1,3-丙二胺通过群体密度感应系统对铜绿假单胞菌生物膜形成的抑制作用

Inhibitory effects of 1,3-diaminopropane on the biofilm formation of Pseudomonas aeruginosa via interaction with quorum sensing system

目的:探讨1,3-丙二胺对铜绿假单胞菌生物膜形成的抑制作用及其潜在的作用机制。方法:将实验分为添加1,3-丙二胺的实验组和不添加1,3-丙二胺的对照组。利用结晶紫染色法检测1,3-丙二胺对铜绿假单胞菌生物膜的抑制作用,比较两组生物膜的形成量;通过初始黏附抑制试验和泳动平板试验检测1,3-丙二胺对铜绿假单胞菌初始黏附和泳动能力的抑制作用,比较两组的细菌初始黏附量和泳动的直径大小;通过蛋白质印迹法比较两组鞭毛蛋白Flagellin的表达;通过real-time RT-PCR比较两组铜绿假单胞菌群体密度感应系统相关基因和鞭毛调控相关基因的表达量;最后,通过分子对接试验检测1,3-丙二胺与靶蛋白LasI的相互作用。结果:与对照组相比,1.0 mmol/L 1,3-丙二胺实验组可使其生物膜的形成量相对值从1.708±0.089减少到1.288±0.080(t=6.07,P<0.01),且生物膜形成量的减少与1,3-丙二胺的浓度呈剂量依赖性;与对照组相比,实验组铜绿假单胞菌的起始黏附能力受到显著抑制,2.0 mmol/L 1,3-丙二胺实验组作用1 h即可使生物膜的黏附量从(0.890±0.389)×10^(6) CFU/mL减少到(0.245±0.076)×10^(6) CFU/mL(t=3.257,P<0.05);与对照组相比,2.0 mmol/L 1,3-丙二胺实验组还能显著抑制铜绿假单胞菌鞭毛介导的泳动能力,使其泳动圈直径从(1.840±0.144)cm减少到(0.756±0.222)cm(t=7.099,P<0.01)。此外,蛋白质印迹法显示:与对照组相比,实验组的鞭毛蛋白Flagellin合成受到抑制,其蛋白质印迹条带明显变淡。分子对接试验表明1,3-丙二胺的潜在作用靶蛋白为LasI。结论:1,3-丙二胺可显著抑制铜绿假单胞菌生物膜的形成,其潜在作用靶点为LasI蛋白。

Objective:To study the inhibitory effects of 1,3-diaminopropane on the biofilm formation of Pseudomonas aeruginosa and the underlying mechanisms.Methods:The experiment was divided into an experimental group and a control group.Crystal violet staining was used to examine the inhibitory effects of 1,3-diaminopropane on the biofilm formation of Pseudomonas aeruginosa,and the biofilm formation was compared between the 2 groups.Initial adherence inhibition assay and swimming plate assay were used to determine the inhibitory effects of 1,3-diaminopropane on the initial adherence and swimming motility of Pseudomonas aeruginosa,and the quantification of adhered cells and swimming diameter were compared between the 2 groups.Meanwhile,Western blotting was used to detect the Flagellin production of Pseudomonas aeruginosa;real-time RT-PCR was used to detect the quorum sensing system relative genes and flagellum regulative related genes expression in the 2 groups.Finally,molecular docking assay was used to calculate the interaction between 1,3-diaminopropane and LasI.Results:Compared with the control group,the biofilm formation of Pseudomonas aeruginosa was significantly inhibited in the experimental group in a dose-dependent manner(t=6.07,P<0.01).Compared with the control group,the initial adherence of Pseudomonas aeruginosa could significantly inhibit from(0.890±0.389)×106 to(0.245±0.076)×106 CFU/mL(t=3.257,P<0.05)in the experimental group(2.0 mmol/L).Compared with the control group,the swimming motility of Pseudomonas aeruginosa flagellar mediation could also inhibit in the experimental group(2.0 mmol/L).The swimming motility diameter was from(1.840±0.144)to(0.756±0.222)cm(t=7.099,P<0.01).Compared with the control group,the Flagellin production was inhibited in the experimental group.Finally,the molecular docking assay showed that the potential target of 1,3-diaminopropane was LasI.Conclusion:1,3-diaminopropane can significantly inhibit the biofilm formation of Pseudomonas aeruginosa,which potentially targets LasI protein.