硫氧还蛋白Lmo1903介导单增李斯特菌抵抗氧化应激的生物学功能研究

The thioredoxin Lmo1903 contributes to oxidative stress tolerance of Listeria monocytogenes

摘要:

【目的】以单增李斯特菌（Listeria monocytogenes，LM）硫氧还蛋白Lmo1903为研究对象，研究其在细菌环境适应过程中的抗氧化应激生物学作用。【方法】使用生物信息学方法分析Lmo1903的进化关系和关键活性位点，使用酶切连接的方法构建Lmo1903蛋白表达载体，获得纯化的重组蛋白，以胰岛素为底物分析其氧化还原酶学活性；同时制备鼠源多克隆抗体，分析其在细胞内的定位；采用核苷酸定点突变技术构建CX₁X₂C基序中的半胱氨酸点突变蛋白，分析关键位点半胱氨酸对Lmo1903酶活的影响；采用同源重组原理构建lmo1903基因缺失株Δlmo1903和回补株CΔlmo1903，研究lmo1903在单增李斯特菌生长、运动和抗氧化应激方面发挥的功能。【结果】生物信息学分析显示，Lmo1903含有CX₁X₂C基序，与枯草芽孢杆菌（Bacillus subtilis）的TrxA的亲缘关系较近，属于硫氧还蛋白家族成员，主要定位在细菌细胞质中，具有较强的还原酶学活性，突变CX₁X₂C基序中的半胱氨酸残基会显著降低Lmo1903的还原酶活能力。缺失lmo1903不影响单增李斯特菌的生长能力，但显著减弱了细菌在铜离子胁迫环境中的氧化应激耐受能力，且影响鞭毛合成相关因子的转录水平和细菌的运动能力。【结论】本研究首次证实了单增李斯特菌硫氧还蛋白Lmo1903具有还原酶学活性，介导细菌运动和对氧化环境的适应。

Abstract:

[Objective] The thioredoxin family plays a key role in the oxidative stress response of the foodborne bacterial pathogen Listeria monocytogenes (LM) during environmental adaptation. Here, we studied the biological role of the thioredoxin Lmo1903 in oxidative stress tolerance.[Methods] The phylogenetic relationship and key active sites of Lmo1903 were analyzed by bioinformatics tools. The recombinant Lmo1903 protein was expressed and purified. The oxidoreductase activity of the recombination protein was determined with insulin as the substrate and the cellular localization was predicted after preparation of mouse polyclonal antibody with the recombinant protein. The oligonucleotide-directed site-specific deletion of cysteine from Lmo1903 protein was carried out to analyze the key sites of enzyme activity. The lmo1903-deleted strain Δlmo1903 was constructed by homologous recombination, and the complementation strain CΔlmo1903was constructed with the integrated complement plasmid pIMK2. The growth, motility, and oxidative stress tolerance of the strains were examined in vitro. [Results] Lmo1903 had a classical CX₁X₂C motif and was close related to the thioredoxin family member TrxA from Bacillus subtilis. It was mainly located in the cytoplasm of bacteria and possessed strong reductase activity. Cysteine was the key site for the enzymatic activity of Lmo1903. Deletion of lmo1903 did not affect bacterial growth, while it significantly weakened the tolerance to oxidative stress in Cu²⁺ stress environment. Furthermore, the deletion of this gene affected the transcriptional levels of the genes involved in bacterial flagellar formation and reduced the swimming motility. [Conclusion] The thioredoxin family member Lmo1903 exhibiting the reductase activity contributes to the oxidative stress tolerance and motility of L. monocytogenes.