单增李斯特菌双元调控系统hssS/hssR缺失株的构建及其生物学特性

doi:10.11843/j.issn.0366-6964.2022.03.019

摘要/Abstract

摘要： 单增李斯特菌(Listeria monocytogenes,LM)是一种重要的食源性人畜共患病原菌,可抵抗外界多种不利因素的影响,如酸、碱、渗透压和氧化应激。双元调控系统(two-component system,TCS)是细菌适应环境的重要调控系统,为了探明双元调控系统HssS/HssR对单增李斯特菌抗应激能力及毒力的影响,本研究以单增李斯特菌10403S为亲本株,利用同源重组技术构建了hssS/hssR基因缺失株,并对其部分生物学特性进行了研究。生长曲线结果显示,在氧化应激(20 mmol·L^-1 H₂O₂)条件下,缺失株ΔhssS/hssR的生长能力减弱;应激存活试验结果显示,在20 mmol·L^-1 H₂O₂条件下应激处理1 h后,缺失株ΔhssS/hssR的生长率显著低于亲本株(P<0.01);小鼠巨噬细胞内吞及增殖试验结果显示,hssS/hssR基因缺失显著增强单增李斯特菌抗巨噬细胞吞噬的能力(P<0.01)并显著增强其在巨噬细胞内的增殖能力(P<0.05);鸡胚毒力试验结果显示,亲本株的LD₅₀为10^2.2,缺失株的LD₅₀为10^1.4,且缺失株对鸡胚肝的定殖能力显著高于亲本株(P<0.01),表明hssS/hssR基因缺失能显著增强单增李斯特菌的毒力。综上表明,双元调控系统HssS/HssR介导单增李斯特菌抗氧化应激作用与毒力。

关键词: 单增李斯特菌, 双元调控系统, 氧化应激, 毒力

Abstract: Listeria monocytogenes(LM)is an important food-borne zoonotic pathogenic microorganism, which can survival under various stress conditions, such as acidic, alkalic, osmotic and oxidative stresses.Two-component systems play important roles in sensing and adaptating to the environments. In this study, we try to explore the roles of the two-component system HssS/HssR in stresses resistance and pathogenicity of Listeria monocytogenes. A hssS/hssR deleted strain was constructed based on Listeria monocytogenes 10403S, and its biological characteristics were investigated. Growth curves showed that deletion of hssS/hssR weakened the growth ability under oxidative stress (20 mmol·L^-1 H₂O₂) condition. Survival assays showed that the growth rate of deletion strain ΔhssS/hssR was significantly lower than that of the parent strain treated with 20 mmol·L^-1 H₂O₂for one hour (P<0.01). Phagocytosis and proliferation assays in macrophage RAW264.7 cells showed that deletion of hssS/hssR significantly enhanced the ability of anti-phagocytosis (P<0.01) and proliferation in macrophages (P<0.05). Virulence assays conducted on chicken embryo showed that the LD₅₀ of the parent strain and deleted strain were 10^2.2and 10^1.4, respectively. And the bacteria load in the liver of chicken embryo infected by the deletion strain was significantly higher than that of parent strain (P<0.01), indicating that deletion of hssS/hssR significantly enhanced the virulence of Listeria monocytogenes. These results indicated that the two-component system HssS/HssR mediates the oxidative stress resistance and virulence of Listeria monocytogenes.

Key words: Listeria monocytogenes, two-component system, oxidative stress, virulence

中图分类号:

安妮, 张钰, 方小伟, 梁雄燕, 刘晶, 杨玉莹, 方春. 单增李斯特菌双元调控系统hssS/hssR缺失株的构建及其生物学特性[J]. 畜牧兽医学报, 2022, 53(3): 867-874.

AN Ni, ZHANG Yu, FANG Xiaowei, LIANG Xiongyan, LIU Jing, YANG Yuying, FANG Chun. Construction of Listeria monocytogenes Two-Component System hssS/hssR Deletion and Research on Its Biological Characteristics[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 867-874.

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