亚MIC庆大霉素下CpxR对鼠伤寒沙门菌生长特性和acrD基因转录的影响

Influence of CpxR on growth characteristics and AcrD gene transcription of Salmonella entericaserovar typhimurium at the subinhibitory concentrations of gentamicin

前期研究发现双组分应答调控子CpxR可调控鼠伤寒沙门菌对氨基糖苷类药物的耐药性。为进一步探索其调控机制,本研究测定了无抗生素和亚MIC庆大霉素下,鼠伤寒沙门菌标准株(JS)、过表达株(JS/pcpxR)、磷酸化位点突变过表达株(JS/pcpxR*)分别在不同能量水平的2种基本培养基中的生长曲线;同时采用LacZ报告基因融合试验方法构建3株acrD启动子融合报告菌株(JS/PacrD、JS△cpxR/PacrD、JS△cpxR/pcpxR/PacrD),通过测定其β-半乳糖苷酶活性,分析无抗生素诱导和亚MIC庆大霉素诱导下CpxR对acrD启动子转录活性的影响。结果显示,JS、JS/pcpxR和JS/pcpxR*在M9-0.2%甘油(+亚MIC庆大霉素)培养基中的对数生长期生长速率均低于各自在M9-0.2%甘油培养基中的对数生长期生长速率,JS和JS/pcpxR*在M9-0.2%葡萄糖(+亚MIC庆大霉素)培养基中的对数生长期生长速率也低于各自在M9-0.2%葡萄糖培养基中的对数生长期生长速率,但JS/pcpxR在M9-0.2%葡萄糖(+亚MIC庆大霉素)培养基与在M9-0.2%葡萄糖培养基中的生长速率基本一致;另外,亚MIC庆大霉素诱导下JS△cpxR/pcpxR/PacrD的β-半乳糖苷酶活性高于JS△cpxR/PacrD,差异极显著(P<0.01),与JS/PacrD的β-半乳糖苷酶活性水平相当。结果表明,在葡萄糖存在的环境中,CpxR的过表达可提高鼠伤寒沙门菌对庆大霉素的抗性,且CpxR对外排泵acrD转录的促进是其发挥对庆大霉素耐药性调控作用的重要机制。

Previous study has confirmed that CpxR can regulate the drug resistance of Salmonella enterica serovar typhimurium to aminoglycosides.To explore the regulatory mechanism of CpxR,the growth curve of S.enterica serovar typhimurium standard strain（JS）,cpxR overexpression strain（JS/pcpxR）and phosphorylation site mutational cpxRoverexpression strain（JS/pcpxR＊）in two different energy levels of minimum medium were determined,and the influence of CpxR on the promoter activities of acrD gene at the subinhibitory concentrations of gentamicin were analyzed by using LacZfusion assay.The results showed that the logarithmic phase growth speed of strains JS,JS/pcpxR and JS/pcpxR＊in M9-0.2% glycerol culture with subinhibitoryconcentrations of gentamicin were much lower than that in M9-0.2%glycerol culture,and the logarithmic phase growth speed of strains JS and JS/pcpxR＊in M9-0.2%glucose culture with subinhibitory concentrations of gentamicin were also lower than that in M9-0.2% glucose culture,but the logarithmic phase growth speed of strain JS/pcpxRin M9-0.2% glucose culture was largely similar to that in M9-0.2% glucose culture with subinhibitory concentrations of gentamicin.In addition,theβ-galactosidase activity of fusion reporter strain JS△cpxR/pcpxR/PacrD was significantly higher than that of JS△cpxR/PacrD（P0.01）,and approached that of JS/PacrD.The results indicated that the overexpression of CpxR can improve the resistance of Salmonella typhimuriumto gentamicin in the presence of glucose,and the enhancing effect of CpxR on the transcription of efflux pump acrDis an important mechanism of cpxR-mediated gentamicin resistance.