机构地区:[1]陆军特色医学中心重症医学科,重庆400042 [2]陆军军医大学/第三军医大学基础医学院微生物学教研室,重庆400038
出 处:《重庆医科大学学报》2024年第10期1067-1073,共7页Journal of Chongqing Medical University
基 金:重庆市自然科学基金资助项目(编号:CSTB2022NSCQMSX1330)。
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12μg/mL下降到8μg/mL,突变菌株Newman-GraS(T136I)对万古霉素的MIC从野生株的1.5μg/mL上升到4μg/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。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 con-centration(MIC)of the reverse strain XN108-GraS(I136T)to vancomycin decreased from 12μg/mL to 8μg/mL,and the MIC of the mutant strain Newman-GraS(T136I)to vancomycin increased from 1.5μg/mL in wild strain to 4μg/mL.The transmission electron mi-croscope 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 signifi-cantly higher than that in XN108-GraS(I136T)(2182.333±104.580 vs.1593.333±179.258,P=0.008),and the binding si
关 键 词:金黄色葡萄球菌 万古霉素 耐药性 GraSR 细胞壁
分 类 号:R378.1[医药卫生—病原生物学]
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