绵马酚抑制多粘菌素耐药关键蛋白MCR-1的机制研究

Study on the mechanism of synergistic action of aspidinol and polymyxin on inhibition of key polymyxin protein MCR-1

mcr-1是近几年发现的一种多粘菌素耐药基因,该基因可随质粒在不同菌株间快速传播,目前已在多个国家及地区被检出,本研究团队前期筛选发现联合使用绵马酚与多粘菌素时可以抑制mcr-1阳性大肠杆菌的生长。为了评价两者联合使用时的抑菌效果,本研究采用棋盘稀释法对mcr-1阳性大肠杆菌进行了绵马酚与多粘菌素联合药敏实验,结果显示,当联合使用绵马酚与多粘菌素时相比单独使用多粘菌素时,多粘菌素的MIC由8μg/m L降低至1μg/m L,FIC指数小于0.5,二者属于协同作用。以终浓度为32μg/m L绵马酚与10μg/m L多粘菌素联合使用,进一步通过平板计数和透射电镜检测其杀菌效果和大肠杆菌菌体的破损程度,结果显示,当联合使用绵马酚与多粘菌素时,大肠杆菌在2 h^8 h逐渐全部死亡,大肠杆菌菌体破损最严重;但不管是单独使用绵马酚还是多粘菌素,大肠杆菌均不能被彻底杀灭。采用氯仿/甲醇/水混合相萃取法分别提取mcr-1阳性大肠杆菌JD08菌株和含有16μg/m L的绵马酚的mcr-1阳性大肠杆菌JD08菌株中类脂A,利用质谱仪检测类脂A的结构,结果显示在绵马酚存在的情况下,MCR-1蛋白的作用受到抑制,发生转移的酰基键含量大大减少。采用双酶切法构建PET-28a-mcr-1重组表达载体,经诱导表达后通过镍柱亲和层析纯化得到MCR-1蛋白,利用Biacore大分子作用仪验证绵马酚与重组MCR-1蛋白的相互作用,结果显示绵马酚与MCR-1蛋白之间的亲和力为1.47μmol/L,属于直接结合。表明单独使用绵马酚对mcr-1阳性大肠杆菌无效,但其与多粘菌素联合时则产生了良好的抑菌及杀菌效果,绵马酚是MCR-1的小分子抑制剂。本研究首次确认了绵马酚是MCR-1的小分子抑制剂,对于开发新型抗多粘菌素耐药菌的药物具有重要意义。

mcr-1 is a polymyxin resistance gene discovered in the four years ago.The gene can be rapidly transmitted among different strains with plasmids.At present,it has been found in many countries and regions.Previous study showed that the combination of mycophenol and polymyxin could inhibit the growth of mcr-1-positive E.coli strain.In order to evaluate the bacteriostatic effect of the above-mentioned compound,the chessboard dilution method was used to test the compound sensitivity to mcr-1 positive E.coli.Compared with polymyxin alone,the MIC of polymyxin decreased from 8μg/m L to 1μg/L,and the FIC index was less than 0.5 belonging to the synergistic effect.The subsequent experiments were carried out with the final concentration of 32μg/m L of gossypol and 10μg/m L of polymyxin.The bactericidal efficacy and the damage degree of E.coli were performed by plate counting and transmission electron microscope.The results showed that the E.coli was gradually killed after interaction 2 h and 8 h,but the E.coli was not completely killed under the treatment of caffeine or colistin alone.Lipoid A was extracted from normal mcr-1 positive E.coli JD08 and E.coli JD08 treated with 16μg/m L gossypol,and its structure was detected by mass spectrometry.The results showed that the effect of MCR-1 protein was inhibited and the content of transferred acyl bond was greatly reduced in the presence of gossypol.The recombinant expression vector of PET-28 a-mcr-1 was constructed via double enzyme digestion.After induction and expression,MCR-1 protein was purified by nickel column affinity chromatography.Biacore macromolecular interaction instrument was used to verify the interaction between gossypol and recombinant MCR-1 protein.The results showed that the affinity between gossypol and MCR-1 protein was 1.47μmol/L,which indicated that the use of gossypol alone was ineffective against mcr-1 positive E.coli.However,when it was combined with polymyxin,it had good bacteriostatic and bactericidal effect,and the macromolecular interaction experiment proved that spongiferol had a direct effect on MCR-1 protein,and it was a small molecule inhibitor of MCR-1.In this study,it was confirmed for the first time that gossypol was a small molecule inhibitor of MCR-1,which was of great significance for the development of new drugs against polymyxin-resistant bacteria.