南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (4): 748-756.doi: 10.12122/j.issn.1673-4254.2024.04.17

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肺炎克雷伯菌钼酸盐转运体modA基因缺失株的构建及其生物学特性

王 慧，姜晓宇，李飞雨   

  1. 黄石市妇幼保健院（湖北理工学院附属妇幼保健院）检验科，湖北 黄石 435000；黄石爱尔眼科医院，湖北 黄石 435000；黄石市中医医院，湖北 黄石 435000；湖北医药学院基础医学院，湖北 十堰 442000
  • 发布日期:2024-04-29

Construction and characterization of a modA gene mutant strain of Klebsiella pneumoniae

WANG Hui, JIANG Xiaoyu, LI Feiyu   

  1. Department of Clinical Laboratory, Huangshi Maternity and Children's Health Hospital (Affiliated Maternity and Children's Health Hospital of Hubei Polytechnic University), Huangshi 435000, China; Huangshi Aier Eye Hospital, Huangshi 435000, China; Clinical Laboratory, Huangshi Hospital of TCM, Huangshi 435000, China; School of Basic Medical Sciences, Hubei University of Medicine, Shiyan 442000, China
  • Published:2024-04-29

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摘要： 目的 利用同源重组技术构建肺炎克雷伯菌NTUH-K2044株钼酸盐转运系统modA基因缺失株和回补株，探讨modA基因对肺炎克雷伯菌厌氧硝酸盐呼吸生长及表型的影响 。方法 利用自杀载体pKO3-Km质粒构建modA基因缺失株，同时扩增包含modA基因启动子、开放阅读框（ORF）和终止子区域的整条序列片段，将其克隆至pGEM-T-easy质粒中获得重组质粒modA-pGEM-T-easy，电转化至modA缺失株中得到回补株 C-modA，通过体外厌氧硝酸盐呼吸生长测定、竞争力指数实验、生物膜结晶紫定量实验、黏度定量实验以及菌株形态Image J测量法比较分析NTUH-K2044野生株、modA基因缺失株、回补株的厌氧硝酸盐呼吸生长、细菌竞争力、生物膜合成能力及超黏表型、菌株形态改变。结果 利用自杀载体pKO3-Km质粒成功构建肺炎克雷伯菌modA基因缺失株ΔmodA；随后将重组质粒modA-pGEM-T-easy电转化入ΔmodA中，得到回补株C-modA；相较于肺炎克雷伯菌野生株（WT）、C-modA，modA基因缺失株厌氧硝酸盐呼吸生长受抑制；ΔmodA相对野生株WT的体外厌氧培养竞争力明显减弱（P<0.05）；ΔmodA厌氧生长生物膜合成减少，与WT、C-modA差异具有统计学意义（P<0.05）；modA基因缺失株厌氧条件下黏液表型明显被削弱，相对WT、C-modA差异具有统计学意义（P<0.01）；厌氧条件下ΔmodA菌体形态由正常的短杆状变为球型，与WT、C-modA长度差异有统计学意义（P<0.0001）。结论 钼酸盐转运系统编码基因modA与肺炎克雷伯菌的厌氧硝酸盐呼吸、细菌体外竞争力、生物膜形成、超黏表型以及菌体形态变化等致病力因素相关。

关键词: 肺炎克雷伯菌 ；ModA蛋白 ；厌氧生长 ；菌体形态

Abstract: Objective To construct a mutant strain of Klebsiella pneumoniae NTUH- K2044 with modA gene deletion and its complementary strain and explore the role of modA gene in modulating anaerobic nitrate respiratory growth and phenotypes of K. pneumoniae. Methods The modA deletion mutant K. pneumoniae strain was constructed by homologous recombination using the suicide vector pKO3-Km. To obtain the complementary strain C-modA, the whole sequence fragment containing the promoter, open reading frame and terminator regions of modA was cloned into pGEM-T-easy and electrically transformed into the modA deletion mutant. The NTUH- K2044 wild- type strain, modA gene deletion mutant and complementary strain were compared by measuring in vitro anaerobic nitrate respiration growth, competitiveness index, biofilm quantification, mucoviscosity assay and morphological measurement using Image J. Results The modA deletion mutant strain ΔmodA and the complementary strain C-modA were successfully constructed. The modA gene knockout strain showed inhibited anaerobic nitrate respiratory growth compared with the wild- type and C-modA strains with significantly weakened competitiveness, reduced capacity of biofilm synthesis during anaerobiosis, and lowered mucoviscosity under anaerobic conditions. The ΔmodA strain showed a spherical morphology in anaerobic conditions as compared with the normal short rod-like morphology of K.pneumoniae, with also distinctly shorter length than the wild- type and C-modA strains. Conclusion The molybdate transport system encoding gene modA is associated with the pathogenic capacity of K. pneumoniae by modulating its anaerobic nitrate respiration, competitiveness, biofilm formation, hypermucoviscous phenotype and morphology.

Key words: Klebsiella pneumoniae; ModA protein; anaerobic growth; bacterial morphology