Construction of hypervirulent Klebsiella pneumoniae wzc-knockout strain and analysis of its biological characteristics

WANG Qing; YANG Rui; ZENG Jie; YU Hongpeng; LU Shouyi; LIANG Xinyun; LIN Huasheng; ZHANG Wei; JIN Min; LI Lu; ZHAO Zuguo

doi:10.20227/j.cnki.2096-3610.2026.01.004

Abstract：Objective To generate a wzc-knockout strain of the hypervirulent Klebsiella Pneumoniae(hvKP)KPN234 and investigate the effect of wzc gene deletion on the bacterial capsule and virulence.Methods A sgRNA targeting the wzc gene was designed,and the CRISPR/Cas9 gene knockout vector was constructed and electroporated into KPN234.After dual antibiotic screening using apramycin and kanamycin and plasmid curing,the wzc gene knockout strain was obtained.PCR and whole-genome sequencing were used to confirm the gene knockout and the absence of off-target effects,and Western blot was utilized to verify the absence of Wzc protein expression in the knockout strain.The growth curve of the wzc gene knockout strain was plotted to assess the effect of gene knockout on bacterial growth.The effects of gene knockout on capsular polysaccharides synthesis were evaluated using string test,viscosity semi-quantitative test,and capsular polysaccharide quantitative test.The virulence of the modified bacteria was subsequently evaluated by Galleria mellonella larva infection test.Results After the gene knockout procedure,the hvKP KPN234 lost the wzc gene with no mutations detected at other sites,confirming the successful gene knockout.The generated wzc gene knockout strain exhibited significantly reduced mucous property of the colony and its capacity to synthesize capsular polysaccharides(P<0.001).Moreover,and the virulence of the wzc-deficient strain was significantly decreased in the Galleria mellonella larval infection test(P<0.001).Conclusion The successful construction of wzc gene knockout hvKP strain establishes a foundation for further research on the mechanism of the wzc gene in hvKP.