单核细胞增多性李斯特菌OpuCA蛋白介导抗氧化应激和宿主感染研究

OpuCA mediates oxidative stress tolerance and host infection of Listeria monocytogenes

摘要:

【目的】本文旨在研究opuCA基因在单核细胞增多性李斯特菌(单增李斯特菌)生长过程及渗透胁迫下发挥的作用，探究opuCA基因参与细菌抗氧化应激和致病力的生物学功能，为阐明OpuCA蛋白介导细菌环境适应和宿主内感染的机制奠定基础。【方法】利用细菌同源重组方法获得opuCA缺失株及回补株后，通过分子生物学、感染生物学和激光共聚焦技术，研究野生株和突变株的生长能力、抗渗透应激能力、抗氧化应激能力、细胞粘附、侵袭以及胞内增殖能力。【结果】缺失opuCA基因后，李斯特菌体外生长能力并没有受到影响，但在渗透条件下生长能力减弱；opuCA缺失株在铜离子和镉离子中抗氧化应激能力降低，但在巯基特异性氧化剂肼应激中无明显变化；opuCA缺失株在细胞中的侵袭能力显著减弱，且缺失该基因导致细菌聚合actin能力下降，进而影响了细菌在胞间迁移。【结论】本研究首次证实了缺失opuCA基因能降低单增李斯特菌抗氧化应激能力和感染宿主能力，并且在渗透胁迫下细菌生长能力减弱，但具体的分子机制有待深入研究。本研究有助于深入理解单增李斯特菌OpuCA蛋白介导的细菌体外环境适应及宿主内感染的分子机制，为防控单增李斯特菌感染提供了新策略。

Abstract:

[Objective] Listeria monocytogenes, the important foodborne pathogen, uses opuCA gene to defend against osmotic stress. In this study, we explored the roles of opuCA gene in growth, pathopoiesis, oxidative stress resistance, and osmotic stress resistance of L.monocytogenes, hoping to lay a basis for clarifying the mechanism of OpuCA-mediated bacterial adaptation and infection. [Methods] We constructed the opuCA deletion mutant (ΔopuCA) and complementary mutant (CΔopuCA) and then compared the growth, oxidative stress tolerance, osmotic stress tolerance, and cellular adhesion and invasion of the wild strain and the mutants by molecular biology technology, infection biology technology, and laser scanning confocal microscopy. [Results] opuCA deletion had no influence on bacterial growth but decreased the osmotic stress tolerance. Additionally, ΔopuCA showed low tolerance to Cu²⁺and Cd²⁺compared with the wild strain, but the tolerance to thiol-specific oxidant diamide demonstrated no obvious variation. Furthermore, loss of opuCA significantly reduced bacterial adhesion and invasion to Caco-2 cells, and actin aggregation, thereby influencing the intercellular migration of bacteria. [Conclusion] opuCA plays a critical role in bacterial osmotic stress tolerance, oxidative stress resistance, and intracellular infection. This study expands our understanding of the OpuCA-mediated in vitro adaption and pathopoiesis of L.monocytogenes, which lays a basis for the prevention and control of the foodborne diseases caused by L.monocytogenes.