The Construction Of CRP Mutant And Analysis Of Biofilm In Klebsiella Pneumoniae

Background: Klebsiella pneumoniae (KP) is a gram-negative bacillibacterium. This type of bacteria is widely distributed in the environmentand is an important opportunistic pathogen, which often causes bothcommunity-acquired and hospital-acquired bacteria infections. In recentyears, among those common gram-negative bacteria infections, K.pneumoniae has become the second important opportunistic pathogen onlynext to Escherichia coli. The infections caused by K. pneumoniae chieflyinclude septicemia, urinary tract infections and respiratory tract infections.Patients who have decreased immunity, such as the use of hormones,immunosuppresants and anti-metabolics can be infected by K. pneumoniae.Moreover, patients with the underlying disease and those who haveundergone invasive surgical treatment are more susceptible to theinfections. Furthermore, K. pneumoniae can be infected by contactsbetween patients, patients and doctors and medical appliances. For betterprevention and treatment of K. pneumoniae infections, there is a necessityto further study its pathogenic mechanism.The formation of biofilm makes it possible for K. pneumoniae toquickly adapt and long-term survive in the host environment so that K.pneumoniae can be protected from killing by those serum factors andphagocytosis. cAMP receptor protein (CRP) can be bined with cAMP toaffect the transcriptional expressions of numerous virulence genes.Therefore, it was speculated that the deletion of crp gene in K. pneumoniae can decrease the capacity of biofilm formation and itsvirulence and pathogenicity can be affected afterwards.Objective: The current study intended to establish a practical geneunmarked deletion method for K. pneumoniae so that it was able to lay thefoundations for further understanding the regulating mechanism of therelated virulence and pathogenicity target genes at molecular levels. Inaddition, the correspondence relationship between the phenotype ofmutants and crp gene deletion was tested so that it could be analyzedwhether the phenotype of the complement strains could be partiallyreturned to the wild type.Methods:1. Firstly, unmarked gene deletion method was used toconstruct the mutant Δcrp. The primers used to amplify the flankingregions were designed based on the genome sequence of K. pneumoniaeNTUH-K2044. Then the homology arm fusion fragments were acquiredby fusion PCR. The PCR products were inserted into the suicide vectorpKO3-km after the enzyme-digesting treatment. After that, the unmarkedmutant was acquired by the method of homology recombination.2. Theestablishment of the complement strain. The fragment of crp gene wasacquired by PCR amplification. After the enzyme-digesting treatment, thecrp fragment was cloned into the vector pGEM-T-easy and thecomplement plasmid pGEM-T-easy-km-crp was constructed. After that,the complement strain C-crp were established by transforming therecombinant plasmid into the mutant crp.3. The measurement of biofilmphenotype. The crystal violet staining method was used to measure thebiofilm production of the mutant, complement and wild-type strainsrespectively and their means and standard deviations were calculated.Results: The unmarked deletion method in K. pneumoniae wasestablished by the current study. Moreover, the crp mutants of K. pneumoniae were successfully constructed and there was no resistentscreening markers left. Furthermore, the complement plasmid wasacquired by applying the pGEM-T-easy-km plasmid. After sequencealignment, the correct plasmid was transformed into the crp mutant.After that, the complement strain C-Δcrp were acquired. In addition, aftermeasuring the biofilm formation of wild-type, mutant and complement, itcould be seen that the biofilm production of mutant was less than thecomplement and wild-type strain. Moreover, the complement strain wasable to partially return the capacity of biofilm formation of the mutant.Conclusion: As the deletion of crp gene could impair the biofilmformation capacity in K. pneumoniae, it was suggested that the crp genepossibly positively involved in the regulation of transcriptional expressionprocess of the genes related to biofilm formation in K. pneumoniae andfurther affect its biofilm formation. As the biofilm phenotype of thecomplement strain C-Δcrp could be partially returned to that of the wildtype strain, it was identified that there was one-to-one correspondencerelationship between the crp gene and biofilm formation. As a result, it ispossible to further study the mechanism regarding how the crp gene affectthe biofilm formation in K. pneumoniae.