agrB/D/C对Agr系统活性及金黄色葡萄球菌毒力的影响

The effect of Staphylococcus aureus agrB/D/C on Agr system and virulence factors expression

目的:构建金黄色葡萄球菌(金葡菌)ΔagrB/D/C/Newman敲除株,分析agrB/D/C对Agr系统活性及金葡菌毒力的影响,为深入研究不同型别Agr系统对金黄色葡萄球菌的致病作用提供依据.方法:利用同源重组技术,以pBT2为敲除载体,构建ΔagrB/D/C/Newman敲除株,检测野生株和敲除株的溶血能力,色素形成能力,生物膜形成能力及毒力基因表达.结果:成功构建了ΔagrB/D/C/Newman敲除株,与野生株Newman相比,敲除株溶血能力显著降低,色素形成能力显著降低,生物膜形成能力显著增加;敲除株培养上清中的蛋白种类和数量较野生株显著减少;实时荧光定量PCR(RT-PCR)结果显示ΔagrB/D/C/Newman敲除株毒力因子的转录水平较野生株显著降低.结论:agrB/D/C能够影响AgrA的表达从而影响Agr系统的功能,agrB/D/C基因敲除后显著改变毒力因子的表达水平.

Objective:To construct the agrB/D/C locus gene knockout mutant of Staphylococcus aureus(S.aureus)and evaluate the effect of agrB/D/C genes on the Agr system and the expression of virulence factors.Methods:The temperature-sensitive S.aureus knock-out vectors are constructed as follows.The left and right homologous arm of agrB/D/C were cloned and inserted into pBT2 vector to construct knock-out plasmid pBT2∷agrB/D/C.Then plasmid pBT2∷agrB/D/C were transferred into S.aureus strain Newman.ΔagrB/D/C/Newman mutant were prepared by homologous recombination.The biological characteristics and virulence genes expression of Newman wild strain and mutant strain were studied.The proteins in the culture supernatant were analyzed with SDS-polyacrylamide gel electrophoresis(SDS-PAGE)analysis and Mass Spectrometry.The transcriptional level of virulence genes were detected using Quantitative Real-time PCR.Results:ΔagrB/D/C/Newman mutant was successfully constructed.Compared with wild Newman strain,the hemolytic ability and the pigment formation of mutant strain was significantly reduced,while the ability of biofilm formation was significantly increased.As compared to that of wild Newman strain,SDS-PAGE and Mass Spectrometry analysis results suggested that the species and quantity of protein expression was significantly decreased in culture supernatant ofΔagrB/D/C/Newman mutant,and RT-PCR results showed that virulence genes transcription was significantly reduced inΔagrB/D/C/Newman strain as well.Conclusion:The agrB/D/C of S.aureus has great influence on the function of Agr system,and a grB/D/C deficiency can significantly alter the expression of virulence factors.