CAJ | 학술논문

目的研究双组分信号调控系统saeRS对金黄色葡萄球菌临床分离株α溶血素基因(hla)和Panton-Valentine杀白细胞素基因(lukS/F-PV)表达的影响.方法利用同源重组技术敲除saeRS基因,获得金黄色葡萄球菌saeRS基因敲除株,并构建saeRS回复突变株.应用SDS-PAGE检测金黄色葡萄球菌saeR/S野生株、敲除株及回复突变株分泌蛋白的变化,采用RT-PCR检测金黄色葡萄球菌saeR/S野生株、敲除株及回复突变株hla mRNA和lukS-PV mRNA.结果成功构建了金黄色葡萄球菌saeRS基因敲除株SA75△saeRS.与野生株SA75相比,SA75△saeRS的生长无明显差异,但分泌蛋白种类和数量有明显减少且溶血能力明显减弱.saeRS基因回复突变株SA75△saeRS-C可以基本恢复敲除株的溶血能力.在3、6和10h3个时间点与野生株相比,SA75△saeRS的hla mRNA均有明显下降,分别是野生株的7.6％、0％和0.1％.lukS-PV mRNA也均有明显下降,分别是野生株lukS-PV表达量的37.2％、19.2％和20.4％.结论 saeRS基因是调节金黄色葡萄球菌分泌蛋白的关键因子.saeRS基因对金黄色葡萄球菌的hla和lukS-PV的表达具有正调控作用.
목적 연구쌍조분신호조공계통saeRS대금황색포도구균림상분리주α용혈소기인(hla)화Panton-Valentine살백세포소기인(lukS/F-PV)표체적영향.방법 이용동원중조기술고제saeRS기인,획득금황색포도구균saeRS기인고제주,병구건saeRS회복돌변주.응용SDS-PAGE검측금황색포도구균saeR/S야생주、고제주급회복돌변주분비단백적변화,채용RT-PCR검측금황색포도구균saeR/S야생주、고제주급회복돌변주hla mRNA화lukS-PV mRNA.결과 성공구건료금황색포도구균saeRS기인고제주SA75△saeRS.여야생주SA75상비,SA75△saeRS적생장무명현차이,단분비단백충류화수량유명현감소차용혈능력명현감약.saeRS기인회복돌변주SA75△saeRS-C가이기본회복고제주적용혈능력.재3、6화10h3개시간점여야생주상비,SA75△saeRS적hla mRNA균유명현하강,분별시야생주적7.6％、0％화0.1％.lukS-PV mRNA야균유명현하강,분별시야생주lukS-PV표체량적37.2％、19.2％화20.4％.결론 saeRS기인시조절금황색포도구균분비단백적관건인자.saeRS기인대금황색포도구균적hla화lukS-PV적표체구유정조공작용.
Objective To investigate regulating effect of saeRS on the expression of hla and lukSPV in Staphylococcus aureus clinical isolate.Methods The homologous recombination technology was used to achieve saeRS knockout S.aureus clinical isolate (SA75 △saeRS),and to construct saeRS reverse mutant (SA75 △saeRS-C).SDS-PAGE and RT-PCR were used for detecting changes of secretory proteins and changes of hla mRNA and lukS-PV mRNA in SA75 wild type (SA75WT),SA75/△saeRS and SA75/△saeRS-C.Results Compared with SA75WT,SA75/△saeRS,the successfully constructed saeRS knockout strain,showed no distinct difference in growth curve,but a significant decrease in secretory protein types and quantity.The hemolytic ability was inhibited in SA75/△saeRS,but could be restored in SA75/△saeRS-C.The hla mRNA levels of SA75/△saeRS at 3,6 and 10 hours were significantly decreased to 92.4 ％,100％ and 99.9％ of those of SA75WT respectively,and the levels of lukS-PV mRNA were decreased to 62.8％,81.8％ and 79.6％ respectively.Conclusion saeRS was a key factor in regulating secretory proteins in S.aureus,and also had some effect in up-regulation of the expression of lukS-PV and hla in S.aureus clinical isolate.