Study On Regulation Mechanism Of KfuABC Operon By Crp In Klebsiella Pneumoniae

Background Klebsiella pneumoniae is a gram-negative enterobacteria.In recent years it almost becomes a secondary conditional pathogenicbacterium next to E. coli, and associates with community-acquired andnosocomial-acquired infections, including liver abscesses, pneumonia,meningitis and endophthalmitis.The virulence factors of K.pneumoniaeinclude capsular polysaccharide, lipopolysaccharide, iron intake and typeⅠfimbriae, and so on. Iron is a vital element for growth and reproduction ofK.pneumoniae. However, iron is located intracellularly, chelated byhigh-affinity iron binding proteins. Consequently K.pneumonia has evolveda large variety of iron acquisition strategies for their survival and growth.KfuABC system which involved in iron intake is one of a number of ironintake systems, which is very important for virulence of K.pneumoniae.cAMP receptor protein (CRP) is an important global regulator ofpathogenic bacteria, involved in regulating the transcription of catabolicgenes and virulence genes. However, the study on regulation mechanism ofkfuABC operon by CRP in K.Pneumoniae is still in the beginning stage.Objective The objective was to investigate the regulation mechanismof regulator CRP on kfuABC operon in K.pneumoniae, which could lay the foundation for controlling the virulence of strain.Methods The effects of CRP on growth rate and virulence phenotypewere determined by growth curve and string test. Real-time PCR wasfirstly used to validate the operon structure of kfuABC by using theintergenic region spanning primers. Total RNAs of the wild-type strainNTUH-K2044(WT) were extracted, which was applied to detect thetranscriptional start site of kfuA using primer extension assay. The entirepromoter-proximal region of kfuA was amplified by PCR and cloned intothe upstream of the beta-galactosidase gene of pHRB309containing apromoterless lacZ reporter gene. The recombinant plasmids weretransformed into WT and the crp null mutant (△crp) to determine theregulation of CRP regulator on kfuA by measuring β-galactosidase activity.Then, total RNAs were extracted from△c rpand WT strains, and thequantitative RT-PCR was carried out to further calculate the transcriptionalvariation of kfuA in the△c rpand WT. The entire promoter-proximalregion of the kfuA gene was amplified by PCR from WT strain and thefusion protein CRP was expressed. Then, the electrophoretic mobility shiftassay and DNase Ⅰ footprinting experiment were applied to analyze theDNA-binding site of His-CRP to kfuA promoter region in vitro.Results The results of the growth curve of K.pneumoniae showed thatthe growth rate of△c rpwas significantly slower than WT strain andcomplemented strain, and hypermucoviscosity of△c rpwas attenuate by string test. The transcription of kfuABC operon was regulated by CRPnegatively. Primer extension assay detected only one transcriptional startsite. The DNase Ⅰfootprinting assays showed that the binding site waslocated from204bp to171bp upstream when the transcription initiationsite is+1.Conclusion The growth rate of△c rpwas significantly slower thanWT strain and complemented strain, and hypermucoviscosity of△c rpwasweaken, as well as less virulence. The transcription of kfuABC operon wasrepressed by CRP via directly combination with promoter region of kfuABCoperon in K.pneumoniae. In our study, we demonstrated the regulationmechanism of kfuABC operon by CRP in K.pneumoniae firstly, whichcould lay the foundation for the further reseach on transcriptionalregulatory networks of virulence factor in K.pneumoniae.