染料木素对MRSA外排蛋白表达的影响

Effect of genistein on MRSA efflux protein expression

摘要:

[目的] 研究染料木素对耐甲氧西林金黄色葡萄球菌（MRSA）外排蛋白的影响。[方法] 通过联合药敏实验检测染料木素影响MRSA对环丙沙星的敏感性；利用等重同位素多标签相对定量蛋白质组学（iTRAQ）技术，检测染料木素作用MRSA41577后菌体蛋白表达量的变化；通过生物信息学方法对差异显著的蛋白进行系统分析；通过qPCR和尼罗红外排实验，探讨耐药相关的蛋白介导细菌耐药的作用机制。[结果] 联合药敏实验结果显示，染料木素能增强MRSA对环丙沙星的敏感性；通过iTRAQ技术检测到差异显著蛋白共有129个，包括60个表达上调的蛋白和69个表达下调的蛋白；生物信息学分析结果显示，与细菌耐药相关的蛋白约有14个，其中，通过主动外排系统介导细菌耐药的蛋白主要有PstB、PstS等；qPCR结果显示，与对照组相比，PstB、PstS的基因表达量分别下降了51.6%和78.6%；尼罗红外排实验结果显示，染料木素与尼罗红之间存在竞争关系，为MRSA41577的竞争性抑制剂。[结论] 染料木素可通过降低MRSA41577外排基因pstB和pstS的mRNA表达量，进而影响PstB、PstS外排蛋白的表达来逆转细菌耐药；此外，染料木素还是MRSA41577的竞争性外排抑制剂，可通过与底物竞争外排的方式，使抗菌药物留在菌体内发挥抗菌作用。

Abstract:

[Objective] To study the effect of genistein on methicillin-resistant Staphylococcus aureus (MRSA) efflux proteins. [Methods] Combined drug sensitivity test was used to detect the effect of genistein on the sensitivity of MRSA to ciprofloxacin. The isobaric tags for relative and absolute quantitation (iTRAQ) technology were used to detect the change of bacterial cell protein expression after genistein was applied to MRSA41577. The bioinformatics method was used to analyze the significantly different proteins. qPCR and Nile Red efflux assay were used to explore the mechanism of drug resistance-related proteins mediating bacterial resistance. [Results] Combined drug sensitivity test showed that genistein enhanced the sensitivity of MRSA to ciprofloxacin. A total of 129 significantly different proteins were detected by iTRAQ technology, including 60 proteins with up-regulated expression and 69 proteins with down-regulated expression. Bioinformatics analysis showed that there were about 14 proteins related to bacterial resistance, of which PstB, PstS and other proteins mainly mediated bacterial resistance through the active efflux system. qPCR results showed that compared with the control group, PstB and PstS gene expression decreased by 51.6% and 78.6%, respectively. Nile Red efflux assay showed there was a competitive relationship between genistein and Nile Red, so that genistein was a competitive inhibitor of MRSA41577. [Conclusion] Genistein reverses bacterial resistance by reducing mRNA expression of the efflux genes pstB and pstS, and affected the expression of efflux proteins PstB and PstS of MRSA41577. Besides, genistein is also a competitive efflux inhibitor of MRSA41577 and plays antibacterial effect by competing with substrates for efflux, which makes antibacterial drugs stay in the bacteria.