万古霉素中介耐药金葡菌（VISA）的发现和流行给临床治疗带来了严峻挑战。研究表明，特定基因的突变与VISA形成相关，且VISA的致病性下降，其机制不清。我们前期报道了一株VISA，基因组测序发现其WalK发生了S221P突变，该新突变与VISA表型相关，预实验发现该菌的毒力基因显著下调，推测WalK突变影响了WalR的激活，通过下调细菌代谢控制蛋白CcpA，抑制了群体感应系统（QS）的功能，导致VISA致病性下降。为证实该假设，本项目拟探讨WalK突变对WalR活化的影响；通过EMSA和lacZ报告实验证实WalKR对ccpA的调控作用；构建WalK回复株，ccpA敲除及回补株，检测这些菌株的QS变化及毒力因子表达水平，再通过细胞和动物实验评价菌株的致病性，阐明WalKR通过CcpA调控VISA致病性的机制，不仅对理解VISA致病性的变化有意义，且可为寻找控制VISA感染的新策略奠定基础。

The discovery and epidemic of vancomycin-intermediate Staphylococcus aureus (VISA) have brought a great challenge to clinical treatment. Most studies have demonstrated that the mutations in some specific genes are associated with the development of VISA and the pathogenicity of VISA was decreased compared to its parent strain, however, the mechanisms for the pathogenic change of VISA remain to be elucidated. We previously isolated and characterized a VISA strain XN108 (MIC=12 µg/ml), and the genomic sequencing revealed that the mutation of S221P in WalK is responsible for the VISA phenotype. The expression of virulence factors was down-regulated in XN108. We suppose that the mutation of WalK may affect the activation of WalR, thereby down-regulates the quorum sensing system (QS) through the catabolite control protein (CcpA), resulting in the decreased pathogenicity of the VISA strain. To test this hypothesis, this project aims to reveal the influence of WalK S221P mutation on the activity of WalR. The regulatory function of WalR on ccpA will be investigated by electrophoretic mobility shift assay (EMSA) and lacZ reporter gene strategy. The gene replacement of WalK S221P, and ccpA gene knock-out and complement experiments will be performed to test the effects of WalK mutation on the expression of virulence factors. The pathogenesis of mutant and wild-type strains will also be evaluated using cell line and animal experiments. The elucidation of WalKR roles in controlling the pathogenicity of VISA via CcpA will not only be important for understanding the pathogenic alteration of VISA strain, but also lay down a great foundation in the development of new strategies to control infections caused by VISA.