芦丁干预S.suis生物被膜的形成及其对cps基因簇和毒力基因表达的影响

发布时间：2018-04-04 17:00

  本文选题：猪链球菌　切入点：生物被膜　出处：《东北农业大学》2017年硕士论文

【摘要】：细菌生物被膜(bacterial biofilms,BF)是指单一或者多种细菌为了适应其周边环境吸附于生物或非生物表面,并分泌大量蛋白类、多糖类、脂类、胞外DNA等复合物,使得细菌之间相互粘连成蘑菇状的膜样结构。它是与浮游态细菌相对的一种生长方式,当细菌形成BF时,其耐药性会增强10~1000倍,对临床用药挑战巨大。猪链球菌(Streptococcus suis,S.suis)是一种重要的人畜共患病病原菌,对人和猪的健康安全危害严重。当S.suis形成BF后,药物很难完全清除,从而造成持续感染。所以,如何抑制S.suis BF的形成已成为兽医临床急需解决的难题。荚膜多糖(Capsular Polysaccharide,CPs)是2型S.suis的毒力因子,受荚膜多糖合成基因(cps)簇调控。它可以通过介导黏附影响S.suis BF的形成。cps基因簇中某些基因表达量变化或突变会导致荚膜多糖含量及结构变化,从而促进细菌的黏附和BF的形成。芦丁(Rutin)是一种黄酮类化合物,在自然界分布广泛且价廉易得到,具有丰富的药理作用。本课题组前期发现丁香叶水提物具有很好的抑制S.suis生物被膜形成的作用,通过谱效关系和灰色关联度分析,确定芦丁为其发挥作用的主要活性成分,但其作用机制尚不明确。本研究通过S.suis体外BF模型,应用扫描电镜技术和结晶紫染色法观察芦丁对S.suis BF的干预作用。并在S.suis BF形成的黏附阶段考察芦丁对BF形成过程中cps基因簇和毒力基因的影响,首先从基因突变层面和mRNA表达水平探讨芦丁对cps基因簇的影响,然后应用荧光定量Real-time PCR检测芦丁对S.suis BF相关的毒力因子及群体感应信号分子Lux S的影响,从黏附的角度阐明其干预猪链球菌生物被膜的机制。结果显示芦丁对S.suis的MIC为0.3125 mg/mL,芦丁在1/2 MIC和1/4 MIC时抑制S.suis BF的形成。在1/4 MIC时,芦丁虽然不影响S.suis的生长但能抑制其黏附。1/4 MIC芦丁上调cps2D、cps2E、cps2Q、cps2S cps2J、cps2C、cps2R、gdh、mrp、gapdh基因mRNA的表达,下调cps2M、cps2V、cps2U、cps2B、cps2K、ef、sly基因m RNA的表达。综上所述,芦丁可通过调节S.suis荚膜多糖的合成以及S.suis毒力基因表达,抑制其黏附,从而抑制S.suis BF的形成,芦丁是一种良好的黏附抑制剂。
[Abstract]:Bacterial biofilms (BFs) refers to a single or multiple bacteria that, in order to adapt to its surrounding environment, adsorb on biological or abiotic surfaces, and secrete a large number of protein, polysaccharides, lipids, extracellular DNA and other complexes.The bacteria form a mushroom-like membrane structure that adheres to each other.It is a growth pattern opposite to that of planktonic bacteria. When bacteria form BF, the drug resistance increases by 101000 times, which is a great challenge to clinical drug use.Streptococcus suis S.suis is an important zoonotic pathogen, which is harmful to human and pig health and safety.When S.suis forms BF, the drug is difficult to remove completely, causing persistent infection.Therefore, how to inhibit the formation of S.suis BF has become an urgent problem in veterinary clinic.Capsular polysaccharide (CPS) is a virulence factor of type 2 S.suis, which is regulated by CPS cluster.It can affect the formation of S.suis BF by mediating adhesion. The change or mutation of some genes in S.suis BF gene cluster will lead to changes in the content and structure of capsule polysaccharides, thus promoting the formation of bacterial adhesion and BF.Our group found that the water extract of clove leaves had a good effect on inhibiting the formation of S.suis biofilm. The main active components of Rutin were determined by spectrum and activity relationship and grey correlation analysis, but the mechanism of its action was not clear.In this study, the effect of rutin on S.suis BF was observed by scanning electron microscopy (SEM) and crystal violet staining by means of BF model of S.suis in vitro.The effects of rutin on the cps gene cluster and virulence genes during the formation of S.suis BF were investigated at the adhesion stage of S.suis BF. The effects of rutin on the cps gene cluster were investigated at the level of gene mutation and mRNA expression.Then the effects of rutin on S.suis BF related virulence factor and colony sensing signal molecule Lux S were detected by fluorescence quantitative Real-time PCR, and the mechanism of rutin interfering with the biofilm of Streptococcus suis was elucidated from the point of view of adhesion.The results showed that the MIC of rutin to S.suis was 0.3125 mg / mL. Rutin inhibited the formation of S.suis BF at 1 / 2 MIC and 1 / 4 MIC.鍦，

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