基于蛋白质组学和代谢组学的mcr-1基因介导的多粘菌素耐药机制研究

In recent years, the rise in infections caused by multidrug-resistant (MDR) Gram-negative bacteria, has led to almost no drug used to cure. And polymyxin was often used clinically as “the last line” to treat the infections caused by MDR Gram-negative bacteria. mcr-1 is a newly discovered plasmid-mediated polymyxin transferable resistance gene and could be transmitted by horizontal transfer from livestock, environment to human beings. The resistant rate of polymyxin caused by mcr-1 gene in animal-derived food was increased year by year and the gene could enter human body through food chain, which seriously threaten to human health and public health security. At present, mcr-1 gene has been disseminated to many countries of four continents. However, there is almost no study on mcr-1 mediated polymyxin resistance mechanisms. This project is going to study mcr-1 gene mediated polymyxin resistant Escherichia coli and susceptible strains using proteomics and metabolomics methods. The aim of this project is to clarify the proteomic and metabolic features related to mcr-1 gene in E. coli and decipher the resistance mechanisms of mcr-1 gene in polymyxin resistance. mcr-1 positive colistin-resistant E. coli strains isolated from livestock which have similar genetic background will be further performed confirmatory analysis of biomarkers. This project will provide scientific basis for avoiding the rapid dissemination of mcr-1 gene and the development of antibiotic agents.

近年来，革兰氏阴性菌多重耐药问题越来越严重，导致革兰氏阴性菌引起的感染几乎无药可用。多粘菌素常作为“最后一道防线”用于临床上多重耐药革兰氏阴性菌感染的治疗。mcr-1是新发现的质粒介导的多粘菌素可转移性耐药基因，可在畜禽养殖场、环境、人医临床水平传播。mcr-1基因引起的多粘菌素耐药率逐年升高，且该基因存在着从食物链进入人体的风险，严重威胁着人类健康和公共卫生安全。目前，四大洲很多国家都有mcr-1报道，但关于mcr-1基因的耐药机制研究仍较少。本项目拟从蛋白质组学和代谢组学角度对mcr-1基因介导多粘菌素耐药大肠杆菌耐药株和敏感株进行研究，明确mcr-1基因引起的蛋白谱和代谢谱特征，并对畜禽养殖场采集的携带mcr-1基因且遗传背景相似的大肠杆菌进行生物标志物验证分析，阐明mcr-1基因介导的粘菌素耐药机制，可为临床上规避mcr-1基因的迅速传播和防止耐药性的产生提供科学依据。

mcr-1是新发现的质粒介导的粘菌素可转移性耐药基因，严重威胁着人类健康和公共卫生安全。本研究采用非标记定量蛋白质组学和非靶向代谢组学对mcr-1基因介导粘菌素耐药大肠杆菌耐药株和敏感株进行研究，获得mcr-1基因引起的细菌蛋白谱和代谢谱变化特征，并进一步阐明其耐药机制。利用圆二色谱比较了MCR-1和MCR-3的二级结构和热稳定性差异，发现它们介导黏菌素耐药的功能差异。采用免疫共沉淀技术对三株不同的E. coli工程菌中MCR-1蛋白相互作用的关键蛋白，并验证其功能。研究结果表明，mcr-1可导致大多数蛋白表达下调以适应药物筛选压力。通路分析表明细菌代谢过程受到影响，主要与甘油磷脂代谢、硫胺素代谢和脂多糖生物合成相关。同时，我们还发现mcr-1介导耐药性的底物磷脂酰乙醇胺在粘菌素耐药E. coli体内大量积聚。值得注意的是，mcr-1不仅可以引起细菌细胞膜脂质A的磷酸乙醇胺修饰，还可以通过干扰阳离子抗菌肽（CAMP）通路中外排泵蛋白的表达，来影响粘菌素耐药性中脂蛋白的生物合成和转运。总体而言，异常的甘油磷脂代谢、脂多糖生物合成和底物磷脂酰乙醇胺的积累与mcr-1介导的粘菌素抗性密切相关。mcr-1和mcr-3介导粘菌素耐药的功能比较发现MCR-3（66.19℃）的热稳定性优于MCR-1（61.14℃），两者的二级结构相似。脂质A的飞行时间质谱分析发现mcr-1和mcr-3均可使细菌细胞膜脂质A发生磷酸乙醇胺修饰。同源模拟和分子对接研究了脂质A头部磷脂酰乙醇胺和MCR-3的分子识别特征，MCR-3蛋白Thr277是负责催化结合的关键氨基酸残基，并且是作为亲核反应部分。基于免疫共沉淀的MCR-1互作蛋白组学在3种工程菌株中共鉴定6种共有蛋白（DnaK、sspB、rpsE、hns、rpsJ 和rpsP）。采用表面等离子共振和基因敲除方法对SspB和DanK与MCR-1的相互作用进行了验证，证实了这两个蛋白均与MCR-1存在互作，可影响其蛋白表达水平。上述研究成果从组学水平阐明了黏菌素耐药基因mcr-1的耐药机理及可能的调控机制，这些发现可为进一步通过阻断该代谢过程来抑制粘菌素耐药性提供有价值的信息，进而为临床上规避mcr-1基因的迅速传播和防止耐药性的产生提供科学依据。