GraSR通过MurC促进金黄色葡萄球菌中等耐受万古霉素的作用研究

Role of GraSR in promoting vancomycin resistance in vancomycin-intermediate Staphylococcus aureus through MurC

摘要

目的 探讨GraS（T136I）突变对万古霉素中等耐受金黄色葡萄球菌（vancomycin-intermediate Staphylococcus aureus，VISA）耐药性的影响及机制。 方法 采用同源重组技术将XN108（VISA）的GraS（T136I）突变位点引入到Newman（VSSA）构建突变菌株Newman-GraS（T136I），利用相同的方法在XN108中将突变位点进行回复，构建回复菌株XN108-GraS（I136T），利用E-test法测定各菌株对万古霉素的耐药性，通过透射电子显微镜观察各菌株细胞壁厚度，采用RT-qPCR检测了金葡菌细胞壁肽聚糖合成相关基因在XN108及其突变回复菌株XN108-GraS（I136T）中的表达水平变化，通过LacZ报告系统检测XN108和XN108-GraS（I136T）中murC基因启动子活性，通过结合位点分析和凝胶迁移阻滞实验（electrophoretic mobility shift assay，EMSA）证实GraSR对murC基因的调控作用。 结果 Newman-GraS（T136I）和XN108-GraS（I136T）替换菌株构建成功。回复株XN108-GraS（I136T）对万古霉素的MIC从原来的12 μg/mL下降到8 μg/mL，突变菌株Newman-GraS（T136I）对万古霉素的MIC 从野生株的1.5 μg/mL上升到4 μg/mL。透射电子显微镜结果显示，XN108-GraS（I136T）的细胞壁（20.097±2.862） nm较野生株XN108（40.283±3.784） nm明显变薄（P=0.000）。通过RT-qPCR实验发现，金葡菌细胞壁肽聚糖合成相关基因murC、murD和mraY在回复菌株XN108-GraS（I136T）中表达下调（P=0.000），且murC的下降水平最多（约1.935倍），LacZ活性检测分析显示XN108中murC基因的启动子活性（2 182.333±104.580）明显强于其在XN108-GraS（I136T）（1 593.333±179.258）中的活性（P=0.008），结合位点检索分析及EMSA实验证实GraS激活的GraR具有直接结合murC基因调控区的功能。 结论 金葡菌GraS（T136I）突变具有促进VISA的形成的作用，该作用可能是通过上调细胞壁合成关键基因murC的表达，导致细胞壁增厚来实现的。

Abstract

Objective To investigate the influence of GraS（T136I） mutation on vancomycin resistance in vancomycin-intermediate Staphylococcus aureus（VISA） and its mechanism. Methods The homologous recombination technique was used to introduce the GraS（T136I） mutation site of XN108（VISA） into Newman（VSSA） to construct the mutant strain of Newman-GraS（T136I），and the same method was used to reverse the mutation in XN108 to construct the reverse strain XN108-GraS（I136T）. The E-test stripes were used to measure the resistance of each strain to vancomycin； a transmission electron microscope was used to observe the cell wall thickness of each strain；RT-qPCR was used to measure the changes in the expression levels of peptidoglycan synthesis-related genes on the cell wall of Staphylococcus aureus in XN108 and its reverse mutant strain XN108-GraS（I136T）；the LacZ report system was used to measure murC gene promoter activity；binding site analysis and electrophoretic mobility shift assay（EMSA） were used to confirm the regulatory effect of GraSR on the murC gene. Results The allelic replacement strains of Newman-GraS（T136I） and XN108-GraS（I136T） were constructed successfully. The minimal inhibitory concentration（MIC） of the reverse strain XN108-GraS（I136T） to vancomycin decreased from 12 μg/mL to 8 μg/mL，and the MIC of the mutant strain Newman-GraS（T136I） to vancomycin increased from 1.5 μg/mL in wild strain to 4 μg/mL. The transmission electron microscope showed that XN108-GraS（I136T） had a significantly thinner cell wall than the wild strain XN108［（20.097±2.862） nm vs.（40.283±3.784） nm，P=0.000］. RT-qPCR showed that the peptidoglycan synthesis-related genes murC，murD，and mraY on the cell wall of Staphylococcus aureus were significantly downregulated in the reverse strain XN108-GraS（I136T）（P=0.000），with the greatest reduction in murC（about 1.935 times）. The LacZ activity test showed that the promoter activity of the murC gene in XN108 was significantly higher than that in XN108-GraS（I136T）（2 182.333±104.580 vs. 1 593.333±179.258，P=0.008），and the binding site analysis and EMSA confirmed that GraR activated by GraS could directly bind to the control region of the murC gene. Conclusion GraS（T136I） mutation in Staphylococcus aureus can promote the formation of VISA，possibly by upregulating the expression of the key murC gene in cell wall synthesis and leading to cell wall thickening.

关键词

金黄色葡萄球菌 / 万古霉素 / 耐药性 / GraSR / 细胞壁

Key words

Staphylococcus aureus / vancomycin / resistance / GraSR / cell wall

基金资助

重庆市自然科学基金资助项目(CSTB2022NSCQ-MSX1330)