铜绿假单胞菌成孔毒素ExlA编码基因无痕缺失突变株的构建及其基本特性研究  

Construction of the pore-forming toxin gene exlA knock-out mutant of Pseudomonas aeruginosa and its basic characteristics

作　　者：张再青 周媛媛 胡凌飞 金秀玉 周冬生 高波[3] 杨慧盈 ZHANG Zaiqing;ZHOU Yuanyuan;HU Lingfei;JIN Xiuyu;ZHOU Dongsheng;GAO Bo;YANG Huiying(State Key Laboratory of Pathogen and Biosecurity,Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China;Center for Disease Control and Prevention of Central Theater Command,Beijing 100042,China;Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]军事科学院军事医学研究院微生物流行病研究所,病原微生物生物安全全国重点实验室,北京100071 [2]中部战区疾病预防控制中心,北京100042 [3]军事科学院军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《军事医学》2024年第2期108-114,共7页Military Medical Sciences

基　　金：病原微生物生物安全国家重点实验室自主研究课题(SKLPBS2207)。

摘　　要：目的构建铜绿假单胞菌NY8755 exlA基因无痕缺失突变株(NY8755ΔexlA),研究成孔毒素ExlA的基本特性。方法利用二次同源重组的方法构建铜绿假单胞菌exlA基因无痕缺失突变株。选取6~8周龄雌性C57BL/6J小鼠,经气溶胶肺递送途径分别感染铜绿假单胞菌NY8755和NY8755ΔexlA,记录感染后7 d小鼠的生存状况和体重变化,检测小鼠肺泡灌洗液中的促炎因子。结果经过测序验证,成功构建铜绿假单胞菌成孔毒素ExlA编码基因无痕缺失突变株。利用气溶胶肺递送途径感染小鼠(1×107 CFU)后,野生株组小鼠48 h内全部死亡,突变株组48 h后开始死亡且7 d后仍有40%存活;突变株组存活小鼠体重先下降,后逐渐恢复;感染12 h后野生株组小鼠肺泡灌洗液明显比突变株组血性渗出物更多(颜色更红),肺泡灌洗液中促炎因子白细胞介素-1β(IL-1β)和白细胞介素-17A(IL-17A)含量有显著差异。结论铜绿假单胞菌成孔毒素ExlA是exlA阳性的新型铜绿假单胞菌的关键毒力因子,可显著影响小鼠的生存状况,引起小鼠体内明显的炎症反应。当前关于外毒素ExlA的致病机制研究较少,所构建的NY8755ΔexlA突变株及野生株和突变株肺炎小鼠模型,可为进一步探索exlA阳性的铜绿假单胞菌致病机制提供研究基础。Objective To construct a non-trace deletion mutant of exlA in Pseudomonas aeruginosa strain NY8755(NY8755ΔexlA)and investigate the basic characteristics of pore-forming toxin ExlA.Methods The NY8755ΔexlA was constructed using the secondary homologous recombination method.C57BL/6J female mice ages 6 to 8 weeks were infected with NY8755 and NY8755ΔexlA via aerosolized intratraheal inoculation respectively.Within 7 days of infection,the survival and weight changes of the mice were observed and recorded before the proinflammatory cytokines in the bronchoalveolar lavage fluid(BALF)of the infected mice in the two groups were detected.Results The sequencing results showed that NY8755ΔexlA was constructed.After 1×107 CFU NY8755 and NY8755ΔexlA were infected,all the mice in the wild-type strain group died within 48 hours,while those in the mutant strain group began to die after 48 hours,and 40%of them remained alive 7 days later.The weight of surviving mice in the mutant strain group decreased but recovered gradually.After 12 hours of infection,there were more bloody exudates(redder in color)in the BALF of the wild-type strain group than in the mutant strain group,and the contents of proinflammatory cytokines interleukin-1β(IL-1β)and interleukin-17A(IL-17A)were significantly different.Conclusion Pseudomonas aeruginosa pore-forming toxin ExlA is the key pathogenic virulence factor of the exlA-positive Pseudomonas aeruginosa,which can significantly affect the survival status of mice and cause obvious inflammation in mice.Very little information is available on the action mechanisms of ExlA.In this study,The NY8755ΔexlA and the C57BL/6J mouse models infected with NY8755 and NY8755ΔexlA have been constructed that may be used for the investigation of pathogenesis of exlA-positive Pseudomonas aeruginosa.

关 键 词：铜绿假单胞菌 基因敲除 成孔毒素 动物模型 

分 类 号：R378[医药卫生—病原生物学] R563.1[医药卫生—基础医学]