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

摘要:

【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200 μmol/L时减少率达24.1% (P＜0.01)。此外，在200 μmol/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。

Abstract:

[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, 10–200 μmol/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, 200 μmol/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.