Recognizing sweet surfaces – structure and function of a siphovirus infection device at the Gram-negative bacterial membrane

细菌病毒（噬菌体）将其基因组运送到细菌细胞中，这是其感染周期的关键步骤。许多噬菌体使用尾巴来吸附和将DNA转移到宿主的细胞质中。尾部噬菌体将核心尾部结构与多种感染部位结合在一起，反映了其宿主所遇到的各种不同包膜组成。长而非收缩的尾型虹膜病毒是一种主要的尾型噬菌体。然而，这些噬菌体从细菌包膜接收的感染信号的性质尚不清楚，需要更多的研究。这个法国-德国项目的目标是用低温电子显微镜（cryoEM）阐明来自沙门氏菌模型噬菌体9NA的聚糖特异性siphovirus尾部和基板感染革兰氏阴性宿主的第一个原型结构。这将定义使用蛋白质受体进行感染的噬菌体的结构差异。利用全内反射荧光（TIRF）显微镜和新的革兰氏阴性模型膜，我们将在单颗粒水平上研究siphovirus 9NA的时间分辨率基因组释放机制，并分析革兰氏阴性膜特性对成功感染启动的影响。存在脂多糖受体或外膜囊泡的低温电镜研究将显示噬菌体结构蛋白如何参与膜接触后感染的启动。因此，我们将进一步了解虹膜病毒尾部导致基因组释放的分子重排。此外，我们将使用这些体外装置来研究在同一宿主上竞争受体的噬菌体混合物的协同作用，并将这些数据与整个细菌细胞的体内感染分析相关联。探索噬菌体-包膜相互作用的动力学将推进我们对噬菌体尾部结构重排的认识，从而导致基因组释放作为噬菌体生命周期的关键步骤。阐明代表广泛类型的革兰氏阴性、甘聚糖特异性噬菌体的模型系统的尾部和基板结构将改进基于结构的基因组注释和噬菌体受体使用预测。我们的工作将为噬菌体群落在特定感染生态系统中的行为提供大量分子知识，这对于开发使用噬菌体的新抗生素治疗非常重要。

Bacterial viruses (bacteriophages) shuttle their genomes into bacterial cells as a key step in their infection cycle. Many bacteriophages use a tail for adsorption and DNA translocation to the host’s cytosol. Tailed phages combine core tail architectures with diversified infection parts, reflecting the various different envelope compositions encountered on their hosts. Long, non-contractile tailed siphoviruses are a predominant tailed phage type. However, the nature of the infection signal these phages receive from the bacterial envelope is not well understood and needs more investigations. Goal of the French-German project is to elucidate, with cryo-elctron microscopy (cryoEM), the first prototype structure for a glycan-specific siphovirus tail and baseplate infecting a Gram-negative host, from Salmonella model phage 9NA. This will define structural differences to phages that use protein receptors for infection. With total internal reflection fluorescence (TIRF) microscopy, and new Gram-negative model membrane set-ups, we will study siphovirus 9NA’s time-resolved genome release mechanism on a single particle level, and analyze the influence of Gram-negative membrane properties on successful infection initiation. CryoEM studies in presence of the lipopolysaccharide receptor or outer membrane vesicles will show how phage structural proteins are involved in infection initiation upon membrane contact. We will thus gain understanding of the molecular rearrangements in the siphovirus tail leading to genome release. We will moreover use these in vitro set-ups to study synergies of phage mixtures that compete for receptors on the same host and correlate this data with in vivo infection analyses on whole bacterial cells. Exploring the dynamics in bacteriophage-envelope interactions will advance our view on phage tail structure rearrangements, leading to genome release as a key step in the phage life cycle. Elucidating the tail and baseplate structure of a model system representing a widespread type of Gram-negative, glycan-specific phages will improve structure-based genome annotation and phage receptor usage prediction from sequence data. Our work will contribute substantial molecular knowledge on how phage communities behave in a given infection ecosystem, important for the development of new antibiotic treatments that employ bacteriophages.