罗非鱼无乳链球菌ΔessA、ΔessB、ΔessC敲除菌株的构建及其特性分析

Construction and characterization research of essA,essB and essC-deleted mutants in Streptococcus agalactiae from tilapia

Ⅶ型分泌系统存在于无乳链球菌(Streptococcus agalactiae)中,其组成膜蛋白对底物蛋白的分泌转运至关重要。为此利用热敏型自杀质粒,构建了含氯霉素筛选标签的膜蛋白重组敲除载体pSET4s-ΔessA、pSET4s-ΔessB、pSET4s-ΔessC,经同源重组后,利用PCR技术筛选到了其缺失突变株-无乳链球菌ΔessA、ΔessB、ΔessC。通过菌株生长曲线分析,ΔessA、ΔessB、ΔessC敲除株均较野生株(WT)生长过程变慢,其中ΔessA、ΔessB敲除株在生长早期有显著性差异(P<0.01)。对ESAT6蛋白分泌影响分析显示,与WT株相比,ΔessA、ΔessB、ΔessC敲除株ESAT6的表达量均显著下调(P<0.01)。结果显示,ΔessA、ΔessB、ΔessC敲除株均较WT株毒力显著降低(P<0.01)。研究表明,essA、essB、essC为无乳链球菌Ⅶ型分泌系统重要的膜蛋白,其基因缺失造成了分泌蛋白ESAT6 mRNA表达水平下降,影响了菌株毒力。

The membrane proteins are critical to substrate protein secretion inⅦsecretion system existing in Streptococcus agalactiae.In this study,we constructed the membrane proteins knockout vectors with chloramphenicol tag including pSET4s-ΔessA,pSET4s-ΔessB and pSET4s-ΔessC,and after the homologous recombination,we screened the mutant S.agalactiaeΔessA,ΔessB andΔessC via PCR assays.Based on the growth curve analysis,S.agalactiaeΔessA,ΔessB andΔessC strains showed signi-ficantly slower growth process than the wild type strain(WT).ΔessA andΔessB strains had significant difference in early stage of growth compared with WT strain(P<0.01).According to the secretion product analysis of ESAT6 protein,S.agalactiaeΔessA,ΔessB andΔessC strains showed significantly lower ESAT6 product than WT(P<0.01).According to challenge test analysis,the deletion of essA,essB or essC gene significantly reduced virulence of S.agalactiaeΔessA,ΔessB andΔessC compared with WT(P<0.01).The results suggest that essA,essB and essC proteins are important component membrane proteins ofⅦsecretion system in S.agalactiae,and these genes deletions cause ESAT6 mRNA expression and virulence decline.