WalKR通过CcpA调控万古霉素中介耐药金黄色葡萄球菌致病性的机制研究

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.

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。

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新挑战。为研究WalK突变通过细菌代谢控制蛋白CcpA，导致VISA致病性降低的作用与机制，本项目开展了WalK突变对WalR活化的影响，WalKR调控ccpA以及agr的分子机制研究。通过制备WalK野生型及其突变体以及WalR的重组蛋白，放射自显影检测目标蛋白的磷酸化，证实WalK(S221P)的自磷酸化水平下降，通过EMSA证实WalK(S221P)突变激活的效应蛋白WalR结合靶基因lytM启动子的能力下降，从生化水平阐明了WalK突变对WalR调控功能的影响。将XN108中的WalK突变进行回复，发现回复菌对万古霉素的耐药水平降低。进一步构建lacZ报告系统，结合EMSA实验证实WalKR对ccpA具有直接调控作用，这种促进作用可能与VISA耐药表型有关。同理，证实WalKR对agr具有直接抑制功能，这可能与VISA的毒力降低有关。通过构建ccpA基因敲除株和回补菌株，发现ccpA缺失导致XN108万古霉素耐药性降低，回复后菌株耐药性升高；这一表型在VISA标准株Mu50中也得到证实，表明CcpA影响金葡菌万古霉素耐药性有一定的普遍性。转录组学和代谢组学分析显示，ccpA敲除改变的通路和产物涉及氨基酸代谢、氧化还原等。实验还证实回复菌株的溶血活性增强，多种毒力因子表达上调。利用RAW264.7细胞模型，观察XN108及其突变菌株感染后，细胞破坏程度及LDH的释放水平，从细胞水平评价了菌株毒力变化；建立小鼠皮肤和血流感染模型，证实回复株的致病性和脏器定植能力增强。发表论文8篇，获授权专利1项，培养研究生5人。