膜蛋白酶介导的多黏菌素耐药机制研究

多粘菌素被认为是治疗革兰阴性菌感染后最后选择的抗生素。近期研究表明，质粒介导的mcr-1基因可致多黏菌素耐药，并在动物和人之间广泛传播, 但Mcr-1具体如何导致多黏菌素耐药的分子机制尚不清楚。因此，解析MCR-1蛋白结构及揭示其生物学功能迫在眉睫。本课题组利用不同表达载体和表达系统对MCR-1膜蛋白进行研究，不断改进纯化和结晶条件，以获得高分辨率蛋白结构。同时结合体外活性实验、质谱定性、定量分析MCR-1对大肠杆菌lipiA的影响，进一步阐明MCR-1的生物学功能；我们利用荧光标记多粘菌素E和MCR-1，借助共聚焦激光扫描显微术观察多粘菌素E与大肠杆菌的相互作用，为揭示MCR-1致多粘菌素耐药机制提供新的选择途径。并通过多粘菌素的药敏试验，从化合物筛选库筛选出能阻断MCR-1介导的多粘菌素耐药的小分子。进一步阐明其逆转多黏菌素耐药的分子机制，为研发新药、延缓多黏菌素耐药奠定重要基础。

Colistin has been regarded as the last resort antibiotic to combat multidrug resistant by Gram negative bacteria. But recent studies showed that the emergence and dissemination of plasmid-mediated colistin resistance enzyme MCR-1 could lead to colistin resistance. Here, we are aiming to investigat the structure and catalytic mechanisms of embedded enzyme MCR-1. Firstly, we executed the crystallization with high pure proteins and keep improving the diffraction of crystal. We then characterized the modification of lipid A extracted from E. coli BL21 by MCR-1 via mass spectrometry to reveal the catalytic mechanisms of MCR-1. Lastly, our findings will elucidate the interaction between colistin and MCR-1 using confocal laser scanning microscopy. Meanwhile, we are screening the putative inhibitors for Mcr-1 based on colistin susceptibility test. Overall, our studies will establish a basis for designing MCR-1 inhibitors and provide an alternative mechanism for MCR-1.