Initial Reseach On Function Of KP1₂626 Gene Of Klebsiella Pneumoniae In Virulence

Objective: Klebsiella pneumoniae?KP?is one of the most important pathogens causing community-acquired infections and nosocomial infections in hospitals.The protein encoded by the KP1₂626 gene belongs to the regulatory protein of the LuxR family.The transcriptional regulators in LuxR family can control virulence and biofilm formation by regulating related genes.To determine the effect of KP1₂626 gene on the virulence of Klebsiella pneumoniae,this paper analyzes the difference of virulence phenotype among wild strain NTUH-K2044,mutant strain Kp-?2626,complement strain Kpc-?2626 and.It provides a basis for further study of the regulation mechanism of KP1₂626 on strain virulence.Methods: The KP1₂626 gene fragment was knocked out by homologous recombination to obtain a mutant Kp-?2626,and then the recombinant plasmid containing the KP1₂626 gene fragment was transformed into a mutant strain to obtain a complement strain Kpc-?2626.The growth curves of three strains were detected to observe the effect of KP1₂626 gene on bacterial growth.The biofilm formation and virulence of the three strains were compared by crystal violet experiment andhyperviscosity experiment.A549 cells were infected with wild type strains,mutant strains,and complement strains,and the toxicity of the bacteria to the cells was evaluated by measuring the amount of LDH released from the cells.Results: 1.The mutant Kp-?2626 and the complement strain Kpc-?2626 were successfully constructed.2.The growth curves of mutant Kp-?2626 and complement strain Kpc-?2626 were similar to those of wild strain NTUH-K2044,and there was no significant difference,indicating that the deletion of KP1₂626 gene had no effect on the growth of the strain.3.After the three strains were cultured for 12 hours,24 hours and 36 hours,the results of biofilm determination showed that the biofilm formation ability of the mutant Kp-?2626 was weakened,and the complement strain Kpc-?2626 was between these two.4.The results of the superviscosity experiment showed that the mutant strain had no obvious superviscosity phenomenon and the superviscosity was weakened.5.Cells were infected with MOI=1000 for the optimal infection index for 21 hours,and the final lactate dehydrogenase?LDH?content was determined by detecting 10-fold dilution reaction supernatant.The cytotoxicity?81.8%?of mutant strain was higher than the cytotoxicity?6.1%?of mutant strain,and the cytotoxicity?68.19%?of complemen strain was between wild strain and mutant strain.Conclusion: Deletion of the KP1₂626 gene does not affect bacterialgrowth.After knocking out the KP1₂626 gene,the superviscosity,biofilm formation and cytotoxicity of the bacteria were significantly reduced,suggesting that this gene may positively affect the virulence of the bacteria.