分子伴侣prsA2基因缺失对单增李斯特菌致病性的影响

doi:10.16431/j.cnki.1671-7236.2024.01.034

摘要/Abstract

摘要： 【目的】构建单增李斯特菌分子伴侣prsA2基因缺失株与回补株，探究其对单增李斯特菌内化素B （InlB）锚定能力以及致病性的影响。【方法】利用穿梭质粒pKSV7和回补质粒pIMK2分别构建prsA2基因缺失株与回补株；通过生长曲线分析亲本株10403S、缺失株ΔprsA2和回补株CΔprsA2生长能力；利用Western blotting检测prsA2基因缺失对单增李斯特菌InlB锚定的影响；通过肠上皮细胞（Caco-2）黏附侵袭试验与免疫荧光试验、小鼠毒力试验分析prsA2基因缺失对单增李斯特菌黏附侵袭能力、在宿主细胞间迁移能力及小鼠致病性的影响。【结果】PCR扩增结果显示，试验成功构建了缺失株ΔprsA2和回补株CΔprsA2。生长曲线结果显示，prsA2基因缺失不影响单增李斯特菌在BHI培养基中的生长能力（P>0.05）。Western blotting结果显示，缺失株ΔprsA2细菌表面的InlB锚定量极显著低于10403S和CΔprsA2（P<0.01），细菌培养上清中InlB锚定量极显著高于10403S和CΔprsA2（P<0.01）。细胞试验结果显示，缺失株ΔprsA2对Caco-2细胞的黏附率、侵袭率均极显著低于10403S和CΔprsA2（P<0.01）；prsA2基因缺失减弱了单增李斯特菌募集肌动蛋白形成肌动蛋白尾巴在细胞间迁移的能力。小鼠毒力试验结果显示，缺失株ΔprsA2在小鼠肝脏与脾脏中的细菌载量均显著低于10403S和CΔprsA2（P<0.05）。【结论】分子伴侣prsA2基因缺失不影响单增李斯特菌在BHI培养基中的生长能力，但缺失株ΔprsA2表面的InlB锚定量显著下降，缺失prsA2基因减弱单增李斯特菌对Caco-2细胞的黏附侵袭能力、在细胞间迁移能力以及对小鼠的致病性。

关键词: 单增李斯特菌; 分子伴侣; prsA2基因; 缺失; 致病性

Abstract: 【Objective】 This study was aimed to construct the chaperone prsA2 gene deletion strain and its complemental strain, and explore the roles of prsA2 gene in the anchoring of virulent factor internalin B (InlB) and pathogenicity of Listeria monocytogenes.【Method】 The deletion strain ΔprsA2 and complemental strain CΔprsA2 were constructed with shuttle vector pKSV7 and pIMK2, respectively.The growth ability of wild type 10403S, ΔprsA2 and CΔprsA2 was analyzed by growth curve.The effect of prsA2 gene deletion on the anchoring of InlB was evaluated by Western blotting.The effect of prsA2 gene deletion on the adhesion and invasion ability, migration ability between host cells and pathogenicity of Listeria monocytogenes were analyzed by intestinal epithelial cell (Caco-2) adhesion and invasion test, immunofluorescence test and mouse virulence test.【Result】 PCR amplification results showed deletion strain ΔprsA2 and its complemental strain CΔprsA2 were constructed successfully.Growth curve results showed that the growth ability of wild type 10403S, ΔprsA2 and CΔprsA2 in BHI broth exhibit no significant difference (P>0.05).The results of Western blotting showed that the InlB anchor content on the surface of the bacteria ΔprsA2 was extremely significantly lower than 10403S and CΔprsA2 (P<0.01), and the InlB anchor content in the bacterial culture supernig was extremely significantly higher than 10403S and CΔprsA2 (P<0.01).The cell test results showed that the adhesion rate and invasion rate of deletion strain ΔprsA2 on Caco-2 cells were extremely significantly lower than 10403S and CΔprsA2 (P<0.01).Deletion of prsA2 gene reduced the ability to recruit actin to form an actin tail for intercellular migration of Listeria monocytogenes.The mouse virulence test showed that the bacterial load of deletion strain ΔprsA2 in liver and spleen of mice was significantly lower than that of 10403S and CΔprsA2 (P<0.05).【Conclusion】 The deletion of chaperone prsA2 gene didn't affect the growth ability of Listeria monocytogenes in BHI broth, but the InlB anchor quantity on the surface of ΔprsA2 strain was significantly decreased, and deletion of prsA2 gene weakened the adhesion and invasion ability on Caco-2 cells, the ability of intercellular migration and the pathogenicity in mice of Listeria monocytogenes.

Key words: Listeria monocytogenes; chaperone; prsA2 gene; deletion; pathogenicity

中图分类号:

S852.61

胡文洁, 方小伟, 郭骞, 杨雨婷, 刘晶, 梁雄燕, 杨玉莹, 方春. 分子伴侣prsA2基因缺失对单增李斯特菌致病性的影响[J]. 中国畜牧兽医, 2024, 51(1): 338-346.

HU Wenjie, FANG Xiaowei, GUO Qian, YANG Yuting, LIU Jing, LIANG Xiongyan, YANG Yuying, FANG Chun. Effect of Chaperone prsA2 Gene Deletion on the Pathogenicity of Listeria monocytogenes[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(1): 338-346.

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