Transcriptional Regulation Of Yersiniabactin Receptor Gene FyuA Gene By Ferric Uptake Regulator (Fur) In Klebsiella Pneumoniae NTUH-K2044

Objective: Klebsiella pneumoniae is one of the main pathogens causing nosocomial infections.Ferric uptake regulator(Fur)is a global transcriptional regulator that affects the expression of virulencerelated genes.The promoter-proximal regions of fyuA contain Fur box-like sequence.Fyu A is the outer membrane receptor for siderophore yersiniabactin.The purpose of this study is to explore the regulatory effect of Fur on the target gene fyuA(yersiniabactin receptor),and analyze the effect of fyuA gene on virulence of Klebsiella pneumonia.To further understand the pathogenic mechanism of Klebsiella pneumoniae NTUHK2044.Methods: The suicide plasmid pKO3-Km was used to construct the recombinant plasmid pKO3-Km-ΔfyuA,which was introduced into Klebsiella pneumoniae NTUH-K2044,and the unmarked fyuA-deletion mutant was obtained through homologous recombination and reverse screening using sucrose.The expression plasmid p BAD33-C-ΔfyuA was constructed and transformed into the mutant,and the complementation strain was obtained by PCR screening and sequencing.The secretion of siderophores and biofilm formation ability of wild strain,fyuA mutant and complementation strain were compared by CAS detection and crystal violet quantitative experiment.The effect of fyuA on the virulence of Klebsiella pneumoniae was analyzed by the virulence test of Galleria mellonella.The GFP reporter gene fusion assays,RT-q PCR,EMSA and DNase I footprint experiment were used to clarify the transcriptional regulation mechanism of Fur on fyuA.Results: The fyuA gene knockout strain and the complementation strain were constructed successfully.CAS plate assay showed that the deletion of the fyuA gene can increase the siderophores secreted of the strain,and the CAS liquid test further confirmed that fyuA can affect the production of siderophores in Klebsiella pneumoniae.The results of crystal violet experiment showed that fyuA can positively affect the formation of biofilms.The virulence test of Galleria mellonella showed that the lack of fyuA can weaken the virulence of the bacteria,so fyuA had a certain influence on the virulence of the strains.The GFP reporter gene fusion experiment and RTq PCR analysis indicated that Fur negatively regulates the expression of fyuA under iron-sufficient conditions.The EMSA experiment showed that Fur can directly bind to the promoter region of fyuA,and the DNase I footprint experiment further obtained the sequence of the specific binding site.Conclusion: Phenotype experiments found that fyuA can affect the virulence of Klebsiella pneumoniae.Molecular experiments have shown that Fur can bind to the promoter region of fyuA,and negatively regulate the transcription of fyuA.These results provide new ideas for studying the pathogenic mechanism of Klebsiella pneumoniae.