Department of Clinical Laboratory, Peking University People's Hospital, Beijing 100044, People's Republic of China.
Department of Clinical Laboratory, China Meitan General Hospital, Beijing 100028, People's Republic of China.
Int J Antimicrob Agents. 2016 May;47(5):362-7. doi: 10.1016/j.ijantimicag.2016.02.003. Epub 2016 Mar 12.
The objective of this study was to comprehensively identify the target genes regulated by the two-component regulatory system VraSR in Staphylococcus aureus and to clarify the role of VraSR in low-level vancomycin resistance. Expression of vraS was determined by real-time quantitative reverse transcriptase PCR (qRT-PCR). A clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) strain B6D and a vancomycin-intermediate S. aureus (VISA) strain D7 that was induced from a meticillin-resistant S. aureus strain were selected to construct vraSR null mutants by allelic replacement. The vraSR-complemented strain B6D_c was also constructed by allelic replacement. Genes differentially expressed in the wild-type, vraSR null mutant and complemented strains were detected using RNA-Seq and were validated by qRT-PCR. Compared with vancomycin-susceptible S. aureus strains, expression of vraS was upregulated in all four isogenic hVISA strains. Vancomycin minimum inhibitory concentrations (MICs) in the vraSR null mutants B6D-ΔvraSR and D7-ΔvraSR were significantly lower than in the wild-type strains B6D and D7 and the complemented strain B6D_c. RNA-Seq and qRT-PCR data showed that expression of genes encoding FmtA protein, foldase protein PrsA, capsular polysaccharide biosynthesis glycosyltransferase, TcaA, a putative membrane protein, and six hypothetical proteins was down regulated in both vraSR-null mutants B6D-ΔvraSR and D7-ΔvraSR. Most of these differentially expressed proteins are involved in cell wall biosynthesis, which is associated with vancomycin resistance in S. aureus. In conclusion, VraSR plays an important role in S. aureus strains with low-level vancomycin resistance. PrsA, FmtA, glycosyltransferase and TcaA are regulated directly or indirectly by VraSR.
本研究的目的是全面鉴定金黄色葡萄球菌中由双组分调控系统 VraSR 调控的靶基因，并阐明 VraSR 在低水平万古霉素耐药中的作用。通过实时定量逆转录 PCR（qRT-PCR）测定 vraS 的表达。选择临床异质性万古霉素中介金黄色葡萄球菌（hVISA）菌株 B6D 和从耐甲氧西林金黄色葡萄球菌诱导的万古霉素中介金黄色葡萄球菌（VISA）菌株 D7，通过等位基因替换构建 vraSR 缺失突变体。还通过等位基因替换构建 vraSR 互补菌株 B6D_c。使用 RNA-Seq 检测野生型、vraSR 缺失突变体和互补菌株中差异表达的基因，并通过 qRT-PCR 进行验证。与万古霉素敏感的金黄色葡萄球菌菌株相比，所有 4 株同源 hVISA 菌株中 vraS 的表达均上调。vraSR 缺失突变体 B6D-ΔvraSR 和 D7-ΔvraSR 的万古霉素最小抑菌浓度（MIC）明显低于野生型菌株 B6D 和 D7 以及互补菌株 B6D_c。RNA-Seq 和 qRT-PCR 数据显示，编码 FmtA 蛋白、折叠酶蛋白 PrsA、荚膜多糖生物合成糖基转移酶、TcaA、一种假定的膜蛋白和六个假定的蛋白质的基因的表达在两个 vraSR 缺失突变体 B6D-ΔvraSR 和 D7-ΔvraSR 中下调。这些差异表达蛋白中的大多数参与细胞壁生物合成，这与金黄色葡萄球菌的万古霉素耐药性有关。总之，VraSR 在具有低水平万古霉素耐药性的金黄色葡萄球菌菌株中发挥重要作用。PrsA、FmtA、糖基转移酶和 TcaA 受 VraSR 直接或间接调控。
