A Study Of HTH Domain Regulatory Protein KbvR In The Pathogenesis Of Klebsiella Pneumoniae

Objective: Klebsiella pneumoniae is an important pathogen for nosocomial infection and community-acquired infection.It is a representative of multi-drug resistant and extensively resistant bacteria.KbvR is a class of transcription regulator containing HTH domain in Klebsiella pneumoniae,which may be related to bacterial pathogenicity.Construct of Klebsiella pneumoniae deletion mutant(?KbvR)and complement strain(C-KbvR),analysis the effect of KbvR on bacterial growth,pathogenicity,biofilm formation,capsular synthesis and fimbriae production,and to elucidate the mechanism of KbvR in pathogenesis of Klebsiella pneumoniae.Methods: KbvR gene deletion mutant was constructed based on homologous recombination by using sucrose-sensitive suicide plasmid pKO3-Km,and then the fragment including KbvR coding region,promoter binding region of about 900 bp and downstream transcription termination region of about 600 bp was amplified and cloned into pGEM-T-easy plasmid and then transferred to the mutant to acquired KbvR complement strain C-KbvR.Bacterias were cultured in LB medium at 37°C to detect the bacterial growth by UV600 spectrophotometer at different time.The effects of KbvR gene on the virulence of Klebsiella pneumoniae were observed by virulence in mice with different strains of LD50,the pathological change in liver and lung was also observed.RNA-seq was used to analyze the changes of the expression profile of mutant and wild plants,and to find the target gene which may be regulated by Kbv R.Combined with RNA-seq results and pathogenicity studies on Klebsiella pneumoniae,some genes were selected for further study on KbvR.Through the quantitative test of crystal violet,the influence of KbvR on bacterial biofilm formation was tested and the effect on bacterial capsular synthesis was detected by string test and centrifugal test.The yeast agglutination test for detection the effect of KbvR gene on the production of type I fimbriae,man-nose-resistant hemagglutination test for type III fimbriae.Using RT-PCR to test the infulence of KbvR gene on structural genes and local regulators that related fimbriae and capsular synthesis,by extracting the total RNA of wild strain and mutant strain.Finally to elaborate the specific mechanism of KbvR regulation on bacterial pathogenicity.Results: The KbvR delection mutant ?KbvR and complement strain C-KbvR were constructed successfully for KbvR gene was only not expressed in KbvR delection mutant detected by RT-PCR.The growth curve of the three strains showed no significant difference,indicating that the KbvR gene did not affect the growth of bacteria.Mice virulence experiments found that virulence decreased significantly after delecting the KbvR gene,the survival rate of mice infected with delection mutant was significantly higher than that of mice infected with WT,as liver and lung tissue have no obvious pathological changes.RNA-seq found that KbvR regulates the transcription of 304 gene,up-regulated by 65,down-regulated by 239.These regulated genes include other transcriptional regulators,capsular and type I fimbriae synthesis-related genes,iron uptake-related genes,urease genes,sugar transport PTS systems and functional unknown proteins.The phenotypic experiments showed that ability of the biofilm formation were significantly lower than that of WT in KbvR delection mutant.Constract to wild strain,the quantitative staining of crystal violet confirmed that the biofilm formation ability of mutant was significantly lower(P < 0.01).And this has nothing with the bacterial growth.Sting test and centrifugal test showed the capsule formation ability of mutation decreased significantly.Yeast agglutination test and mannose-resistant hemagglutination test showed the synthesis of type I fimbriae and type III fimbriae were significantly increased.RT-PCR results showed that the expression of magA was decreased after delection of KbvR,and the expression of the local regulators rmpA and rcs A was increased.While,expression of type I fimbriae structure gene fimA and regulator fimK,type III fimbriae structure gene mrkA and regulator mrkH were increased.Conclusion:(1)KbvR gene is positively regulated bacterial pathogenicity,the virulence of KbvR delection mutant decreased significantly.The KbvR gene does not affect the growth ability of bacteria,indicating that the effect of KbvR on pathogenicity of bacteria has nothing with the bacterial growth,so the KbvR gene may regulates some pathogenic factors.(2)KbvR positive regulates biofilm formation which is the important pathogenic factors of Klebsiella pneumoniae.(3)KbvR positive regulates the formation of capsules and negative regulation of type I fimbriae and type III fimbriae synthesis.(4)KbvR infulences the bacterial virulence by regulating the capsule,fimbriae synthesis and biofilm formation.