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

Construction of Listeria monocytogenes Two-Component System hssS/hssR Deletion and Research on Its Biological Characteristics

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) H_(2)O_(2))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) H_(2)O_(2)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102.2,缺失株的LD50为101.4,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。

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) H_(2)O_(2))condition.Survival assays showed that the growth rate of deletion strainΔhssS/hssR was significantly lower than that of the parent strain treated with 20 mmol·L^(-1) H_(2)O_(2)for 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 102.2and 101.4,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.