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

摘要:

【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 .0l）；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。

Abstract:

[Objective] 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.