铜绿假单胞菌噬菌体PaP3的ORF48通过RpoD“关闭宿主”功能的分子机制研究

The proteins encoded by bacteriophage can interact with bacterial proteins to shut off host functions to facilitate bacteriophage replication.At present little known is about the mechanism of this "shut-off" functions imposed by bacteriophages. In our previous experiments, ORF48 encoded by Pseudomonas aeruginosa bacteriophage PaP3 was found to have the "shut-off" functions and could interact with a host sigma factor (RpoD) screened through a transcription activation-based bacterium two-hybrid assay. The interaction between ORF48 and RpoD was confirmed by Co-IP.Simutaneously level of many genes expression under the control of RpoD within bacterium were obviously reduced after bacteriophage infection. To begin with this project，the inhibitory effect of ORF48 on RpoD as a sigma factor is verified by Single-round in vitro transcription assay. The directly interaction and binding site of amino acid between ORF48 and RpoD will be analyzed through in vitro reconstruction of RNA polymerase and protease footprint method , together with EMSA analysis of combination of ORF48 and promoter control by RpoD, which will elucidate the molecular mechanism of ORF48. Further investigation will be carried out to inspect the inhibitory effect of ORF48 on target genes controled by RpoD with the analysis of regulatory pathays under the action of ORF48, which will preliminary illuminate the molecular role of ORF48 on "shut-off" host function. The results of this project will provide new experiment evidence of bacteriophage and bacterium interaction for in-depth research, and will offer novel ideas for screening new antibacterial drug target .

为创造有利的复制增殖环境，噬菌体感染宿主菌后其自身编码蛋白能与宿主菌蛋白发生相互作用而实施"关闭宿主"功能。目前，对噬菌体"关闭宿主"的机制知之甚少。我们在前期实验中发现铜绿假单胞菌噬菌体PaP3的ORF48具有"关闭宿主"功能，通过细菌双杂交技术筛选到宿主菌σ因子（RpoD）可与之发生相互作用并通过Co-IP验证该相互作用，同时观察到在噬菌体感染后RpoD所控制的基因表达水平下降。因此本课题拟首先体外验证ORF48蛋白对RpoD作为σ因子功能的抑制作用，然后分析ORF48蛋白与宿主菌RpoD在生理条件下的直接相互作用及氨基酸结合位点，并检测ORF48是否可直接与启动子序列结合，以阐明ORF48作用的分子机理；进一步考察ORF48蛋白对RpoD所调控的哪些靶基因具有抑制作用，并分析其调控途径，初步阐明其关闭宿主的分子机制。其结果可为筛选新的抗菌药物作用靶点提供新的思路。

铜绿假单胞菌是医院感染的重要病原菌，但该菌天然对许多抗生素不敏感，并且可通过多种途径产生多重耐药性，迫切需要寻找抗铜绿假单胞菌药物作用的新靶点。噬菌体是能感染细菌的病毒，噬菌体基因组编码产生某些特殊的蛋白分子具有“关闭宿主”功能。铜绿假单胞菌噬菌体PaP3是我们分离的一株新的噬菌体，PaP3感染其宿主菌PA3后，在一定时间内可引起细菌裂解或发生溶原性转换，可见噬菌体和宿主菌两套基因组的各种编码产物之间必然存在着广泛的相互作用，但我们对噬菌体与宿主菌之间这种具有时序性和系统性的相互作用还知之甚少。.本课题利用表达谱芯片、RNA-seq、蛋白质二维电泳-质谱技术考察了铜绿假单胞菌噬菌体PaP3与其宿主-铜绿假单胞菌PA3在感染后早期、中期及晚期不同时间的全基因组表达情况，并绘制了噬菌体-宿主基因之间的时间依赖性的相互作用网络；利用细菌双杂交技术及GST pull down从噬菌体PaP3早期基因中筛选并鉴定到一个具有抑菌活性的基因- orf70.1；通过表达谱芯片技术、RT-qPCR分析对orf70.1的关闭宿主功能进行了综合的分析，结果显示在细菌生长的延迟期、对数期和稳定期一共有178个宿主菌基因受到gp70.1的影响，这些差异表达的基因主要涉及细菌的细胞外功能和代谢功能；核磁共振（NMR）质谱分析发现gp70.1对细菌的氨基酸代谢具有显著的抑制作用；表型分析显示，gp70.1对PA3的基本形态没有影响，但是能强烈地抑制细菌的胞外蛋白酶，胞外多糖，胞外纤维素酶，绿脓色素以及运动功能。表明Gp70.1可能是一个抗-sigma因子，可通过RpoS抑制铜绿假单胞菌对环境压力的应激反应。迄今为止，我们发现的gp70.1是第一个能与RpoS相互作用并抑制其控制的应激反应的噬菌体蛋白。.本课题为深入了解噬菌体基因表达模式及噬菌体与宿主相互作用机制提供了基础，并为进一步筛选抗铜绿假单胞菌感染的候选药靶提供新的思路与视角。