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

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。

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.