基于RNA-Seq技术分析白藜芦醇对副溶血弧菌生物被膜的抑制作用

Inhibition analysis of resveratrol against Vibrio parahaemolyticus biofilm based on RNA-Seq technology

【目的】利用可食用植物素抑制致病菌生物被膜受到广泛的关注。本文研究分析白藜芦醇对水产品致病菌副溶血性弧菌(Vibrio parahaemolyticus)生物被膜形成的影响及重要的调控基因。【方法】研究测定亚抑菌浓度白藜芦醇对V.parahaemolyticus生物被膜形成和胞外多糖分泌的影响,采用RNA-Seq技术分析白藜芦醇作用下V.parahaemolyticus基因表达水平变化,并利用荧光定量PCR方法验证部分差异表达基因。【结果】白藜芦醇对V.parahaemolyticus的最小抑菌浓度为20μg/m L,亚抑菌浓度5μg/m L和10μg/m L作用下能显著抑制该菌的生物被膜形成和胞外多糖产量(P<0.05),扫描电镜观察发现细菌的粘附量和胞外分泌物显著减少。V.parahaemolyticus在10μg/m L白藜芦醇作用下共检测到106个基因表达量发生显著变化(P<0.05),其中上调基因约占22.6%,下调基因约占77.4%。这些基因主要在7条代谢通路中被显著富集,其中外膜蛋白(W,Yed S,Omp K)、群体感应(Lux S)、鞭毛蛋白(Fla A)、菌毛蛋白(Pil Q)、溶血素分泌蛋白等14个调控基因可能与V.parahaemolyticus生物被膜有关,呈现显著下调表达。荧光定量PCR发现lux S、trh、tlh和fla A 4个基因在白藜芦醇作用后表现不同程度的显著下降,与转录组结果一致。【结论】白藜芦醇抑制V.parahaemolyticus生物被膜形成是一个多基因参与、多个生物过程协同调控的过程,其主要通过干扰V.parahaemolyticus新陈代谢过程、群体感应系统、膜蛋白分泌通路,抑制细胞的粘附和生物被膜形成。研究为揭示白藜芦醇抗生物被膜的分子机制提供参考。

[Objective] Food phytochemicals as biofilm inhibitor of pathogens have been highlighted. Our study aimed to investigate the effects of resveratrol on biofilm formation of an aquatic pathogen Vibrio parahaemolyticus, and to elucidate the important regulatory genes. [Methods] In the subinhibitory concentrations, the inhibition of resveratrol aganist biofilm and exopolysaccharides of V. parahaemolyticus was detected, and the differentially expressed genes were analyzed based on RNA-Seq. Four genes involved in biofilm formation was validated by q RT-PCR. [Results]The minimum inhibitory concentration of resveratrol against V. parahaemolyticus was 20 μg/m L. Resveratrol at the subinhibitory concentration of 5 μg/m L and 10 μg/m L significantly decreased the biofilm development and exopolysaccharides production in V. parahaemolyticus（P〈0.05）. Scanning electron microscopy micrographs showed a significant reduction of adherence and extracellular polymeric substances. RNA-Seq analysis revealed that 22.6%up-regulated and 77.4% down-regulated gene（P〈0.05） after treatment by 10 μg/m L resveratrol among 106 differential gene expressions. These differential genes in V. parahaemolyticu focused on 7 metabolic pathways, and 14 genes involved in biofilm formation were down-regulated by resveratrol, such as outer membrane protein（W, Yed S,Omp K）, quorum sensing（Lux S）, flagellin（Fla A）, fimbrial assembly protein（Pil Q）, hemolysin secreted protein. q RTPCR confirmed that the expressions of lux S, trh, tlh and fla A, was significantly repressed in the presence of resveratrol, which was consistent with transcriptomics data. [Conclusion] Inhibitory activity of resveratrol on biofilm formation was assicated with multiple genes and diverse cellular processes in V. parahaemolyticus. These findings suggest that resveratrol would disturb various metabolic pathways, particularly quorum sensing system, adhesion process and membrane proteins secretion, resulting in inhibition of attachment and biofilm development. The present work provided valuable information to explore molecular mechanism of resveratrol as an novel anti-biofilm compound.