细菌IV型分泌系统Trb蛋白促进IncX4-mcr-1质粒传播的分子机理研究

mcr-1 is the first plasmid-mediated colistin resistance gene, which has been proven to disseminate ubiquitously. The applicant has proved that mcr-1 can exist in the IncX4 plasmid stably and IncX4 has become the predominant plasmid type to be harboring mcr-1 (JAC, 2018, 2016; AAC, 2017, et al). However, the molecular mechanism of transmission of mcr-1-carrying IncX4 plasmid (IncX4-mcr-1,IM) is unknown. Our preliminary data demonstrated that: (i) colistin up-regulated mRNA expression of the trb gene family that consists of the type IV secretion system (T4SS) and transcriptional regulatory gene ymoB, respectively. (ii) YmoB can bind to the 5' upstream of trb genes. Furthermore, studies have confirmed that T4SS was strongly related to plasmid transfer. According to all these pieces of information, we propose the scientific hypothesis of this project: the selective pressure of colistin can promote the expression of ymob gene, whereafter Ymob up-regulate trb genes by binding to the 5' upstream of trb genes. This will activate T4SS to accelerate the transmission of IncX4-mcr-1. Our further studies in the project are: (i) building genomic database of IncX4-mcr-1 plasmid to analyze the diversity of T4SS and Ymob, respectively; (ii) knocking out trb genes of T4SS to identify pivotal trb genes that increase plasmid transmission; (iii) determining the binding motif of YmoB protein to the 5' upstream of trb genes using CHIP-seq and other technologies. We hope to provide evidence of finding the target to prevent mcr-1 transmission.

首个发现的质粒介导多粘菌素耐药基因mcr-1已引起广泛传播。申请人已证实mcr-1能稳定存在于IncX4型质粒，且IncX4已成为介导mcr-1传播的主要质粒（JAC,2018,2016;AAC,2017等），但携带mcr-1的IncX4质粒（IncX4-mcr-1,IM）传播的分子机理不明。预实验进一步发现：多粘菌素可上调IM编码的IV型分泌系统（T4SS）trb家族基因和转录调控基因ymoB表达；且YmoB可结合到trb的5'端。而研究已证实T4SS与质粒传播密切相关。据此提出假说：多粘菌素作用下YmoB通过结合到trb的5'端上调其表达，进而激活T4SS促进IM的传播。本项目将构建IM基因组数据库以分别分析T4SS和YmoB多样性；构建trb各家族基因敲除株以鉴定促进质粒传播的关键trb；利用ChIP-seq等方法揭示YmoB和trb结合位点。为确定防控mcr-1传播的靶点提供依据。

细菌耐药性是世界普遍关注、长期存在的重要公共安全问题。多粘菌素是临床治疗碳青霉烯类等多重耐药阴性菌感染的“最后一线”药物。mcr-1是首个发现可传播的多粘菌素耐药基因，而IncX4是介导mcr-1传播的最主要质粒类型，但IncX4-mcr-1的遗传多样性和传播机制不清楚。本项目构建了IncX4-mcr-1 质粒全基因组数据库；阐明了IncX4-mcr-1质粒宿主基因组和质粒遗传多样性研究，包括解析了全国范围内携带mcr-1基因的大肠埃希菌的基因组结构以及遗传分化；解析了携带mcr-1基因的IncX4等质粒流行和遗传环境。本项目通过敲除多个IV型分泌系统基因，利用多种试验技术，鉴定了IncX4-mcr-1上促进质粒传播的关键基因。本项目的研究成果为阻断mcr-1的传播扩散提供了直接靶点。