单核细胞增多性李斯特菌硫氧还蛋白Lmo1609的应激生物学功能研究

Stress response of the thioredoxin Lmo1609 from Listeria monocytogenes

摘要:

[目的]本研究旨在构建单核细胞增多性李斯特菌(Listeria monocytogenes)硫氧还蛋白Lmo1609的基因缺失株,分析Lmo1609的氧化还原酶学活性,及其在细菌生长、运动过程中发挥的作用,并探究了Lmo1609参与细菌抗氧化应激和致病的生物学基础。为阐明其抗应激生物学作用以及完善李斯特菌的感染机制奠定分子基础。[方法]利用同源重组原理构建lmo1609基因缺失株及回补株。通过分子生物学、应激生物学和感染生物学等手段,对Lmo1609的生物学功能进行探索。以胰岛素为底物分析其氧化还原酶学活性;通过构建lmo1609缺失株和回补株,比较野生株和突变株在运动性、生长能力、抗氧化应激、细胞黏附、侵袭和增殖能力等方面的差异,进而鉴定Lmo1609的生物学功能。[结果]缺失lmo1609后,单增李斯特菌在生长能力上无明显变化,而运动能力明显减弱;对H₂O₂的敏感性增强;对细胞的黏附侵袭能力没有差异;对小鼠的致病力没有显著影响。[结论]本研究首次证实了单增李斯特菌硫氧还蛋白Lmo1609具有还原酶学活性,参与调控细菌的运动和对H₂O₂的氧化应激耐受,不介导单增李斯特菌的致病性。

Abstract:

[Objective] The thioredoxin family plays an important role in the response of the foodborne bacterial pathogen Listeria monocytogenes to the oxidative stress during the adaptation to environment.Here,we explored the biological characteristics of the thioredoxin Lmo1609 in the oxidative stress tolerance and infection of L.monocytogenes.[Methods] We constructed the lmo1609-deleted and-complemented strains and then studied the growth,motility,oxidative stress tolerance,and pathogenicity of the strains both in vitro and in vivo.Moreover,we purified the recombinant Lmo1609 protein and then studied the oxidoreductase activity of the protein in vitro.[Results] The deletion of lmo1609 did not affect the bacterial growth but significantly reduced the swimming motility in vitro.Surprisingly,it enhanced the tolerance of this bacterium to H₂O₂ stress while showing no impact on bacterial infection.[Conclusion]This study indicates that the thioredoxin family member Lmo609 exhibiting the oxidoreductase activity contributes to the motility and oxidative stress tolerance but not in vivo infection of L.monocytogenes.