。因此，他们开发了各种信号通路，负责 发现并适应内部和外部环境的变化。这些信号可以是 通过合成小分子传播，也称为第二信使，放大信号 并可与下游效应器结合。最近发现一类酶，cGAS/DncV- 相关的核苷酸转移酶负责合成各种各样的环二核苷酸和三核苷酸， 它们被证明扮演着第二信使的角色。在细菌中，二核苷酸调节过多的行为。 从生物膜的形成，到细胞内离子浓度，再到对噬菌体感染的防御。使用 细菌对小分子药物抗药性的增加和对替代方法的兴趣增加，包括 在噬菌体治疗中，我们必须了解对噬菌体感染的反应。此外，还有 越来越多的证据表明，宿主在感染时会感应到环二核苷酸，这导致了对 细菌环核苷酸信号转导。在这项研究中，我们建议研究分子机制和 最近在不同环境中发现的一种新的噬菌体防御途径的生物学作用 和病原菌菌株。这条途径的组成部分包含在操纵子中，它有一种酶， CDNC可合成环三AMP(CAAA)，并与最早发现的细菌蛋白共存，其中包含 霍尔马结构域，在真核生物的许多关键信号通路中发现的一个多肽结合域。这个 操纵子还编码AAA+ATPase Pch2的同源基因，AAA+ATPase Pch2是真核细胞荷尔蒙的重要调节因子 结构域蛋白。我们大量的初步数据表明，cdnC本身是不活跃的，但可以合成CAAA 当以封闭的构象和DNA结合到荷马上时。CAAA进而结合并激活一种DNA 核酸内切酶NucC也存在于操纵子中。我发现这条途径使人对噬菌体具有免疫力。 ，而这种免疫需要CDNC、HORMA和NucC蛋白。在这个项目中，我将确定 噬菌体蛋白(S)被Horma识别来启动信号，并定义噬菌体的光谱 由操纵者赋予的豁免权。我将进一步定义NucC的活动，并确定它是否具体 靶向噬菌体DNA，或者更确切地说，降解细菌自己的基因组。最后，我将确定CDNC如何 操纵子是通过DNA结合蛋白/金属肽酶对的活性进行转录调控的 与葡萄球菌转录控制蛋白的同源性。总体而言，这项工作将决定对 细菌中新的三核苷酸信号通路，以及对其机制的进一步了解 它提供噬菌体免疫力。

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.