和厚朴酚对大肠埃希氏菌生物被膜形成的抑制机制

Mechanism of honokiol in inhibiting Escherichia coli biofilm formation

摘要:

【目的】研究不同浓度的和厚朴酚(honokiol)抑制大肠埃希菌(Escherichia coli)的供试菌株10389生物被膜(biofilm，BF)形成的作用机制。【方法】用氯化三苯基四氮唑比色法(TTC)和四唑盐减低法(XTT)测定honokiol抑制E.coli10389生物被膜形成的药物最低抑菌浓度(MIC)和最低杀菌浓度(MBC)及其抑制作用与时间的关系；通过qRT-PCR法检测不同浓度的honokiol对E.coli10389生物被膜形成基因和群体感应系统相关基因表达量的影响；通过生物发光法和qRT-PCR法检测亚-MIC honokiol对E.coli10389呋喃糖基硼酸二酯(AI-2)及其调控的与生物被膜形成相关的下游基因表达量的影响。【结果】Honokiol能抑制E.coli10389生物被膜的形成，但不同浓度的honokiol抑制E.coli10389 BF形成的作用机制不同。其中，与对照组相比，MIC的honokiol能使E.coli10389 BF形成相关基因编码毒素(hha)和细菌酸性调节因子(ariR) mRNA的表达量显著提高，抗毒素(ybaJ)的mRNA表达量显著降低。亚-MIC的honokiol则能抑制E.coli10389分泌AI-2的量，降低由其调控的与BF形成相关的下游基因的mRNA表达量。与对照组相比，16 mg/mL的honokiol可使荚膜异多糖酸基因mqsR、类黏蛋白基因mcbR和鞭毛形成基因csrA、flhD、flhC和flic的mRNA表达量分别降低65.21%、55.01%、73.16%、62.01%、60.30%和59.71%。【结论】Honokiol能抑制E.coli10389 BF的形成，但不同浓度的honokiol其抑制E.coli10389 BF形成的作用机制不同。其中，MIC的honokiol主要是通过影响BF形成的相关基因表达量来抑制E.coliBF的形成；而亚-MIC的honokiol则主要是通过抑制Luxs/AI-2系统的AI-2合成酶luxs基因的表达量，降低AI-2的分泌量，进而影响荚膜多糖、类黏蛋白和鞭毛等合成抑制E.coli BF的形成。

Abstract:

[Objective] To investigate the mechanism of honokiol at different concentration in inhibiting Escherichia coli 10389 biofilm (BF) formation. [Methods] We employed triphenyltetrazolium chloride (TTC) method to determine the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of honokiol for the test strain and tetrazolium salt (XTT) reduction assay to investigate the influence of honokiol concentration on BF formation of the test strain and the influence over time. Through qRT-PCR, we examined the effect of honokiol concentration on the expression of genes related to BF formation and quorum sensing of the test strain. We detected the effect of honokiol at sub-MIC on the expression of furanosyl borate diester (AI-2) in E.coli 10389 and its regulated downstream genes associated with BF formation by bioluminescence and qRT-PCR. [Results] Honokiol inhibited the BF formation of E.coli 10389, but the mechanism was different for different concentration of honokiol. Among them, honokiol at MIC significantly increased the mRNA expression of toxin gene hha and bacterial acid regulator ariR involved in the BF formation of E.coli 10389 and significantly decreased the mRNA expression of toxin overexpression-modulating gene ybaJ compared with the control group. Honokiol at sub-MIC can suppress AI-2 secretion by E.coli 10389 and decrease the mRNA expression of its regulated downstream genes related to BF formation. Compared with the control, 16 mg/mL honokiol reduced the mRNA expression of colanic acid gene mqsR, mucoid gene mcbR, and flagellum formation-related genes csrA, flhD, flhC, and flic by 65.21%, 55.01%, 73.16%, 62.01%, 60.30%, and 59.71%, respectively. [Conclusion] Honokiol suppresses the BF formation of E.coli 10389, but the mechanism is different for honokiol of different concentration. Among them, honokiol at MIC mainly inhibits BF formation by affecting the expression of related genes, and honokiol at sub-MIC by suppressing the expression of the AI-2 synthase luxs gene of Luxs/AI-2 system, decreasing AI-2 secretion, and further influencing the synthesis of capsular polysaccharide, mucoid, and flagellin.