茶树油对SarA介导的金黄色葡萄球菌生物被膜形成的干预机制研究

Staphylococcus aureus is a common food-borne pathogenic bacterium. It has the ability to form biofilm. Bacteria living in a biofilm are more resistant to food preservatives and more difficult to remove from food compared to free-floating bacteria. Thus, research on pathogenic bacteria biofilm has important academic value in the fields of food processing and preservation. Natural plant extracts, tea tree oil, being a potential preservative, has the ability to disrupt the Staphylococcus aureus biofilm. At present, due to its resistance to biofilm mechanism has not yet clear. Because the mechanism of tea tree oil against the bacteria biofilm is not chear, its development and application as food preservatives was limited. In this program, we firstly plan to analyze the transcriptional profiles of Staphylococcus aureus grown in biofilm under tea tree oil stress using DNA microarray, in order to assay the the effect of tea tree oil on the genes and signal pathways of biofilm fomation mediated by SarA, a transcriptional regulator, and reveal the molecular mechanism of tea tree oil against the biofilm. To verify the microarray results by the technology such as knock-out, real time RT-PCR, western-blot. Moreover, flow cytometry and the laser confocal microscopy technique will be applied to analyse the relativity between gene variation and phenotype, which conforms the molecular intervention mechanism of the potential preservative tea tree oil. These results lay the foundation to futher develop and utilize tea tree oil, and meanwhile provide new strategies to study the antibacterial mechanism of the other food preservatives.

金黄色葡萄球菌（简称金葡菌）是一种常见食源性致病菌，具有形成生物被膜的能力。与浮游菌相比，被膜态细菌对防腐剂更具有耐性，更难从食品中清除。因此，研究致病菌生物被膜对食品加工和保藏具有重要意义。天然植物提取物- - 茶树油，作为一种潜在的防腐抗菌剂具有较好的干预金葡菌生物被膜形成的能力。目前由于该油抗生物被膜机制尚未清楚，限制了其作为食品防腐抗菌剂的开发应用。本项目拟首先借助生物芯片技术，分析茶树油压力下被膜态金葡菌的转录图谱，考察茶树油对转录调节因子SarA介导的生物被膜形成相关基因及信号通路的影响，揭示茶树油对SarA介导的金葡菌生物被膜形成的干预机制；再利用基因敲除技术、实时荧光定量PCR及免疫印迹技术验证芯片结果的正确性，运用激光扫描共聚焦显微成像系统和流式细胞术分析基因变化与表型的相关性。本研究将为茶树油的进一步开发应用提供理论依据，为其他天然食品防腐剂的抗菌机制研究提供新思路。

金黄色葡萄球菌（金葡菌）是一种常见食源性致病菌，具有形成生物被膜的能力。与浮游菌相比，被膜态细菌对防腐剂更具有耐性，更难从食品中清除。因此，研究致病菌生物被膜对食品加工和保藏具有重要意义。本研究发现茶树油可以通过抑制细胞间粘附素（PIA）的合成及胞外DNA（eDNA）的释放量从而有效抑制金葡菌生物被膜的形成，同时随着药物浓度的增加PIA的合成、eDNA的释放量逐渐减少，且呈剂量依赖性。同时本项目通过RNA测序（RNA-Seq）研究了天然植物提取物——茶树油抗金葡菌的转录组学影响。实验表明，悬浮菌加药组与悬浮菌对照组相比，共有715个基因表达显著差异，其中294个基因的表达显著下调(≤-2fold)，421个基因的表达显著上调(≥2fold)；被膜菌加药组与被膜菌对照组相比，共有304个基因表达显著差异，其中104个基因的表达显著下调(≤-2fold)，200个基因的表达显著上调(≥2fold)，并对差异表达的基因进行了GO和KEGG富集分析；其中SarA表达倍数降低1.3倍，表明茶树油通过抑制SarA降低生物被膜。同时，荧光实时定量RT-PCR试验结果显示icaR，sspA，lrgA，fnbB，lytM，glpT，msmX，agrA，sarA，cidA基因的差异表达趋势与RNA测序结果一致。利用激光共聚焦扫描显微镜的绿色荧光蛋白荧光成像和流式细胞术从显微结构更深入分析茶树油的抗金葡菌生物被膜机制，进一步明确基因表达变化与表型变化的相关性。.在此基础上，我们发现亚抑菌浓度的茶树油作用后的金葡菌上清对兔血红细胞的溶血效果具有抑制作用，并通过western blot法从蛋白水平证明了亚抑菌浓度的茶树油对促进生物被膜形成的α-溶血素具有较好的抑制作用，并通过荧光实时定量RT-PCR从转录水平分析了茶树油对icaA、sarA、cidA、hla这些生物被膜相关基因表达的抑制作用。.本研究首次在转录组学水平分析了茶树油抑制被膜态金葡菌的分子机制，为茶树油的进一步开发应用提供理论依据，为其他天然食品防腐剂的抗菌机制研究提供新思路。.本项目研究过程中还建立的多个筛选细菌生物被膜和胞外毒力蛋白的平台，同时取得了多项成果。共发表SCI论文12篇，申请专利3项，培养硕士研究生2名，在读硕士研究生2名。