肺炎克雷伯菌转录调控子CRP对菌株粘附能力及细胞活性的影响  

Influence of Klebsiella pneumoniae CRP protein on bacterial adhesion and virulence in vitro

作　　者：谭斌[1] 白群华[1] 罗美[1] 杨世亚[1] 薛健[1] 周锡鹏[1] 李迎丽[1] 邱景富[1] 

机构地区：[1]重庆医科大学公共卫生与管理学院,重庆400016

出　　处：《第三军医大学学报》2014年第22期2259-2262,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(31071093;31170129;31200064);重庆市自然科学基金(CSTC2010BB5354;CSTC2010BB5103)~~

摘　　要：目的分析肺炎克雷伯菌临床分离株WT(wild type)、回补株(c-Δcrp)和突变株(Δcrp)对人肺癌上皮细胞A549细胞的粘附能力及细胞活性的影响。方法肺炎克雷伯菌WT株、c-Δcrp株和Δcrp株感染人肺癌上皮细胞A549,经裂解液裂解后平板计数计算粘附的菌量。LDH释放法检测细菌对细胞的毒性,优化感染时间和感染指数。结果 WT株及c-Δcrp株粘附的菌量分别为logCFU=5.145和logCFU=4.915,均高于Δcrp株(logCFU=4.122),差异有统计学意义(F=8.366,P=0.004)。以MOI=1 000(细菌∶细胞=1 000)的菌量感染靶细胞,37℃孵育8 h,加底物液避光显色10 min,离心所得上清稀释10倍进行测定为最佳反应条件。WT株对细胞的毒性百分比(70.69%)明显高于Δcrp株(19.54%),差异有统计学意义(t=8.843,P=0.001)。结论 crp基因敲除后,突变株的细胞毒性和粘附力明显下降,肺炎克雷伯菌转录调控子CRP对菌株粘附力及细胞活性具有正向调控作用。Objective To analyze the adhesion and cell virulence of Klebsiella pneumonia wild type（ WT） strain,complemented strain c-Δcrp（ c AMP receptor protein） and mutant strain Δcrp,in order to investigate crp gene on the adhesion and cell toxicity of Klebsiella pneumonia. Methods After infection of A549 cells by Klebsiella pneumonia WT strain,c-Δcrp strain and Δcrp strain,the cells were lysed and the bacteria were quantified by plating appropriate dilutions on Luria-Bertani agar plates. LDH release was detected to estimate cell activity. Infection time and MOI were optimized. Results The adhesion ability of Klebsiella pneumonia WT（ log^CFU= 5. 145） and c-Δcrp strain（ log^CFU= 4. 915） was higher than that of Δcrp strain（ log^CFU= 4. 122）（ P = 0. 004）. The optimal conditions to determinate the LDH release included infected cells incubation for 8 h at 37 ℃,the developing time for 10 min in dark,and 1∶ 10 dilution of the supernatant for test. The virulence of WT strain（ 70. 69%） was significantly higher than that of Δcrp strain（ 19. 54%）（ P = 0. 001）. Conclusion Knocking-out of crp gene causes obvious decrease of cellular toxicity and adhesion,comparing with the WT strain and c-Δcrp strain. Klebsiella pneumonia CRP protein positively regulates bacterial virulence and adhesion.

关 键 词：肺炎克雷伯菌 cAMP受体蛋白 A549 粘附力 细胞活性 

分 类 号：R378.996[医药卫生—病原生物学] R394.3[医药卫生—基础医学]