clpE基因缺失对肺炎链球菌毒力的影响

Effect of clpE gene deletion on virulence of Streptococcus pneumoniae

【目的】探索clpE基因缺失对肺炎链球菌毒力的影响。【方法】用长臂同源多聚酶链式反应(LFH-PCR)方法失活clpE基因,用PCR、测序鉴定缺失菌株,通过动物实验观察clpE基因缺失株毒力改变情况,同时用细胞实验比较clpE基因缺失株和野生菌对宿主细胞的粘附和侵袭能力,最后用实时荧光定量PCR分析自溶素(major autolysin A,lytA)、表面黏附素A(pneumococcal surface adhesion A,psaA)、溶血素(pneumolysin,ply)、肺炎球菌表面蛋白A(pneumococcal surface protein A,pspA)和神经氨酸酶(neuraminidase,nanA)的表达。【结果】小鼠毒力实验表明野生菌株半数致死时间54h,而缺失株半数致死时间为21d,两者比较有统计学差异(P<0.01);缺失菌在对宿主细胞的粘附能力明显低于野生菌株(P<0.05)。实时荧光定量PCR显示clpE缺失株的五个毒力因子mRNA表达水平均低于野生菌,两者比较有统计学差异(P<0.05);【结论】ClpE通过调控肺炎链球菌多种毒力因子表达,而影响其毒力。

[Obejctive]To study the effect of clpE gene deletion on the virulence of Streptococcus pneumoniae. [Methods] The clpE-deficient strain was constructed by LFH-PCR and identified by PCR and sequencing. The impact of clpE mutant on the virulence of S. pneumoniae was evaluated in a mouse model. In addition,we also studied the effect of clpE mutant on adherence and invasion of host cells. Real time RT-PCR was used to measure the mRNA expression levels of autolysin A,pneumococcal surface adhesion A,pneumolysin,pneumococcal surface protein A and neuraminidase. [Results] The clpE gene was replaced completely by erm cassette. Mice virulence experiments showed that the median lethal time of the wide-type was 54 h,whereas that of clpE mutant was 21d （P〈0.01）. Cell culture infection experiments indicated that adherence and invasion of clpE mutant were strongly reduced （P〈0.05）. The expression of virulent factors in clpE mutant was lower than that of the wild-type （P〈0.05）. [Conclusion] ClpE is involved in virulence by modulating the expressions of virulence factors.