lambda噬菌体与其宿主大肠杆菌相互作用的新机制的研究

Bacteriophages or phages are bacterial viruses that invade bacterial cells and, in the case of lytic phages, cause the bacterium to lyse. Phage may provide new treatment regimes in bacterial infection. The emergence of pathogenic bacteria resistance to most currently available antibiotics makes people reconsider to apply phage to antibacterial treatment. Interaction about phages and their hosts must be clear before lytic phages can be widely endorsed for antibacterial application. Bacteriophage lambda is one of the most extensively studied organisms and has been a primary model for understanding basic modes of genetic regulation. The virulent mutants of lambda phage (only entering lytic cycles) can kill Escherichia coli (E.coli) in vivo. In our previous studies, we examined the progress of lambda gene expression during phage development by ribosome profiling and, thereby, provide a very high-resolution view of its host gene expression. Most of host genes were down-regulated. We also discovered some E.coli genes' expression level being upregulated during phage development. These up-regulated genes may play key roles during lambda phage life cycle. In this study, we propose to confirm the novel functions of these unregulated genes from E.coli and establish the interaction network between lambda phage and its host. Further, we will monitor the interaction between lambda phage and its host in a real-time manner during their lysis cycles for virulent mutants of lambda phage. Our studies may reveal novel mechanisms of the interaction between lambda phage and host bacterial, which will give us some useful information in using phage against bacterial infection.

噬菌体是细菌的病毒，有望用于抗感染，对杀灭耐药菌具有重要意义。但要将噬菌体真正应用，还需更全面了解其与宿主之间的相互作用。作为模式生物的lambda噬菌体其毒性突变株具有抗感染的广泛应用前景。我们前期采用核糖体谱技术检测了lambda噬菌体在诱导活化过程中及其宿主大肠杆菌基因翻译表达水平变化。结果显示，在lambda噬菌体活化过程中宿主很多基因表达水平发生明显变化，尤其是表达上调的基因可能在感染裂解细菌中发挥了重要作用。为此，本项目拟深入研究lambda噬菌体促使宿主基因上调的新机制，丰富lambda噬菌体与其宿主菌之间相互作用的网络。同时将继续采用核糖体谱技术检测lambda噬菌体毒性突变株在感染裂解大肠杆菌时基因表达变化。分析lambda噬菌体毒性突变株和宿主的相互作用。本项目的实施将有助于深入了解lambda噬菌体和宿主菌的相互作用机制，为噬菌体有效对抗细菌感染奠定理论基础。

噬菌体是细菌的病毒，对杀灭耐药菌具有重要意义。但要将噬菌体真正应用，还需更全面了解其与宿主之间的相互作用。作为模式生物的lambda噬菌体其毒性突变株具有抗感染的广泛应用前景。我们前期采用核糖体谱检测了lambda噬菌体在诱导活化过程中基因翻译水平变化。其中宿主上调的基因可能在感染裂解细菌中发挥了重要作用。课题将初步阐明噬菌体具体调控这些基因的机制。同时得到lambda噬菌体毒性突变株，了解其和宿主相互作用，为其后续应用于对抗细菌感染奠定基础。我们用Q蛋白抗体做了染色质免疫共沉淀测序。发现λ噬菌体编码的Q蛋白在pR’区开始结合到噬菌体基因组后调控晚期蛋白的表达。Q还能够结合到部分大肠杆菌基因，如上调基因raiA和epd。发现过表达Q后提高了raiA基因的mRNA水平，同时RNA polymerase在raiA基因的结合也升高。并且 Q蛋白能够和raiA基因的启动子区DNA特异结合。采用RNA-seq 检测5’端磷酸化的小RNA，发现pR’启动子区有16和17碱基长的转录暂停小RNA的存在。同时在raiA等数百个启动子区发现了此类小RNA存在。其中raiA等几十个基因启动子区的小RNA受到greA和sigma70的影响，这和pR’区的转录暂停小RNA的变化相似。综上所述认为 Q在raiA等基因可能通过和pR’区相似的抗转录暂停的方式来调控这些基因的转录表达。体外转录实验也证实了这几十个基因应该是sigma region 2依赖的转录暂停。同时当我们在宿主菌中敲除raiA基因后，lambada噬菌体的诱导表达受到了一定的抑制，核糖体谱发现敲除raiA后很多基因出现了差异表达，可能多种途径协同raiA共同参与噬菌体的活化过程。得到lambda毒性噬菌体能快速有效裂解宿主菌，核糖体谱实验看到其能快速夺取宿主的基因表达系统来完成自身的基因表达，对宿主的基因表达产生巨大的影响。通过我们的实验对λ噬菌体作用于宿主菌的过程有了具体的认识;分离得到更多的裂解性强的大肠杆菌毒性噬菌体，通过基因重组得到λ噬菌体毒性突变株及重组突变株能更有效的裂解临床分离得到的多耐药的大肠杆菌，为噬菌体抗细菌感染提供理论依据与实验基础。