高毒力肺炎克雷伯菌hfq基因缺失株的构建及其生物学特性分析

Construction of hypervirulent Klebsiella pneumoniae strain with hfq gene deletion and study on its biological characteristics

目的构建高毒力肺炎克雷伯菌NTHU-K2044菌株hfq基因缺失株(△hfq),并通过表型试验分析hfq基因对高毒力肺炎克雷伯菌生长特性、环境适应能力和毒力等生物学特性的影响。方法利用同源重组技术对NTUH-K2044菌株进行hfq基因敲除;通过表型试验,比较高毒力肺炎克雷伯菌野生株和△hfq突变株在细菌生长速度、抗环境胁迫能力、生物膜形成、荚膜形成中性粒细胞杀菌活性和大蜡螟幼虫感染致死率等方面的差异性。结果成功构建高毒力肺炎克雷伯菌NTUH-K2044△hfq缺失株,表型试验表明△hfq缺失株的生长速度显著减慢,菌落变小和超黏性下降;在pH9、pH5.5、0.7 mmol/L SDS、5%NaCl、0.1%H2O2环境和50℃高温等环境胁迫条件下,△hfq缺失株生长受到显著抑制;在毒力方面,△hfq缺失株生物膜形成能力下降,荚膜形成能力下降且荚膜合成基因magA和rmpA表达量明显下降,中性粒细胞杀菌试验存活率下降,大蜡螟幼虫致死能力显著降低。结论Hfq蛋白作为RNA伴侣分子能够参与转录后调控进而对高毒力肺炎克雷伯菌生理、环境适应性及毒力致病性具有重要的调控作用。这为寻找治疗和控制高毒力高耐药肺炎克雷伯菌新靶标提供了基础。

Objective To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics.Methods The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct△hfq mutant strain.Its biological characteristics including growth rate,environmental stress tolerance,biofilm formation,capsular polysaccharide synthesis,resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments.Results The△hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed.Phenotypic experiments showed that the△hfq mutant strain had significantly slower growth rate,smaller colonies and decreased hypermucoviscosity.Its growth was significantly inhibited under different environmental stress conditions such as pH9,pH5.5,0.7 mmol/L SDS,5%NaCl,0.1%H2O2 and high temperature of 50℃.In terms of virulence and pathogenicity,the△hfq mutant strain showed decreased ability to form biofilm and capsule,significantly down-regulated expression of magA and rmpA genes required for capsule synthesis,lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae.Conclusions As a RNA chaperone,Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology,environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae.This study provided reference for further study on hypervirulent Klebsiella pneumoniae.