CodY在单核细胞增生李斯特菌抗氧化胁迫中的作用

doi:10.7506/spkx1002-6630-20200507-072

摘要/Abstract

摘要： 目的：探究全局性转录调控因子CodY在单核细胞增生李斯特菌（Listeria monocytogenes，Lm）抗氧化应激中的作用。方法：利用H2O2对处于对数中期（OD600 nm=0.65）的Lm野生株EGDe和CodY缺失株EGDeDcodY进行氧化胁迫，比较两菌株氧化耐受能力、抗氧化应激物（过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）和谷胱甘肽（glutathione，GSH））活性或含量差异；通过随机扩增多态性DNA技术比较两菌株基因组模板稳定性（genomic template stability，GTS）；利用实时聚合酶链式反应（real-time polymerase chain reaction，real-time PCR）检测两菌株抗氧化应激物编码基因以及DNA损伤诱导反应（SOS反应）重要基因的转录表达变化。结果：1）H2O2对EGDe的最低抑菌浓度和最低杀菌浓度大约是EGDeDcodY的2 倍，当H2O2浓度超过20 mmol/L时，EGDeDcodY抑菌圈直径极显著大于EGDe（P≤0.01）。2）200 mmol/L H2O2完全抑制EGDeDcodY生长而EGDe生长速率则有所减慢。3）H2O2胁迫使EGDe和EGDeDcodY中CAT活力均下降，但转录水平并没有显著变化；SOD活力在两菌株内无明显变化，但转录表达在EGDe和EGDeDcodY中差异高度显著（P≤0.001）；值得注意的是，GSH编码基因的转录表达和其含量在EGDe和EGDeDcodY中变化趋势基本一致，随H2O2胁迫时间延长呈先显著下降后略微上升，而且与EGDe相比，GSH在EGDeDcodY中的转录表达水平具有极显著差异（P≤0.01）。4）EGDe和EGDeDcodY的GTS分别为93.1%和80.0%；real-time PCR显示EGDeDcodY中参与SOS反应的重要基因（recA、lexA、recR、lmo1302、lmo1975）转录表达受到高度显著抑制（P≤0.001），而EGDe中recA、lmo1302和lmo1975的转录表达被激活。结论：CodY缺失导致细菌在H2O2胁迫下的耐受能力和生长速率显著降低，GSH含量下降，GTS降低，参与SOS反应的重要基因表达受到显著抑制，表明CodY在Lm抵御氧化胁迫中发挥了重要的调控作用。

关键词: 单核细胞增生李斯特菌；CodY；氧化应激；SOS反应

Abstract: Objective: The purpose of this study was to explore the role of the global transcriptional regulator CodY in the oxidative stress response in Listeria monocytogenes. Methods: Oxidative stress tolerance and antioxidant parameters (CAT, SOD and GSH) were compared between the wild-type strain EGDe and the isogenic CodY deletion strain EGDeDcodY at the mid-logarithmic growth stage (OD600 nm= 0.65) under H2O2 stress. Genomic template stability (GTS) of the two strains was compared by random amplified polymorphic DNA (RAPD) method and the transcriptional expression of the genes encoding antioxidants and those involved in SOS response were assessed by real-time PCR. Results: 1) The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of H2O2 against EGDeDcodY were about twice higher than against EGDe, and the diameter of inhibition zone of EGDeDcodY was significantly greater than that of EGDe when the concentration of H2O2 was over 20 mmol/L (P ≤ 0.01); 2) The growth of EGDeDcodY was completely inhibited by 200 mmol/L H2O2, while it just slightly slowed down the growth of EGDe; 3) H2O2-induced oxidative stress decreased CAT activity in both EGDe and EGDeDcodY, but did not significantly alter the transcription level of this enzyme. SOD activity was not changed significantly, but its mRNA expression presented significant differences between the two strains (P ≤ 0.001). Notably, the general trend of transcription and concentration level of GSH in the two strains was almost same, which decreased markedly and then increased slightly with increasing H2O2 exposure time. Moreover, the transcription level of GSH in EGDeDcodY was always lower than that in EGDe (P ≤ 0.01); 4) GTS of EGDe and EGDeDcodY were 93.1% and 80.0%, respectively. In addition, the expressions of recA, lexA, recR, lmo1302, and lmo1975, which are important for SOS response, were significantly inhibited in EGDeDcodY (P ≤ 0.001), while the expressions of recA, lmo1302 and lmo1975 in EGDe were activated. Conclusion: The deletion of CodY can reduce bacterial oxidative stress tolerance, growth rate and GSH content as well as GTS and inhibit SOS response-related gene expressions, corroborating that CodY plays an important role in the oxidative stress response in Listeria monocytogenes.

Key words: Listeria monocytogenes; CodY; oxidative stress; SOS response

中图分类号:

TS201.3

杨诗怡，林巍，杨丽玉，左成，罗勤. CodY在单核细胞增生李斯特菌抗氧化胁迫中的作用[J]. 食品科学, 2021, 42(14): 46-53.

YANG Shiyi, LIN Wei, YANG Liyu, ZUO Cheng, LUO Qin. The Global Transcriptional Regulator CodY Promotes Oxidative Stress Resistance in Listeria monocytogenes[J]. FOOD SCIENCE, 2021, 42(14): 46-53.

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CodY在单核细胞增生李斯特菌抗氧化胁迫中的作用

The Global Transcriptional Regulator CodY Promotes Oxidative Stress Resistance in Listeria monocytogenes

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