Transcriptional Regulation Of GalF By RcsAB Affects Capsular Polysaccharide Formation In Klebsiella Pneumoniae NTUH-K2044

Objective The purpose of this study is to explore the effect of RcsAB,a transcription regulator of Klebsiella pneumoniae(K.pneumoniae),on the phenotype of capsular polysaccharide and the regulation mechanism of galF which is the capsular polysaccharide related gene.Methods The wild-type K.pneumoniae strain NTUH-K2044 and the rcsA,rcs B,and rcs AB gene knockout and complemented strains previously constructed in the laboratory were subjected to virulence and capsular polysaccharide related phenotype experiments.To assay the influence of RcsAB on the formation of capsular polysaccharide of K.pneumoniae,cytotoxicity assay,quantitative test of capsular polysaccharide and electron microscope observation of capsular polysaccharide were carried out.At the same time,qRT-PCR was used to verify the regulation relationship of galF and RcsAB protein..Then,the binding fragment was identified by EMSA and DNaseI footprint experiments.Results The results of Capsular polysaccharide-related experiment showed that rcsAB genes were involved in the function of capsular polysaccharide,and cytotoxicity assay showed that rcsAB genes affected the virulence of the strain.qRT-PCR results suggested that RcsAB positively regulated the transcription of galF gene.EMSA results indicated that only RcsAB proteins complex rather than individual RcsA or RcsB could bind to galF gene directly..DNaseI footprinting experiments explored that RcsAB protein complex could bind to the upstream of the galF promoter from 177 bp to 151 bp.Conclusions The RcsAB protein complex positively regulated the formation of the capsular polysaccharide by directly regulating the capsular polysaccharide-related gene gal F to affect the formation of strain virulence.