摘要
目的对肺炎克雷伯菌(Klebsiella pneumoniae,Kpn)Ⅵ型分泌系统(typeⅥsecretion system,T6SS)结构基因vgrG进行敲除,评估缺失vgrG后的细菌体外生长能力和致病力。方法根据NCBI公布的Kpn vgrG序列和前后基因序列设计引物,经PCR扩增出vgrG上下游同源臂序列并搭桥后克隆至载体pKO3-Km,重组载体导入Kpn感受态细胞,经同源重组获得vgrG缺失株ΔvgrG。通过PCR扩增出vgrG启动子加完整基因片段并克隆至载体pBAD33,导入缺失株ΔvgrG感受态细胞获得回补株CΔvgrG。将野生株WT、ΔvgrG株和CΔvgrG株在LB(Luria-Bertani)液体培养基中培养并比较生长速度;不同菌株感染人肺上皮细胞A549比较黏附细胞能力;不同菌株感染小鼠巨噬细胞Raw264.7比较胞内存活能力;不同菌株感染小鼠观察小鼠的存活率(n=10)和肺脏细菌数量(n=6)。组间比较采用t检验。结果经过同源重组获得了ΔvgrG株,经特异性引物鉴定相比WT株缺失了完整vgrG片段(2487 bp),并在此基础上获得了CΔvgrG株。Kpn缺失vgrG前后细菌的体外生长能力没有显著变化[(1.40±0.10)vs(1.20±0.30),t=0.63,P>0.05],WT株黏度显著高于ΔvgrG株[(0.96±0.04)vs(0.38±0.05),t=9.72,P<0.05],CΔvgrG株黏度也显著高于ΔvgrG株(P<0.05)。细胞层面,野生株WT对A549细胞的黏附菌量显著多于ΔvgrG株[(5367.00±318.00)CFU vs(4067.00±88.19)CFU,t=3.94,P<0.05],CΔvgrG株黏附菌量也显著高于ΔvgrG株(P<0.05)。细菌感染12 h后,WT株在巨噬细胞中的存活率显著高于ΔvgrG株[(69.00±1.00)%vs(47.50±2.50)%,t=7.99,P<0.05]。动物层面,致死剂量感染小鼠后WT株组存活率显著低于ΔvgrG株组[(16.67±8.82)%vs(53.33±6.67)%,t=3.32,P<0.05];半致死剂量感染小鼠后WT株组小鼠肺脏细菌数显著多于ΔvgrG组[(4.97±0.06)lg CFU/g vs(4.05±0.04)lg CFU/g,t=12.27,P<0.01],CΔvgrG株组小鼠肺脏细菌数也显著多于ΔvgrG株组(P<0.01)。结论vgrG不影响Kpn的体外生长,但参与Kpn对上皮细胞的黏附、抵抗巨噬细胞杀伤和对小鼠的致病力。
Objective To investigate the role of the structural gene vgrG of the typeⅥsecretion system(T6SS)of Klebsiella pneumoniae(Kpn),and evaluate the growth ability in vitro and pathogenicity of the bacteria after vgrG was deleted.Methods Using sequences published by the National Center for Biotechnology Information(NCBI),primers were designed to amplify the upstream and downstream homology arms of vgrG via PCR.These fragments were cloned into the vector pKO3-Km after overlapping,the recombinant vector pKO3-Km-vgrG was transferred into Kpn competent cells,and the vgrG deletion strain ΔvgrG was obtained through homologous recombination.The vgrG promoter with the complete gene fragment was amplify by PCR and cloned into the pBAD33 vector.The pBAD33-vgrG was then transferred into ΔvgrG competent cells to obtain the complemented strain CΔvgrG.The wild-type strain(WT),ΔvgrG strain and CΔvgrG strain were cultured in LB(Luria-Bertani)liquid medium to compare growth rates.Adhesion to human lung epithelial A549 cells and intracellular survival in macrophages Raw264.7 cells were assessed.In vivo experiments included mouse survival analysis(n=10)and lung bacterial load quantification(n=6).Statistical comparisons were performed using the Student t-test.Results The ΔvgrG strain was obtained through homologous recombination.It was identified by specific primers that compared with the WT strain,the complete vgrG fragment(2487 bp)was deleted.On this basis,the CΔvgrG strain was obtained.Deletion of vgrG did not significantly affect Kpn growth in vitro growth ability of bacteria before on after Kpn deleted vgrG[(1.40±0.10)vs(1.20±0.30),t=0.63,P>0.05].The viscosity of WT strain was significantly higher than that of theΔvgrG strain[(0.96±0.04)vs(0.38±0.05),t=9.72,P<0.05],the viscosity of the CΔvgrG strain was also significantly higher than that of the ΔvgrG strain(P<0.05).At the cellular level,the amount of adherent bacteria of the WT strain to A549 cells was significantly greater than that of the ΔvgrG strain[(5367.00±318.00)CFU vs(4067.00±88.19)CFU,t=3.94,P<0.05],the amount of adherent bacteria of CΔvgrG strain was also significantly higher than that of ΔvgrG strain(P<0.05).After 12 h infection,the WT strain survival rate in macrophages was significantly higher than that of theΔvgrG strain[(69.00±1.00)%vs(47.50±2.50)%,t=7.99,P<0.05].At the animal level,the survival rate of WT strain group after lethal dose infection of mice was significantly lower than that of ΔvgrG strain group[(16.67±8.82)%vs(53.33±6.67)%,t=3.32,P<0.05];mice infected with semi-lethal dose and the number of bacteria load in the lungs of WT strain group was significantly higher than that of the ΔvgrG strain group[(4.97±0.06)lg CFU/g vs(4.05±0.04)lg CFU/g,t=12.27,P<0.01],the amount of bacteria in the lungs of mice in CΔvgrG strain group was also significantly higher than that in ΔvgrG strain group(P<0.01).Conclusions The vgrG gene does not affect the growth of Kpn in vitro,but it is involved in the adhesion of Kpn to epithelial cells,resistance to macrophage killing and pathogenicity to mice.
作者
徐双依
张筱薇
韩雨佳
李笑眉
徐刚
Xu Shuangyi;Zhang Xiaowei;Han Yujia;Li Xiaomei;Xu Gang(Department of Plastic and Burn Surgery,Northern Jiangsu People's Hospital Affiliated to Yangzhou University/Northern Jiangsu People's Hospital,Yangzhou 225001,China;Graduate School of Yangzhou University,Yangzhou 225001,China)
出处
《中华微生物学和免疫学杂志》
北大核心
2025年第8期643-648,共6页
Chinese Journal of Microbiology and Immunology
基金
杭州市医药卫生科技项目(B20200041)。
关键词
肺炎克雷伯菌
细菌Ⅵ型分泌系统
结构蛋白
致病性
Klebsiella pneumoniae
TypeⅥsecretion system
Structural protein
Pathogenicity
