单核细胞增多性李斯特菌谷胱甘肽还原酶GR的生物学特性

Characterization of the glutathione reductase from Listeria monocytogenes

摘要:

[目的] 本文旨在构建单核细胞增多性李斯特菌谷胱甘肽还原酶（glutathione reductase，GR）基因lmo1433（gr）缺失株，并研究GR在细菌生长和运动过程中发挥的作用及与谷氧还蛋白（glutaredoxin，Grx）系统间的调控关系，探究GR参与细菌抗氧化应激和致病力的生物学功能，为阐明氧化还原蛋白介导细菌环境适应和宿主内感染的机制奠定分子基础。[方法] 利用细菌遗传操作系统构建获得gr缺失株及回补株后，通过分子生物学和感染生物学手段，比较野生株和突变株的运动性、生长能力、抗氧化应激、细胞黏附、侵袭和增殖能力；利用整合型质粒构建带GR启动子的荧光报告系统，并结合荧光定量分析GR受Grx调控的情况。[结果] 缺失gr后李斯特菌在体外培养基中的生长能力未受明显影响，但在半固体培养基中的运动能力却显著增强；缺失gr后细菌在铜离子、镉离子以及肼中抗氧化应激能力增强，在H₂O₂中无差异；缺失gr后细胞黏附、侵袭和增殖能力均显著增强；荧光报告系统定量分析发现grx缺失后gr的启动子活性显著增强，表明Grx参与对GR的转录负调控。[结论] 本研究首次证实了单增李斯特菌谷胱甘肽还原酶GR能调控细菌的运动能力，并且缺失GR增强了李斯特菌的抗氧化应激和感染宿主能力；首次证实了GR的自身转录受Grx负调控，但具体分子机制有待于深入探究。本研究有助于深入理解单增李斯特菌氧化还原蛋白的调控关系以及通过参与诸多生物学过程介导细菌体外环境适应及宿主内感染的分子机制，为防控胞内菌感染提供了新策略。

Abstract:

[Objective] The important foodborne bacterial pathogen Listeria monocytogenes uses the glutaredoxin (Grx) system to defend the oxidative stress during environmental adaption. Here, we explore biological characteristics of the glutathione reductase (GR) during bacterial infection. [Methods] We constructed the gr deletion and complementation strains to compare the abilities in bacterial growth, motility, oxidative tolerance and cellular infection. Besides, the gr promoter-based fluorescent report system (FRS) was genetically obtained to further study regulation of gr by Grx. [Results] Our data show that deletion of gr did not affect bacterial growth but enhanced the capability of swarming. Additionally, lack of gr significantly increased oxidative tolerance of bacteria under Cu²⁺ and Cd²⁺ stress, as well as increased efficiencies of intracellular infection in Caco-2 and RAW264.7 cells. Importantly, transcription of gr was negatively regulated by Grx. [Conclusion] The glutathione reductase plays a critical role in bacterial flagella-based swarming motility and correlates a regulatory relationship with the Grx system. Also, GR plays a non-classical role in bacterial oxidative resistance and intracellular infection. This study will expand our understanding of the redox regulatory mechanisms exploited by intracellular bacteria during adaption outside the environment and inside the host, which provides novel strategies to prevent and control the foodborne diseases.