单增李斯特菌双元调控系统lmo1060/lmo1061缺失株的构建及其抗酸应激能力

Construction and acid resistance of the lmo1060/lmo1061 deletion strain of Listeria monocytogenes

为了探明双元调控系统Lmo1060/Lmo1061是否影响单增李斯特菌(LM)的抗应激作用及致病性,本研究以LM 10403S为亲本株,通过同源重组技术构建了Δlmo1060/lmo1061缺失株。应激存活试验结果表明,lmo1060/lmo1061缺失显著增强LM在酸应激(pH=2.5)条件下的存活率,但不影响该菌在10%NaCl和10 mmol/L H;O;中的存活能力。绿色荧光蛋白(GFP)报告基因筛选显示,lmo1060/lmo1061缺失不影响谷氨酸脱羧酶系统gadD1/gadT1和gadD3报告基因的表达,但显著上调gadT2/gadD2报告基因的表达。Western blot结果证实,缺失株Δlmo1060/lmo1061中GadD2蛋白水平显著高于亲本株,回补株CΔlmo1060/lmo1061中GadD2蛋白水平与亲本株则无显著性差异。小鼠毒力试验显示,缺失株Δlmo1060/lmo1061与亲本株感染24 h后在小鼠肝脏和脾脏中的载菌量并无显著差异。研究表明,双元调控系统Lmo1060/Lmo1061可能感应环境pH值变化,并通过调控gadT2/gadD2表达影响LM的抗酸应激作用。

In order to find out whether the two-component system(TCS)Lmo1060/Lmo1061 TCS affects the stress resistance and pathogenicity of Listeria monocytogenes,L.monocytogenes 10403 S was used as the parental strain in this study,and a TCS-deficient strainΔlmo1060/1061 was constructed through homologous recombination.The results of the stress survival test showed that the deletion of lmo1060/lmo1061 significantly enhanced the survival rate of L.monocytogenes under acid stress(pH 2.5)but it didn’t affect the survival ability under the conditions of 10%NaCl and 10 mmol/L H;O;.GFP reporter gene screening showed that the deletion of lmo1060/lmo1061 significantly increased the expression level of the gadT2/gadD2 reporter gene,rather than those of the gadD1/gadT1 and gadD3 reporter genes.Western blot assay confirmed that the GadD2 protein level ofΔlmo1060/lmo1061 was significantly higher than that of the parent strain,but there was no significant difference between the complementary strain CΔlmo1060/lmo1061 and the parent stain.However,the virulence assay of the tested mice showed thatΔlmo1060/lmo1061 and the parent strain showed no significant difference in bacteria loads in the liver and spleen of the rodents after 24 hours of infection.The above results indicated that the Lmo1060/Lmo1061 TCS might respond to changes in the environmental pH and mediate the acid resistance of L.monocytogenes by regulating the glutamate decarboxylase system gadT2/gadD2.