转录调控子CRP对碳青霉烯类耐药肺炎克雷伯菌entC的调控机制

Mechanism of transcriptional regulator CRP in regulating carbapenem-resistant Klebsiella pneumoniae entC

目的确定环腺苷酸(cAMP)受体蛋白(CRP)对碳青霉烯类耐药肺炎克雷伯菌(CRKP)肠杆菌素铁载体相关基因entC的调控机制。方法以CRKP-27野生株构建crp基因缺失株Δcrp和回补株c-Δcrp。利用铬天青S(CAS)定量试验观察CRP对CRKP铁载体分泌量的影响。RT-qPCR试验和lacZ报告基因融合试验研究CRP对entC表达及对其启动子的调控。凝胶阻滞试验(EMSA)确定CRP是否与entC启动子区结合,DNaseⅠ足迹试验进一步确定结合序列。结果成功构建Δcrp和c-Δcrp菌株;在正常条件和缺铁条件下,Δcrp菌株的铁载体分泌量均高于野生株和c-Δcrp菌株,但仅在正常环境中有统计学意义(P<0.05);正常条件和缺铁条件下,Δcrp菌株中entC基因的mRNA相对表达量较野生株和c-Δcrp菌株明显降低(P均<0.05);正常条件和缺铁条件下,Δcrp菌株中entC基因的启动子活性低于野生株和c-Δcrp菌株(P均<0.05);EMSA结合试验显示随着CRP蛋白量的增加,entC探针朝正极移动的距离都相应缩短,出现阻滞现象;DNaseⅠ足迹试验进一步检测到entC启动子区与CRP特异性结合位点为:5′-AAGGTGATAAATGCGTCTCATTTTCAA-3′。结论CRKP中CRP能特异地结合到entC启动子区并直接促进其转录表达。

ObjectiveTo investigate the mechanism of cyclic AMP receptor protein(CRP)in regulating the siderophore enterobactin-related gene entC of carbapenem-resistant Klebsiella pneumoniae(CRKP).MethodsA mutant strain with crp gene deletion strain(Δcrp)and a complementary strain(c-Δcrp)were constructed using CRKP-27 as the wild-type strain.The influence of CRP on the secretion of siderophore by CRKP was analyzed by chrome azurol sulfonate(CAS)quantitative assay.RT-qPCR and lacZ reporter gene fusion assay were used to detect the regulatory effect of CRP on entC gene expression and its promoter.Electric mobility shift assay(EMSA)was performed to detect the binding of CRP to the entC promoter region and the binding sequence was analyzed by DNaseⅠfootprinting assay.ResultsTheΔcrp and c-Δcrp strains were successfully constructed.Compared with the wild-type and c-Δcrp strains,theΔcrp strain could secrete more siderophore under both normal and iron-deficient conditions,but the difference was statistically significant only under normal condition(P<0.05).The relative expression of entC gene at mRNA level was significantly lower in theΔcrp strain than that in the wild-type and c-Δcrp strains under both normal and iron-deficient conditions(both P<0.05).The promoter of entC gene in theΔcrp strain was less active than that in the wild-type and c-Δcrp strains under both normal and iron-deficient conditions(both P<0.05).EMSA showed that with the increase of CRP protein,the distance of entC probe from the positive pole was shortened and blocked.DNaseⅠfootprinting assay further identified the specific binding site of the entC promoter region to CRP as 5′-AAGGTGATAAATGCGTCTCATTTTCAA-3′.ConclusionsThe CRP protein in CRKP could specifically bind to the entC promoter region and directly promote its expression at transcriptional level.