双元调控系统LisRK对单增李斯特菌抗酸应激与侵袭力作用的研究

doi:10.16431/j.cnki.1671-7236.2022.02.002

摘要/Abstract

摘要： 【目的】试验旨在阐明双元调控系统LisRK在单增李斯特菌(Listeria monocytogenes)抵抗不利环境应激以及感染过程中的作用。【方法】以参考菌株10403S为亲本株，利用穿梭质粒pKSV7通过同源重组方法构建lisRK基因缺失株，并基于表达质粒pIMK2构建了lisRK回补株；比较了亲本株10403S、缺失株ΔlisRK与回补株CΔlisRK在酸(pH 4.5)、渗透压(5% NaCl)和氧化(10 mmol/L H₂O₂)应激条件下的生长与存活能力及其对胃腺癌细胞MGC803和肠上皮细胞Caco-2的侵袭率，进一步比较了亲本株10403S、缺失株ΔlisRK与回补株CΔlisRK经腹腔注射感染的小鼠肝脏和脾脏中的细菌载量。【结果】应激试验结果显示，lisRK基因缺失显著降低单增李斯特菌在酸应激(pH 4.5)条件下的生长能力，但不影响该菌在5% NaCl和10 mmol/L H₂O₂中的生长能力；缺失株ΔlisRK在强酸应激条件下(pH 2.5)的存活率(0.57%)极显著低于亲本株(2.07%)与回补株(1.97%)(P < 0.01)。细胞侵袭试验显示，缺失株ΔlisRK对肠上皮细胞Caco-2和胃腺癌细胞MGC803的侵袭率分别为2.04%和0.13%，极显著或显著低于亲本株与回补株(P < 0.01；P < 0.05)。小鼠感染试验显示，在感染后48 h，缺失株ΔlisRK在小鼠肝脏和脾脏中的细菌载量分别为7.42×10⁷和1.87×10⁷ CFU，均低于亲本株和回补株感染小鼠肝脏和脾脏中的细菌载量，但并无显著性差异(P > 0.05)。【结论】双元调控系统LisRK缺失减弱单增李斯特菌的抗酸应激能力和对宿主细胞的侵袭能力。

关键词: 单增李斯特菌; 双元调控系统; 抗酸应激; 侵袭力

Abstract: 【Objective】 This study was conducted to clarify the roles of two-component system LisRK of Listeria monocytogenes in stress resistance and infection.【Method】 The deletion strain ΔlisRK was constructed by homologous recombination with shuttle vector pKSV7 at the background of wild type strain 10403S and the complemental strain CΔlisRK was constructed with plasmid pIMK2.The growth and survival ability of the strains 10403S, ΔlisRK and CΔlisRK under various stresses including acidic (pH 4.5), osmotic (5% NaCl) and oxidative (10 mmol/L H₂O₂) conditions were analyzed.Then the invasion rate to MGC803 cells and Caco-2 cells, and bacteria load in the liver and spleen of infected mice of strains 10403S, ΔlisRK and CΔlisRK were compared.【Result】 The results of stress test showed that the deletion strain ΔlisRK significantly reduced the growth ability of Listeria monocytogenes under acid stress (pH 4.5), but did not affect the growth ability of Listeria monocytogenes in 5% NaCl and 10 mmol/L H₂O₂.The survival rate (0.57%) of the deletion strain ΔlisRK under severe acid stress (pH 2.5) was extremely significantly lower than that of strains 10403S (2.07%) and CΔlisRK (1.97%) (P < 0.01).Cell invasion test showed that the invasion rates of the deletion strain ΔlisRK on intestinal epithelial cell Caco-2 and gastric adenocarcinoma cell MGC803 were 2.04% and 0.13%, respectively, which were extremely significantly or significantly lower than that of strains 10403S and CΔlisRK (P < 0.01;P < 0.05).The mouse infection test showed that bacteria load in the liver and spleen of ΔlisRK infected mice were 7.42×10⁷and 1.87×10⁷CFU, which were lower than those of 10403S and CΔlisRK, respectively, but there were no significant difference (P > 0.05).【Conclusion】 These data demonstrated that knocking out the LisRK impaired the acid resistance and invasiveness of Listeria monocytogenes.

Key words: Listeria monocytogenes; two-component system; acid resistance; invasiveness

中图分类号:

S852.61

陈凌波, 张钰, 方小伟, 吉俊芝, 方春, 杨玉莹. 双元调控系统LisRK对单增李斯特菌抗酸应激与侵袭力作用的研究[J]. 中国畜牧兽医, 2022, 49(2): 416-423.

CHEN Lingbo, ZHANG Yu, FANG Xiaowei, JI Junzhi, FANG Chun, YANG Yuying. Effects of the Two-component System LisRK on Acid Resistance and Invasiveness of Listeria monocytogenes[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(2): 416-423.

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