肺炎克雷伯菌 wza基因缺失对荚膜形成与噬菌体敏感性的研究  

The effect of wza gene deletion in Klebsiella pneumoniae on capsule formation and bacteriophage sensitivity

作　　者：范政 刘洪伯 陈雨晨 崔晓虎 李周霏 付彤彤 袁静[1] Fan Zheng;Liu Hongbo;Chen Yuchen;Cui Xiaohu;Li Zhoufei;Fu Tongtong;Yuan Jing(Department of Bacteriology,Capital Institute of Pediatrics,Beijing 100020,China;Graduate School of Peking Union Medical College,Beijing 100730,China)

机构地区：[1]首都儿科研究所细菌学研究室,北京100020 [2]北京协和医学院研究生院,北京100730

出　　处：《中华预防医学杂志》2024年第7期992-997,共6页Chinese Journal of Preventive Medicine

基　　金：国家自然科学基金(32200159)。

摘　　要：目的探讨肺炎克雷伯菌wza基因缺失对细菌荚膜形成能力及对噬菌体敏感性的影响。方法以野生型肺炎克雷伯菌株W14为背景,利用温敏型质粒介导的同源重组技术构建Δwza基因缺失菌株。在溶菌肉汤(LB)培养基固体平板观察菌落形态并在液体LB培养基中测定细菌的生长曲线。通过透射电镜,糖醛酸含量测定的方法检测wza基因缺失对肺炎克雷伯菌株荚膜形成的影响。并通过观察噬菌斑明确wza基因缺失是否影响菌株W14对噬菌体phiW14的敏感性。使用t检验比较菌株W14与Δwza 缺失菌株荚膜多糖的糖醛酸含量差异情况。结果通过PCR技术明确Δwza基因缺失菌株构建成功。LB固体平板上观察到Δwza基因缺失菌株的菌落较小,而生长曲线未观察到wza基因缺失对菌株生长速度的影响。透射电镜结果显示,wza基因缺失后菌株荚膜多糖缺失。糖醛酸含量检测表明Δwza基因缺失菌株的糖醛酸含量为(45.963±2.795)μg/ml,远低于野生型菌株W14的(138.800±5.201)μg/ml,t=27.233,P<0.001。通过观察噬菌斑发现wza基因缺失后,菌株失去了对噬菌体phiW14的敏感性。结论基因wza的缺失损害了细菌的荚膜形成能力并使得菌株失去了对噬菌体phiW14的敏感性。Objective To investigate the effects of wza gene deletion in Klebsiella pneumoniae on capsule formation ability and bacteriophage sensitivity.Methods The wza deletion mutant strain was constructed through a temperature-sensitive plasmid-mediated homologous recombination.The growth curves of W14 andΔwza were detected by measuring the optical density OD600.In order to analyze the effect of gene wza on bacterial capsule formation,wild-type strain W14 andΔwza mutant strain were detected by transmission electron microscope,and their capsule contents were measured by quantifying the uronic acid contents.The plaque assay was used to detect bacterial sensitivity to bacteriophage in wild-type strain W14 andΔwza mutant strain.The t test was used to compare whether there were differences in the contents of uronic acid in the capsules of wild-type strain W14 andΔwza mutant strain.Results The PCR results revealed that theΔwza mutant strain was successfully constructed.Compared with wild-type strain W14,the growth curves ofΔwza on the solid plates demonstrated a slightly slower growth.However,no difference in growth was observed among wild-type strain W14 andΔwza mutant strains in LB broth.The transmission electron microscope results showed that wza gene deletion resulted in the loss of capsule in bacteria.The uronic acid content assay suggested that the capsule content was significantly decreased inΔwza mutant strain(45.963±2.795)μg/ml compared with wild-type strain W14(138.800±5.201)μg/ml.There was a statistical difference between the two groups(t=27.233,P<0.001).The plaque assay indicated that bacteria lost its sensitivity to bacteriophage when gene wza was deleted.Conclusion Deletion of the wza gene impairs bacterial capsule formation ability and can affect bacterial sensitivity to bacteriophage phiW14.

关 键 词：肺炎克雷伯菌 wza 基因敲除 荚膜 噬菌体 

分 类 号：R446.5[医药卫生—诊断学]