肺炎链球菌dnaJ基因缺陷菌株的构建及毒力变化的初步研究

Construction of dnaJ-defecient mutant strain of Streptococcus pneumoniae and preliminary study of its virulence

目的构建肺炎链球菌(Streptococcus pneumoniae,S.pn)dnaJ基因缺陷菌株,并对其毒力作初步研究。方法采用长臂同源多聚酶链式反应(long flanking homology polymerase chain reaction,LFH-PCR)技术将dnaJ基因替换为红霉素耐药基因(erm)后同源重组于肺炎链球菌,在含红霉素的血平板上筛选出dnaJ缺陷菌株。用PCR鉴定缺陷菌株,以细菌的吸光度值D(600 nm)观察体外缺陷菌株生长情况。将44只BALB/c小鼠采用随机数字表法分为2组,分别用野生菌株和缺陷菌株处理,腹腔攻毒100μl高(2×108cfu)、低(2×106cfu)剂量的菌液,各处理组的不同剂量组均有11只小鼠。记录小鼠死亡时间,计算存活率。60只BALB/c小鼠采用随机数字表分为2组,分别鼻腔滴注含菌量为2×107 cfu的30μl D39菌液和△dnaJ菌液,感染后6、12、24、36、48 h处死每组中的5只小鼠,分别取鼻咽灌洗液、肺组织(匀浆)和心脏血培养,根据每组平均菌落计数结果绘制细菌在宿主体内的定植曲线。结果 PCR结果显示dnaJ基因完全被erm基因所替代,构建dnaJ缺陷菌成功;单个菌落培养基生长情况表明体外热休克状态抑制dnaJ缺陷菌生长;小鼠毒力实验显示腹腔感染缺陷菌株的小鼠存活率可达到100%,而感染野生菌株的小鼠全部死亡,两者比较有统计学差异(P<0.01)。小鼠鼻腔感染模型中,缺陷菌株在各时间点鼻咽灌洗液和肺部的细菌载量均显著低于野生菌株,而且入血后可在感染24 h内被宿主清除,上述差异有统计学意义(P<0.01)。结论采用LFH-PCR技术作基因突变完全替代dnaJ基因,方法简便快捷;dnaJ的缺陷影响细菌在体外应激条件下的生长,并显著降低细菌在宿主体内的毒力和定植。

Objective To lay a foundation for further exploration of host defense responses to DnaJ through the construction of dnaJ-defecient mutant strain of Streptococcus pneumoniae（S.pn） and the preliminary study of its virulence.Methods The dnaJ gene was replaced with an erythromycin resistance gene（erm） by long flanking homology polymerase chain reaction（LFH-PCR）,and the recombinant fragment was transformed into S.pn.The dnaJ-defecient mutant strain of S.pn was screened out from the blood plates containing erythromycin and was verified by PCR.The growth of dnaJ-defecient mutant strain in vitro was observed based on its OD value（600 nm）.Forty-four BALB/c mice were randomly divided into 2 groups,a wild-type strain group and a dnaJ-deficient mutant strain group.Each group was subdivided into a high dose（2×108 cfu） and a low dose（2×106 cfu） group（11 mice in each subgroup）.The mice of the two groups were intraperitoneally given a high and a low dose of 100 μl D39 and △dnaJ bacteria liquid separately.The mice death time was recorded,and the survival rate was calculated.Sixty BALB/c mice were randomly divided into 2 groups,the mice of which were intranasally given D39 and △dnaJ bacteria liquid（30 μl,2×107 cfu） separately.After 6 h,12 h,24 h,36 h and 48 h infection,the mice were put to death（5 mice of each time point）.The mice nasopharyngeal lavage fluid,lung tissue（homogenate） and heart blood were collected and cultured seperately.The colonization curve of the bacteria was drawn according to the average colony counting in each group.Results The PCR result showed that the dnaJ gene was completely replaced by the erm gene,and the dnaJ-deficient mutant strain was successfully constructed.The growth of the single colony in culture medium showed that the dnaJ-deficient bacteria growth was inhibited by heat shock in vitro.The virulence test suggested that the survival rate of mice with abdominal infection of the deficient strain was 100%,while that of mice with abdominal infection of wild-type strain was 0%（P0.01）.In the intranasal infection experiment,the loading amount of the deficient strain,which could be cleared in the blood within 24 h after infection,was significantly lower than that of the wild-type strain in the nasopharyngeal lavage fluid and lung tissue at each time point（P0.01）.Conclusion It is a convenient way to completely substitute for dnaJ gene using LFH-PCR.The dnaJ deficiency inhibits the growth of dnaJ-deficient bacteria under the heat shock in vitro and simultaneously reduces the bacterial virulence and colonization in vivo.