甲型副伤寒杆菌抗噬菌体及生物膜形成基因的筛选及鉴定  

Screening and Identification of Genes of Salmonella paratyphi A Related to Anti-phage and Biofilm Formation

作　　者：毛普加 冯梦蝶[1] 洪愉[3] 许泽仰 赵继华[3] 杨洪文[3] 宋武战[3] 黄芬[1] 井申荣[1] 曾韦锟[1,2] 

机构地区：[1]昆明理工大学医学院,昆明650500 [2]昆明学院医学院,昆明650214 [3]成都军区昆明总医院核医学科,昆明650032

出　　处：《生物化学与生物物理进展》2015年第6期583-588,共6页Progress In Biochemistry and Biophysics

基　　金：云南省教育厅科学研究基金资助项目(2010Y398);云南省应用基础研究面上项目(2010ZC055,2012FB135);国家自然科学基金资助项目(31160193)~~

摘　　要：细菌生物膜(bacterial biofilm,BF)与大部分的细菌感染相关,有助于病原菌抵抗外部不利的环境,包括抗生素和抗噬菌体等.为了研究生物膜和抗噬菌体的作用机制,本文以6株抗噬菌体甲型副伤寒杆菌(副甲菌)突变菌作为研究对象,在Rif+(利福平)(200 mg/L)平板中划线并滴加噬菌体验证能否抗噬菌体,将6株突变菌接种于96孔板中,每组3个重复,观察其成膜能力以及生物膜的形态,定点突变和互补实验验证突变菌的噬菌体抗性是否由突变基因所引起.结果显示:划线平板中野生型副甲菌在滴加1.2×106个噬菌体处出现空缺,而6株突变菌在滴加2.4×109个噬菌体后仍能生长,表明6株突变菌具有抗噬菌体特性;6株突变菌中,σ-54依赖的翻译调节器突变菌成膜能力(A595=1.1±0.2)较野生型副甲菌(A595=0.5±0.1)显著性增强,且差异显著(P<0.05),光学显微镜下菌体聚集成粗大的不规则团块;同源重组敲除野生型副甲菌σ-54依赖的翻译调节器,突变菌出现噬菌体抗性,将表达σ-54依赖的翻译调节器的载体转化该突变菌,突变菌又恢复了噬菌体敏感性.结果表明,σ-54依赖的翻译调节器是抗噬菌体和生物膜形成相关的基因.Biofilm （bacterial biofilm, BF） are associated with most bacterial infections, which can help pathogens against adverse environment, such as antibiotic and phages. In order to exploid the mechanism involved with BF formation and anti-phage, six Salmonella paratyphi A （S. paratyphi A） mutants resistant to phage were analyzed in this study. Phages were dropped on bacteria line on LB plate containing Rif^＋ （200 mg/L）to confirm phage resistance. Six mutants were cultured in 96 well plate with three replications to monitor biofilm formation and morphology. Site-directed mutagenesis and complementation test were used to confirm whether phage resistance was caused by gene mutant. Results of this study showed that all six mutants were resistant to phage, because those could growth with dropped 2.4×10^9 phages and wild bacteria could not with dropped 1.2×10^6 phages. Among six mutants, BF formation of mutant of σ-54-dependent translation regulator was significantly enhanced contrast with wild bacteria （P 〈 0.05, 1.1 ±0.2 vs 0.5±0.1）. Bacteria aggregated to large irregular mass. Site-directed mutation of σ-54-dependent translation regulator with homologous recombination led to be resistant to phages. This resistance could be recovered by supplemented with plasmid expressed σ-54-dependent translation regulator. All those showed that σ-54-dependent translation of the regulator was associated with anti-phage and BF formation.

关 键 词：甲型副伤寒沙门菌 转座 抗噬菌体 生物膜 

分 类 号：R378.24[医药卫生—病原生物学]