鼠伤寒沙门菌中甲基化修饰调控PhoP活性的机制研究

Role of methylation in regulating PhoP activity in Salmonella enterica serovar Typhimurium

目的·探究鼠伤寒沙门菌转录调控因子PhoP的甲基化修饰与其活性的相关性,筛选PhoP的甲基转移酶。方法·Westernblotting检测不同培养条件下鼠伤寒沙门菌中PhoP的甲基化水平变化。通过生物信息学预测候选的甲基转移酶,体内过表达预测的甲基转移酶后检测phoP及其下游基因的转录水平变化,找到可能的PhoP甲基转移酶,并体外验证该甲基转移酶能否甲基化PhoP。使用real-time PCR检测高浓度Mg^2+、弱酸性等培养条件下,phoP与其甲基转移酶在转录水平的相对表达变化。结果·随着培养基中Mg^2+浓度升高,PhoP甲基化水平上升;而经过弱酸刺激后,PhoP甲基化水平下降。生物信息学预测并筛选到9个甲基转移酶,其中体内过表达STM14_0023后,phoP及其下游基因的转录水平下降且PhoP蛋白水平也下降,但敲除STM14_0023对上述分子的表达无影响。体外甲基化酶促反应显示STM14_0023能够甲基化PhoP,体内过表达STM14_0023后也可提高PhoP的甲基化水平,且在抑制phoP表达的高浓度Mg2+培养条件下STM14_0023的转录水平升高。结论·STM14_0023是PhoP的甲基转移酶,甲基化修饰抑制PhoP活性。

Objective·To investigate the correlation between methylation and the activity of transcriptional regulator PhoP in Salmonella enterica serovar Typhimurium(S.Typhimurium),and screen for the methyltransferase of PhoP.Methods·The methylation level of PhoP in S.Typhimurium under different culture conditions was determined by Western blotting.The transcription levels of phoP and the genes regulated by phoP were examined to screen for the methyltransferase of PhoP after overexpressing methyltransferase candidates predicted by bioinformatics.And then methyltransferase assay was verified in vitro.The transcription levels of phoP and its methyltransferase were determined by real-time PCR in high concentration of Mg^2+or weak acid conditions.Results·As the concentration of Mg^2+increased in the medium,the methylation level of PhoP increased,and its methylation level decreased after S.Typhimurium was stimulated by weak acid.In the screening of 9 methyltransferases predicted by bioinformatics,overexpression of STM14_0023 reduced the transcription level of phoP and its downstream genes and the protein level of PhoP in vivo,but knockout of STM14_0023 had no effect on the expression of phoP and its downstream genes.STM14_0023 was capable of methylating PhoP in vitro and could also increase the methylation level of PhoP after overexpression of STM14_0023 in vivo.Under the condition of high concentration of Mg2+when the expression of phoP was inhibited,the transcriptional level of STM14_0023 was reduced.Conclusion·STM14_0023 is the methyltransferase of PhoP,and methylation inhibits PhoP activity.