N-十一烷酰基环戊酰胺对铜绿假单胞菌生物被膜及毒力的影响

N-Undecanoyl cyclopentamide affects the biofilm and virulence of Pseudomonas aeruginosa

【背景】铜绿假单胞菌(Pseudomonas aeruginosa)耐药性问题日趋严重的重要原因之一是细菌生物被膜的产生,群体感应(quorum sensing,QS)系统在其生物被膜形成过程中发挥了重要作用。QS抑制剂能够抑制生物被膜的形成和毒力因子的分泌,成为解决细菌耐药性问题的新策略。【目的】通过化学方法对las系统信号分子N-(3-氧十二烷基)-L-高丝氨酸内酯[N-3-(oxododecanoyl)-L-homoserine lactone,OdDHL]的母核和酰基侧链同时改变,合成N-十一烷酰基环戊酰胺,命名为Y0-C11-HSL,探讨其对P.aeruginosa生物被膜形成和毒力因子分泌的作用潜力和分子机制。【方法】采用结晶紫染色和扫描电子显微镜(scanning electron microscope,SEM)评价Y0-C11-HSL对生物被膜形成和结构的影响,通过测定毒力因子的产生和运动试验评析Y0-C11-HSL的抑制活性,通过傅里叶红外光谱(Fourier transform infrared spectrometer,FT-IR)研究Y0-C11-HSL对胞外聚合物(extracellular polymers,EPS)表面化学基团的影响,采用分子对接进一步解析Y0-C11-HSL的作用机制。【结果】与对照组相比,在10−200μmol/L浓度梯度下,Y0-C11-HSL能够减少P.aeruginosa生物被膜形成,且在200μmol/L时减少率达24.1%(P<0.01)。此外,在200μmol/L处理下,Y0-C11-HSL能够显著抑制绿脓菌素、鼠李糖脂、胞外多糖和水解蛋白酶的分泌,抑制率分别为34.7%(P<0.01)、33.1%(P<0.01)、27.3%(P<0.01)和37.3%(P<0.01),抑制swarming和twitching运动,抑制率分别为45.6%(P<0.01)和51.7%(P<0.01),影响了EPS表面化学基团。分子对接结果表明,Y0-C11-HSL能与OdDHL结合的LasR受体蛋白竞争性结合。【结论】Y0-C11-HSL能与OdDHL结合的LasR受体蛋白竞争性结合,对转录蛋白产生影响,进而下调P.aeruginosa QS相关基因的表达。

[Background]Biofilm formation is one of the major reasons for the increasing drug resistance of Pseudomonas aeruginosa,and the quorum sensing(QS)system plays a key role in biofilm formation.[Objective]QS inhibitors can inhibit the formation of biofilm and the secretion of virulence factors,serving as a new approach to address drug resistance.We chemically altered both the parent nucleus and acyl side chains of the las QS signaling molecule N-(3-oxododecanoyl)-L-homoserine lactone(OdDHL)to synthesize N-undecanoyl cyclopentamide,named Y0-C11-HSL,aiming to reveal the effects of Y0-C11-HSL on the biofilm formation and virulence factor secretion of P.aeruginosa and the underlying molecular mechanism.[Methods]Cystal violet staining and scanning electron microscopy(SEM)were employed to evaluate the effects of Y0-C11-HSL on the biofilm formation and structure.The inhibitory activity of Y0-C11-HSL was assessed by measuring the production and movement of virulence factors.The effect of Y0-C11-HSL on the surface chemical groups of extracellular polymers(EPS)was investigated by Fourier transform infrared spectrometry(FT-IR),and the mechanism of action of Y0-C11-HSL was studied by molecular docking.[Results]Compared with the control group,10–200μmol/L Y0-C11-HSL reduced the biofilm formation of P.aeruginosa,and the reduction rate reached 24.1%at 200μmol/L(P<0.01).In addition,200μmol/L Y0-C11-HSL inhibited the secretion of pyocyanin,rhamnolipid,extracellular polysaccharide,and hydrolytic protease by P.aeruginosa,with the inhibition rates of 34.7%(P<0.01),33.1%(P<0.01),27.3%(P<0.01),and 37.3%(P<0.01),respectively.Furthermore,Y0-C11-HSL inhibited the swarming and twitching of P.aeruginosa,with the inhibition rates of 45.6%(P<0.01)and 51.7%(P<0.01),respectively,and it affected the EPS surface chemical groups.The molecular docking results showed that Y0-C11-HSL competitively bound to the OdDHL-bound LasR receptor.[Conclusion]Y0-C11-HSL could competitively bind to OdDHL-bound LasR receptor to affect the transcriptional proteins and thus down-regulate the expression of P.aeruginosa QS-related genes.