葡萄球菌ccrC2基因影响甲氧西林抗性菌间传播的分子机制研究

Currently, the epidemic and threat to healthcare of methicillin resistant Staphylococcus aureus (MRSA) from livestock have become hot research topic. The spread of methicillin resistance among staphylococci, which is mediated by the cassette chromosome recombinase (Ccr), is one of the primary reasons leading to high incidence of MRSA. Previously, we have identified a novel recombinase CcrC2 in MRSA which was widely distributed and its mechanism of action remains elusive, as well as another protein with unknown function, designated CRP, which can bind to the promoter of ccrC2. Moreover, antibiotic exposure could up-regulate their expressions. Herein, around the issue-horizontal gene transfer of methicillin resistance, we choose the CcrC2 as a research object and a cut-in point, and aim to determine the recombination efficiency of SCCmec mediated by CcrC2, activity range and functional domain of CcrC2, by utilizing the methods such as promoter pull-down, gene knock-out, and color-plate screening technologies. This project sought to check the effect of CRP on the regulation of ccrC2 expression under antibiotic exposure and excavate the upstream regulator of the crp gene. Another purpose of this project is to reproduce the transition process from MSSA to MRSA mediated by CcrC2 in lab condition. This work will improve the molecular basis of the horizontal gene transfer of methicillin resistance in staphylococci, and explain the emergence of MRSA facilitated by antibiotics, as well as laid a theoretical foundation for reducing the incidence of MRSA.

家畜源耐甲氧西林金黄色葡萄球菌（MRSA）的流行及其对公共卫生的影响已成为近年来的研究热点，而重组酶Ccr介导的甲氧西林抗性菌间传播是造成MRSA流行的主要原因之一。我们曾从MRSA中发现了一个广泛存在而作用机制不明晰的新型重组酶CcrC2，及一个能结合ccrC2启动子的功能未知蛋白CRP，且抗生素能上调它们的表达。本项目拟围绕甲氧西林抗性菌间传播这一科学问题，以CcrC2重组酶为切入点和研究对象，通过启动子免疫共沉淀、基因敲除、显色平板筛选等技术手段，明确CcrC2的重组效率、作用范围和功能结构域，揭示抗生素刺激下CRP对ccrC2基因表达的调控机制，挖掘crp基因的上游调控因子，并在实验室条件下重现CcrC2介导敏感菌株向MRSA耐药菌株的转变过程。本项目将揭示甲氧西林抗性在葡萄球菌间传播的分子机制，进而阐明抗生素滥用催生MRSA耐药菌的机理，为降低MRSA耐药菌的发生率奠定理论基础。

动物源耐甲氧西林金黄色葡萄球菌（MRSA）的流行对公共卫生造成极大困扰，而Ccr重组酶家族是介导MRSA流行的主要内因之一。目前，尚缺乏特异的控制手段来消除MRSA的耐药性并防范其传播。故以Ccr重组酶为靶标、研究消除MRSA的耐药性具有重要的现实意义。CcrC2是本研究发现的新型重组酶，具有高适应性与高活性，是一种极为理想的候选靶标。本项目围绕甲氧西林抗性菌间传播这一科学问题，以CcrC2重组酶为切入点和研究对象，成功构建ccrC2基因敲除菌和过表达菌；通过qRT-PCR和Western blot发现CcrC2重组酶能介导SCCmec耐药元件在葡萄球菌间进行水平转移，CcrC2重组酶的工作效率主要和其识别的att位点的保守性有关；通过构建一系列CcrC2重组酶截短突变子和定点突变子，利用凝胶迁移阻滞实验检测CcrC2重组酶与att位点的结合效率，发现S12、R84、K149三个位点是CcrC2重组酶发挥作用的关键氨基酸；通过启动子pull-down实验、EMSA实验和抗生素诱导实验，证实CRP蛋白为调控ccrC2转录的转录因子；进而结合CcrC2重组酶和CRISPR技术，实现了甲氧西林耐药金黄色葡萄球菌中SCCmec耐药元件的100%消除，使耐药菌变异为敏感菌，并在实验室条件下重现CcrC2介导敏感菌株向MRSA耐药菌株的转变过程，为消除病原菌MRSA的耐药性提供了良好的理论依据和技术手段。此外，本项目利用全基因组测序和比较基因组学分析法，分别完成了表皮葡萄球菌NW32耐药基因岛和奶牛乳腺炎葡萄球Y24菌株的精氨酸分解代谢移动元件的进化生物学研究，并阐明了葡萄球菌新木质素降解酶Mco的功能与应用潜力。项目负责人以第一完成人荣获陕西省科学技术奖二等奖1项（2019年），发表全国性会议论文1篇，SCI论文3篇，联合培养博士研究生3名，硕士研究生1名。