HtrA基因缺陷对肺炎链球菌毒力影响探究.docVIP

HtrA基因缺陷对肺炎链球菌毒力影响探究 　 作者：黄远帅，许颂霄，朱旦，阳强，刘明芳，陈淑惠，张雪梅，尹一兵 【关键词】 肺炎链球菌；毒力因子；密度感应系统；HtrA基因 　　comEffect of HtrA gene deletion on virulence of Streptococcus pneumoniae 　　【Abstract】 AIM: To investigate the effect of HtrA gene deletion on the virulence of Streptococcus pneumoniae. METHODS: HtrAdeficient strain was constructed by insertion, replication and inactivation, and identified by PCR. Relative quantitative RTPCR was used to measure the expression levels of virulent factors and study the virulence change by mice virulence experiments. RESULTS: The expression of virulent factors in HtrAdeficient strain was lower than that of the widetype (P＜0.05). Transformation capability of HtrAdeficient strain obviously decreased. Mice virulence experiments showed the median lethal time of the widetype was 22 h, while that of HtrA mutant was 8 d (P＜0.01). The elimination of HtrA mutant in mice blood was faster than that of the widetype. CONCLUSION: The deficiency of HtrA gene decreases the expression of virulent genes and the transformation capability, which finally reduced the bacterial virulence. 　　【Keywords】 Streptococcus pneumoniae; virulence factor; quorum sensing; HtrA gene 　　【摘要】 目的：HtrA基因可编码肺炎链球菌的一种重要蛋白酶，本文目的是探索HtrA基因缺陷对细菌毒力的影响. 方法：用插入复制失活的方式使HtrA基因缺失，通过RTPCR分析毒力因子表达，并通过动物试验观察HtrA基因缺陷株毒力改变情况. 结果：RTPCR显示HtrA缺陷株毒力因子表达低于野毒株，两组比较Plt;0.05；转化试验发现缺陷菌株转化力下降；小鼠毒力试验表明野毒株半数致死时间22 h，而缺陷株半数致死时间8 d，Plt;0.01，有显著性差异；缺陷菌在小鼠体内被清除速度也显著快于野毒株. 结论：HtrA基因缺陷使肺炎链球菌多种毒力因子表达减弱，自然转化能力下降，毒力降低. 　　【关键词】 肺炎链球菌；毒力因子；密度感应系统；HtrA基因 　　0引言 　　肺炎链球菌（Streptococcus pneumoniae, S.pn）是世界范围内引起感染和死亡的重要病原菌之一，是引起肺炎、脑膜炎和中耳炎最常见的病原菌［1］. 目前广泛使用的23价肺炎链球菌多糖疫苗免疫原性弱，对老人、幼儿和免疫缺陷疾病患者预防保护效果较差［2］. 以破伤风和白喉类毒素作载体的11价S.pn结合疫苗仅对2岁以下的婴幼儿有较好的免疫原性［3］. 最新研制的S.pn溶血素等蛋白类疫苗也只是处于早期临床试验阶段［4-5］. 要彻底解决S.pn感染的防治关键在于进一步阐明肺炎链球菌致病的分子机制. 　　HtrA (high temperature requirement A)是S.pn的一种热休克诱导的丝氨酸蛋白酶，又叫做DegP或DO蛋白酶［6］. HtrA同时具有一般的分子伴侣功能和蛋白水解活性，环境温度升高时其蛋白酶活性变得明显［7］. 目前，已经在多种生物中发现了HtrA的相似体，比如细菌、酵母、植物［8］和人类［9］. 在这些生物中HtrA的作用大多表现为帮助它们逃避高氧浓度、高温和高渗等环境变化. HtrA存在于多