Molecular Mechanism Of RcsAB And Fur Coregulate The Rgulation Of EntC In Klebsiella Pneumoniae Ntuh-k2044

Objective:The iron acquisition system is significant for virulence and is under tight regulatory control in dozens of pathogens.Ferric-uptake regulator?Fur?is an Fe²⁺-responsive transcription factor and maintains iron balance in bactiera,and the regulator of capsule synthesis?Rcs?can modulatethe expression of exopolysaccharide.The iron acquisition system is known to be transcriptionally regulated by Fur,but very little is known regarding the mechanism by which RcsAB controls the iron-acquisition system.Enterobactin as an siderophore belongs to the iron acquisition system of bactria.The entC gene encodes isochorismate synthetase,which participates in enterobactin synthesis.The promoter-proximal regions of entC contain RcsAB and Fur box-like sequence.The purpose of this study is to investigate the molecular mechanism by which RcsAB and Fur coregulate the iron-acquisition system via entC in Klebsiella pneumonia NTUH-K2044 in response to iron availability.Methods:The secretion of siderophore from NTUH-K2044,Kp:?rcsA,Kp:?rcsB,Kp:?rcsAB and complement strains were detected by CAS assay.The transcriptional activity of entC promoter was under different conditions through lacZ fusion assay.The lacZ fusion assay,RT-qPCR,EMSA and DNase?footprinting assay were conducted to study the transcriptional regulation of entC by RcsAB under iron limitation.The fur gene was knocked out by homologous recombination using pKO3-Km suicide plasmid.RT-qPCR,EMSA and DNase?footprinting assay were performed to explore the transcriptional regulation of entC and rcsA by Fur under iron repletion.Results:CAS assay showed that RcsAB can affect the expression of iron-acquisition system.ThelacZ fusion assay showed iron limitation enhanced the activity of the entC promoter and RcsAB can regulate entC regulation under iron restricted conditions.RT-qPCR indicated entC regulation can be regulated by RcsAB and Fur under iron restricted conditions and iron sufficient conditions;and Fur depressed rcsA regulation under iron sufficient conditions.EMSA indicated that RcsABproteins complex and Fur could bind to promoter of entC directly.DNase?footprinting assay showed the location of RcsAB and Fur binding to promoter of entC.Conclusion:RcsAB and Fur can regulate the transcription of entC under iron-depltion and iron-repletion,respectively.In addition,a regulatory cascade was identified in which Fur repressd rcsA expression and reduced RcsAB and entC expression.