铜绿假单胞菌生物被膜中PF4噬菌体生命周期的调控

Biofilm-associated growth of pseudomonas aeruginosa (PA) plays a key role in the development of antibiotic resistance. Disruption of its biofilm can lead to greater antibiotic susceptibility of PA infections. Recent studies suggested that the filamentous ssDNA bacteriophage Pf4 is involved in PA biofilm disruption. In the late mature stage of PA biofilms, these Pf4 phages can further become “superinfective” and lyse P-lysogenized PA cells, which are normally resistant to the infection. Although the molecular basis of this fascinating phenomenon has not been well investigated, recent analysis of the Pf4 prophage genome in P. aeruginosa PAO1 strain uncovered an open reading frame (ORF) on the complementary intergenic region between gene PA0716 and PA0717, which has 42% homology to the sequence encoding the repressor C protein of phage P2 (9), suggesting that the Pf4 phage might use a mechanism similar to that of the classical tailed phage to initiate the lytic cycle. We hypothesize that in PA biofilms, Pf4 phage uses repressor-like element(s) to control the expression of genes involved in the lytic cycle, and as the biofilm ages, specific mutations occur in non-superinfective lytic Pf4 phage, allowing it to switch to the superinfective form. Here, we propose research designed to test this hypothesis. Specific goals of the project will be as follows: 1)Characterize the genetic components causing the switch of Pf4 phage from lysogenic to lytic. 2)Characterize the genetic components causing the switch of Pf4 phage to its superinfective form. 3)Determine the physiological factors within PA biofilms that trigger the switch of Pf4 phage to its superinfective form. .Insight into this mechanism of superinfective phage-mediated cell death and the consequent biofilm disruption can promote the development of appropriate targets for the treatment of biofilm-associated PA infections.

破坏生物被膜可增强铜绿假单胞菌（pseudomonas aeruginosa PA）对抗生素的敏感性。晚期PA生物被膜中Pf4噬菌体具有溶解包含有该噬菌体PA细胞的超感染能力，参与了生物被膜的破坏过程。这一现象的分子机制尚未阐明。分析PA菌株PAO1中Pf4噬菌体基因组发现了一个与编码噬菌体P2抑制物C蛋白的基因序列有42%同源性的开放读码框，提示Pf4噬菌体可能采用与P2相似机制启动溶菌周期。我们提出假设： Pf4噬菌体使用抑制物样原件控制参与溶解循环的基因表达，随着生物被膜的成熟，Pf4噬菌体突变为超感染噬菌体。本研究中我们从如下几方面对该假设进行验证：探寻Pf4噬菌体溶源期向溶解期转化的控制基因；探寻Pf4噬菌体由普通相向超感染相转化的控制基因；确定PA生物被膜中触发Pf4噬菌体向超感染相转变的生理因素。认识调控Pf4噬菌体生活周期的分子基础将为治疗生物被膜相关PA感染提供新思路

铜绿假单胞菌是多种结构性肺病患者合并感染的常见病原菌，其临床治疗药物有限，且耐药情况日益严重。生物被膜导致的抗菌药物浓度局部下降在铜绿假单胞菌耐药过程中发挥了重要作用。PF4是一种丝状噬菌体，和普通噬菌体一样以溶原状态存在于铜绿假单胞菌PAO1，当PF4噬菌体感染包涵有PF4噬菌体细菌时，其不能产生溶菌效应，称之为溶原免疫，但其机制一直未能完全明确，根据在其他噬菌体中的研究，提出假设可能溶原状态噬菌体分泌阻遏蛋白与进入的噬菌体结合阻碍其基因复制，使其不能产生溶菌效应。我们通过构建PF4噬菌体敲除的WT PAO1菌株, 并将PF4噬菌体9个未知功能基因候选区基因分别过表达，然后通过提前1日龄生物被膜中噬菌体进行噬菌斑试验，筛选出了可能编码阻遏蛋白的基因区PAO1-719并进行测序。该结果验证了前述假设，并为后续进一步研究阻遏蛋白的功能结构提供了依据和工具，也为耐药铜绿假单胞菌治疗提供了一个新的方向。与普通噬菌体不同，在生物被膜中的PF4噬菌体存在一种特殊现象，就是晚期生物被膜中的PF4噬菌体能够突破溶原免疫，能在包含有溶原状态PF4噬菌体的PAO1菌苔中产生噬菌作用。这提示晚期生物被膜PF4噬菌体与早期生物被膜PF4噬菌体相比可能存在阻遏蛋白结合位点基因突变，从而突破溶原免疫，产生噬菌作用，我们称之为超感染噬菌体。本研究中我们通过提取不同生物被膜期的噬菌体，进行噬菌斑实验验证其功能，并进行二代测序，发现其突变位点，后续进一步可以针对突变位点，在WT PAO1中构建突变株。超感染PF4导致的细菌死亡可能是治疗生物被膜中耐药铜绿假单胞菌的一个可能方向。