Tn6330介导mcr-1传播的分子机制研究

Recently, the worldwide spread of the plasmid-mediated colistin resistance gene mcr-1 will pose the great threat to the clinical treatment. The horizontal distribution of mcr-1 is not only due to the effects of plasmid transfer, but it is also closely related to the transposition of ISApl1. The composite transposon Tn6330 (ISApl1-mcr-1-orf-ISApl1) can generate the circular intermediate (ISApl1-mcr-1-orf) by hypothesized excision mechanism. However it is obscure that the formation mechanism of the circular intermediate and whether the intermediate can facilitate the insertion of mcr-1 among different genetic loci at a higher frequency. In addition, based on the preliminary data, intact Tn6330 cannot be found in IncI2 plasmids naturally, and the IncI2 plasmids carrying mcr-1 have diffused widely in many countries. What has caused this phenomenon? Whether the existence of Tn6330 can affect the stability of IncI2 plasmids or make IncI2 plasmids to produce the fitness cost. Based on these knowledge gap, in our present project, the methods including Red recombination system, conjugation/transformation, competition experiments in vivo and in vitro, subculture, PCR, gene clone, site-directed gene mutagenesis, S1-PFGE and southern blot hybridization will be performed to analyze the molecular mechanism of Tn6330 related to the transfer of mcr-1. In conclusion, our project would like to provide new strategies and new ideas for resistance control.

目前，质粒介导的黏菌素耐药基因mcr-1在全球范围内广泛传播，给临床治疗带来极大挑战。它的传播不仅在于质粒的水平转移，而且与ISApl1密切相关。位于Tn6330（ISApl1-mcr-1-orf-ISApl1）中的mcr-1能够借助环形中间体（ISApl1-mcr-1-orf）而脱离所在的基因位置，而该环形中间体形成的机制以及能否以较高的频率将mcr-1基因插入细菌基因组中仍不清楚。另外，目前在多个国家流行的携带mcr-1基因的IncI2型质粒中仍未发现完整的Tn6330，这是否由于Tn6330的存在能够影响IncI2型质粒的稳定性并使其产生适应性代价所致？鉴于此，本项目通过Red重组系统、接合转移、体内外竞争实验、传代培养、PCR、基因克隆、定点突变、S1-PFGE及southern杂交等实验方法，探讨Tn6330介导mcr-1传播的分子机制，进而为探索耐药控制方案提供新策略和新思路。

本研究共获得了四个质粒，包括携带Tn6330的IncI2质粒、携带不完整Tn6330的IncI2质粒、丢失Tn6330的IncI2质粒以及携带Tn6330的IncHI2质粒。稳定性实验显示，以上四个质粒及质粒中的Tn6330在无抗性的条件下传代10天均能够保持近100%的稳定性，这暗示了mcr-1阳性IncI2和IncHI2质粒以及Tn6330有相对高的稳定性，其能够介导mcr-1的稳定遗传。.为了评估Tn6330在mcr-1阳性IncI2和IncHI2质粒对宿主菌适应性影响中的作用，本研究将分别携带以上四个质粒的四个E. coli DH5a转化子与受体菌E. coli DH5a进行体外竞争实验，结果显示，在共培养24h、48h、72h、96h后，携带IncI2质粒的转化子数量超过了受体菌E. coli DH5a的数量，三个IncI2质粒均对宿主呈现出相似的适应性优势，在24h-96h内适应性逐渐升高；相反地，携带IncHI2质粒的转化子数量少于DH5a的数量，在24h-96h内适应性降低了20%左右，即IncHI2质粒对宿主产生了轻微的适应性代价。利用小鼠体内模型进行的体内竞争实验结果与体外竞争实验的结果一致。以上研究揭示了Tn6330不影响所在质粒的稳定性和适应性，IncI2质粒对宿主的适应性优势可能利于mcr-1的快速传播。.Tn6330被成功克隆入低拷贝质粒载体pTWV228中，得到携带重组质粒pTWV228-Tn6330的DH5a转化子，反向PCR检测有环形中间体存在。借助转座实验和三代MinION测序技术发现，Tn6330能够以较低的转座频率（~10-8）介导单拷贝或双拷贝的mcr-1插入染色体或质粒的AT富集区。研究发现Tn6330中ISApl1片段的缺失或截断能够抑制环形中间体的形成，为了探索ISApl1及其两侧反向重复序列（IRs）中在环形中间体形成中的重要位点，通过定点突变实验对重组质粒pTWV228-Tn6330中的ISApl1进行改造，结果发现ISApl1中某个位点的有义突变不影响环形中间体的形成，而哪些位点的共同作用能够抑制环形中间体的形成需要进一步研究。本研究系统地阐明了Tn6330介导mcr-1传播的分子机制，为有效控制mcr-1的快速传播提供新思路和理论依据。