噬菌体NM1的Gp29 通过NadE参与控制金黄色葡萄球菌的机制研究

Bacteriophages have developed proteins encoded by early genes to specifically and optimally shut down their host’s biosynthetic pathways and enslave its machinery to establish an efficient infection cycle. However, the mechanisms by which these phage proteins manipulate host functions remain obscure. In our previous work, we found that Gp29 of phage NM1 could inhibit S.aureus growth. By screening bacterial two-hybrid library, we also found that there is interaction between Gp29 and NadE which have been confirmed by pull-down and that the inhibitory effect of Gp29 could be rescued by overexpressing of nadE. In the current project, we will apply several different approaches, e.g., phage genome editing, bacterial two-hybrid assay, protein complex crystallization, gene knock-out and complementation tests, to detail the inhibitory effects of Gp29, and to identify Gp29-NadE interaction sites, and to systematically characterize the role and mechanism of manipulating host of Gp29 through interacting with NadE, both in vitro and in vivo. We aim to biologically characterize bacteriophage early proteins’ fuctions and elucidate their mechanisms by which they manipulate host, which would provide novel concepts for phage application against bacterial pathogens.

噬菌体侵染宿主菌后其早期基因编码产生的一些蛋白能迅速控制宿主菌的生长及代谢，为自身的增殖创造有利环境。目前噬菌体这些蛋白的功能及作用机制仍不清楚，限制了对噬菌体基因资源的挖掘。我们在前期实验中发现表达噬菌体NM1的Gp29对金黄色葡萄球菌具有抑制作用，通过细菌双杂交文库筛选到Gp29与宿主菌的NadE有相互作用并已经Pull-down验证，同时发现过表达nadE可拯救Gp29对宿主菌的抑制效果。本项目拟采用噬菌体基因组编辑、细菌双杂交、蛋白复合体结晶、基因敲除及回补等不同方法，在菌体内和体外两个层面系统分析噬菌体NM1的Gp29蛋白对金黄色葡萄球菌的控制效应、Gp29与NadE的相互作用位点、Gp29通过与NadE相互作用实现的控制功能及其具体作用机制等。本项目将阐释噬菌体早期基因编码的未知蛋白的功能，揭示其参与控制宿主菌的作用机制，为利用噬菌体基因资源控制病原菌提供新的理论依据。

耐药性病原菌的难以控制已成为全世界面临的重大挑战。噬菌体的一些早期蛋白可以非常高效地控制宿主菌，但其具体的作用机制不很清楚。本项目通过对金黄色葡萄球菌噬菌体NM1的Gp29的功能探讨，从体内和体外两个层面揭示其与宿主必需蛋白NadE互作参与控制宿主菌的作用机制，主要进展为：1.首次构建了基于腺苷酸环化酶的金黄色葡萄球菌必需基因的细菌双杂交文库，确定了噬菌体早期蛋白互作宿主靶标的筛选流程。2.建立了不同诱导体系的基于CRISPR的金黄色葡萄球菌必需基因敲降系统以及噬菌体的基因编辑系统，为研究噬菌体与宿主菌之间的相互作用提供了良好的支撑。3.通过诱导表达对宿主菌的生长抑制确定了噬菌体NM1的Gp29参与控制宿主菌，通过细菌双杂交文库筛选和必需基因敲降系统确定了其互作的必需蛋白靶标NadE，通过位点突变互补、蛋白生化等方法探讨了二者互作参与控制宿主菌的作用机制。4.根据Gp29与NadE二者研究建立的流程，对噬菌体NM1的具有生长抑制作用的早期蛋白进行了广泛的筛选研究，绘制了潜在的全局互作网络关系图。本研究有助于噬菌体早期基因编码的未知蛋白的功能阐释，揭示其参与控制宿主菌的作用机制，为系统深入地理解噬菌体早期蛋白控制病原菌的作用机理奠定了基础。