铜绿假单胞菌中噬菌体感染必需基因的筛选及功能研究

Currently, due to the dearth of new antibiotic, it is very important using bacteriophages in bacterial infection control, espcially in the cases caused by multiple drug resistance bacteria. Clinical trial studies have been conducted for several bacteriophage therapies.However, molecular basis of host and bacteriophage interaction remains largely undisclosed, and it limited the widely clinical applications of phage therapy. In the previous studies, we constructed transposon mutagenesis bank of Pseudomonas aeruginosa for screening phage resistant mutants. Most phage resistant mutants exhibited decreased phage adsorption rates, suggesting the disrupted genes were related with bacterial phage receptor synthesis or expression; while the other mutants showed similar phage adsorption rates to the wild type strain, and the disrupted gene, which was tentavely assigned pirR (phage infection related regulator), encoded a putative regulator protein. Our results demonstrated that pirR was a novel gene essential for phage infection and this is the first evidence indicating the existence of this infection related genes. Based on the preliminary data obtained, this project is aimed at investigation of pirR gene function and its related regulation pathways; study of the role and expression of the remaining disrupted genes of phage resistant mutants; and analysis the relationships between the known mechanisms and pirR gene. Finally, a molecular model for phage infection will be proposed based on our results from this project. Our data will make contributions to the phage-host interaction mechanisms and aid the selection and modification of candidate bacteriophages for clinical therapy.

目前新的抗菌素研发停滞不前，利用噬菌体治疗耐药性细菌感染具有重要意义。若干噬菌体治疗已经进入临床试验阶段，然而细菌与噬菌体相互作用的分子机制尚未完全清晰，限制了噬菌体治疗的广泛应用。我们通过构建铜绿假单胞菌转座子突变文库，获得了噬菌体耐受突变株。大部分突变子吸附噬菌体能力显著下降，突变基因与噬菌体受体合成或表达相关；少数突变子吸附噬菌体能力与野生型菌株相同，被阻断基因（暂命名为pirR）与噬菌体受体合成或表达无关，pirR基因编码假定调节因子,这是首次发现此类基因的存在，预示噬菌体感染过程可能存在新的分子机制。本项目拟在此基础上，研究pirR基因编码蛋白的功能及调控途径；分析其它突变基因的功能及表达方式；研究已知噬菌体耐受机制及其与pirR基因的关系；建立相应的理论模型。研究结果将完善噬菌体与细菌相互作用的分子机制，并有助于筛选和改造适用于临床治疗的噬菌体。

目前新的抗菌素研发停滞不前，利用噬菌体治疗耐药性细菌感染具有重要意义。若干噬菌体治疗已经进入临床试验阶段，然而细菌与噬菌体相互作用的分子机制尚未完全清晰，限制了噬菌体治疗的广泛应用。我们通过构建铜绿假单胞菌转座子突变文库，获得了噬菌体耐受突变株。大部分突变子吸附噬菌体能力显著下降，突变基因与噬菌体受体合成或表达相关；少数突变子吸附噬菌体能力与野生型菌株相同，被阻断基因（暂命名为srpA）与噬菌体受体合成或表达无关，srpA基因编码假定调节因子,这是首次发现此类基因的存在，预示噬菌体感染过程可能存在新的分子机制。研究结果显示，SrpA蛋白是一个只有84 aa的小调节蛋白，是噬菌体K5基因组复制所必需的因子。SrpA蛋白通过结合噬菌体RNA聚合酶基因启动子区域的(NTATCN(0,9)GATAN)回文结构位点，控制该基因的转录，从而控制噬菌体基因组相关基因的转录。调节蛋白SrpA的结合位点同样广泛存在于细菌的基因组上，初步统计共有66个基因启动子区域具有SrpA的结合位点，其中15个基因编码调节蛋白。我们进一步进行了转录组学分析，发现SrpA能够调节多种关键细胞过程，与细菌的毒力与致病性密切相关。生物信息学分析显示，SrpA同源蛋白的功能未知，广泛地存在于各种微生物中，我们的结果有助于这些蛋白的功能研究。本项目研究了srpA基因编码蛋白的功能及调控途径，分析了其它突变基因的功能及表达方式，研究了已知噬菌体耐受机制及其与srpA基因的关系，揭示了SrpA蛋白是一个全局调控因子，最后建立了SrpA蛋白调节的理论模型。另一方面，研究结果完善了噬菌体与细菌相互作用的分子机制，有助于筛选和改造适用于临床治疗的噬菌体。