单核细胞增生李斯特菌hfq基因缺失株的构建及其生物学特性

Construction and characterization of Listeria monocytogenes Δhfq mutant strain

【目的】单核细胞增生李斯特菌(Listeria monocytogenes,Lm)为革兰氏阳性的短杆菌,营腐生和寄生生活,是重要的食源性人兽共患病原菌,对低温、酸碱和高渗透压等环境具有较强的抵抗力。Lm在各种环境中适应、生存并表现致病力,是与调控因子的网络调控密切相关,本文初步研究了细菌调控因子hfq的生物学特性。【方法】利用同源重组技术对血清型为1/2 a的EGDe菌株进行hfq基因的敲除,对获得的突变株EGDe△hfq进行生化鉴定及生物学特性研究。【结果】实验结果表明:在4℃低温环境下,缺失株生长显著减慢(P<0.05);在含7%Na Cl高渗BHI培养基及含4.5%乙醇的BHI培养基中生长受到抑制;缺失株形成生物被膜的能力显著下降(P<0.05);对Caco-2细胞系的侵袭能力下降;与野生型菌株相比,EGDe△hfq对BALB/c小鼠的感染能力减弱、半数致死剂量提高。【结论】由此表明,Hfq蛋白对细菌抗胁迫、生物被膜形成及细菌毒力具有重要的调控作用。此缺失株的构建为进一步研究Hfq的功能提供了材料,为研究其在Lm胁迫环境生存及疾病预防控制奠定了基础。

[Objective] Gram-positive brevibacterium Listeria monocytogenes（ Lm） is an important zoonotic foodborne pathogen,engaged in both saprophytism and parasitism. It could adapt,survive and display pathogenicity under different environmental stress challenges,which is associated with the regulatory network consisting of regulating factors. The biological characterizations of regulator hfq was evaluated in this study. [Methods]hfq deleted serovar 1 /2 a strain EGDe was constructed with homologous recombination,the biological characteristics of the mutant strain was compared with its parental strain. [Results]The growth of EGDe△hfq was significantly inhibited under cold temperature（ P 0. 05）,salt medium containing 7% Na Cl and the medium containing 4. 5% ethanol. The ability of biofilm formation of the mutant strain in Bacto TM Brain Heart Infusion（ BHI） was reduced significantly（ P 0. 05）; notably,the invasion rate to Caco-2 cell lines was obviously reduced. Infection capacity of EGDe△hfq to BALB / c mice decreased and the LD50 was 6 times higher than EGDe. [Conclusion] Hfq protein of Listeria monocytogenes plays an important role in regulating bacterial virulence,biofilm formation and stress response. This deletion strain provided material to further study the function of Hfq and provides the possibilities to elucidate the mechanisms of Lm in resisting the stress and paves ways to the development of novel strategies for the prevention and control of Lm infections.