哌立福新通过结合PqsE蛋白抑制铜绿假单胞菌生物膜的形成

Perifosine inhibits biofilm formation of Pseudomonas aeruginosa by interacting with PqsE protein

旨在探讨哌立福新对铜绿假单胞菌生物膜形成的影响及其作用机制。本研究通过24孔板静置孵育24 h培养铜绿假单胞菌生物膜,再通过结晶紫染色法检测哌立福新对铜绿假单胞菌培养基底部生物膜的抑制作用,将未加哌立福新的孔设置为对照组;通过玻璃试管静置孵育24 h构建气-液交界面生物膜,并通过结晶紫染色法检测哌立福新对铜绿假单胞菌气-液交界面生物膜形成的影响,将未加哌立福新的孔设置为对照组;将铜绿假单胞菌重悬于含有系列浓度的哌立福新培养基中,置于恒温摇床,连续检测其生长浊度随时间的变化,绘制时间-生长曲线,检测哌立福新对铜绿假单胞菌浮游菌增殖的影响,将未加哌立福新的孔设置为对照组;通过分子对接中的Glide超精确模式将哌立福新与PqsE蛋白进行柔性对接,预测哌立福新与靶蛋白PqsE的结合力及结合模式;通过检测PqsE蛋白的催化底物硫醇的生成量,验证哌立福新对PqsE蛋白催化功能的抑制能力,将未加哌立福新的孔设置为对照组;最后,通过等离子表面共振试验,验证哌立福新与PqsE蛋白的结合能力。结果显示,与未加哌立福新的对照组相比较,4~8μg/ml的哌立福新可有效抑制铜绿假单胞菌培养基底部和气-液交界面生物膜的形成;哌立福新对铜绿假单胞菌浮游菌的增殖无影响;分子对接试验提示哌立福新与PqsE蛋白具有良好的结合力,对接分数为-10.67 kcal/mol;哌立福新还能有效抑制PqsE蛋白的催化功能,与未加哌立福新的对照组相比,随着哌立福新浓度增高,PqsE蛋白酶受抑制程度越高;通过等离子表面共振试验发现PqsE蛋白与哌立福新具有良好的结合能力,且其结合常数为6.65×10^(-5) mol/L。综上,哌立福新可结合PqsE蛋白,干扰PqsE蛋白的催化功能并能抑制铜绿假单胞菌生物膜的形成。

To explore the biofilm inhibitory efficacy of perifosine against Pseudomonas aeruginosa(P.aeruginos)and its mechanisms.Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against P.aeruginosa,the wells without perifosine was set as control group.Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine,the wells without perifosine was set as control group.Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of P.aeruginosa,the wells without perifosine was set as control group.The interaction model between perifosine and PqsE was assessed by molecular docking assay.The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols,the wells without perifosine was set as control group.Binding affinity between perifosine and PqsE was detected by plasma surface resonance.The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8μg/ml.There was no influence of perifosine on the cells growth of P.aeruginosa.The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of-10.67 kcal/mol.Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner.The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10^(-5)mol/L.Perifosine could inhibited the biofilm formation of P.aeruginosa by interacting with PqsE.