With the treatment failure by vancomycin and poor clinical outcomes, the emergence and spread of vancomycin intermediate-resistant Staphylococcus aureus (VISA) has raised more concerns in recent years. While most VISA strains are isolated from methicillin-resistant S. aureus (MRSA), the mechanism underlying the generation of VISA from methicillin-susceptible S. aureus (MSSA) is still largely unknown. Here, we identified a total of 10 mutations in 9 genes through comparative genome analysis from laboratory-derived VISA strain. We verified the role of a novel mutation of WalK (I237T) and our results further indicated that the introduction of WalK (I237T) by allelic replacement can confer vancomycin resistance in MSSA with common VISA characteristics, including thickened cell walls, reduced autolysis, and attenuated virulence. Consistent with these phenotypes, real-time quantitative reverse transcription-PCR revealed the altered expression of several genes associated with cell wall metabolism and virulence control. In addition, electrophoretic mobility shift assay indicated that WalR can directly bind to the promoter regions of oatA, sle1, and mgt, fluorescence-based promoter activity and β-galactosidase assays revealed WalK (I237T) can alter promoter activities of oatA, mgt, and sle1, thus regulating genes expression. These findings broaden our understanding of the regulatory network by WalKR system and decipher the molecular mechanisms of developmental VISA resistance in MSSA with point mutations.

随着万古霉素治疗失败和临床效果差，近年来对万古霉素中等耐药性金黄色葡萄球菌（VISA）的出现和传播引起了更多关注。虽然大多数VISA菌株是从耐甲氧西林的金黄色葡萄球菌（MRSA）中分离出来的，但其机制却是由对甲氧西林敏感的金黄色葡萄球菌产生VISA的基础（MSSA）仍然未知。在这里，我们通过实验室衍生的VISA菌株的比较基因组分析确定了9个基因中的10个突变。我们验证了WalK（I237T）新型突变的作用，我们的结果进一步表明，通过等位基因置换引入WalK（I237T）可以赋予MSSA具有常见VISA特征的万古霉素耐药性，包括细胞壁增厚，自溶减少和减弱毒力。与这些表型一致，实时定量逆转录-PCR显示与细胞壁代谢和毒力控制相关的几个基因的表达发生了改变。此外，电泳迁移率变动分析表明，WalR可以直接结合到燕麦，sle1和mgt，基于荧光的启动子活性和β-半乳糖苷酶分析表明，WalK（I237T）可以改变oatA，mgt和sle1的启动子活性，从而调节基因表达。这些发现拓宽了我们对WalKR系统调控网络的理解，并破译了具有点突变的MSSA中发展的VISA抗性的分子机制。