单增李斯特菌lmo2300介导的氧化应激和感染生物学作用

Oxidative stress resistance and infection mediated by lmo2300 of Listeria monocytogenes

摘要:

目的 研究单增李斯特菌lmo2300基因在EGD-e株抗氧化应激和感染生物学特性中的作用。方法 以单增李斯特菌1/2a血清型EGD-e为亲本株，利用同源重组法构建lmo2300的缺失株和回补株，检测Δlmo2300在体外的生长、运动能力，对氧化环境的抵抗能力，还原酶活性，以及抗生素最小抑菌浓度；检验突变株感染细胞后的黏附、侵袭、增殖和细胞间迁移能力；使用荧光定量PCR技术检测lmo2300基因缺失后单增李斯特菌硫氧还蛋白相关基因和主要毒力基因的转录水平变化。结果 经PCR鉴定及测序证明缺失株Δlmo2300和回补株CΔlmo2300构建成功。lmo2300基因缺失后，单增李斯特菌的生长、运动和对抗生素的敏感性均无显著变化，黏附、侵袭和增殖能力也未受显著影响。Lmo2300具有还原酶活性，Δlmo2300中硫氧还蛋白基因lmo1903和grx转录水平显著升高，对H₂O₂、Diamide、CdCl₂和MnSO₄的氧化应激抵抗能力显著增强；且该菌细胞间迁移能力显著提高，Δlmo2300中与细胞间迁移相关的actA基因转录水平上调约8倍。结论 本研究表明lmo2300在细胞间迁移和抵抗氧化应激中发挥关键作用，这一发现对于深入理解单增李斯特菌的抗氧化应激和感染生物学机制具有重要意义。

Abstract:

Objective To investigate the role of lmo2300 in oxidative stress resistance and infection of Listeria monocytogenes.Methods With L. monocytogenes 1/2 a serotype EGD-e as the parent strain, the lmo2300-deleted strain and complementary strain were constructed by homologous recombination. The growth, motility, oxidative stress resistance, minimum inhibitory concentration and reductase activity of Δlmo2300 were evaluated. The adhesion, invasion, proliferation, and intercellular migration abilities of the Δlmo2300 strain was assessed using cell models. The transcriptional levels of thioredoxin-related genes and major virulence genes in Δlmo2300 were detected with RT-qPCR.Results PCR identification and DNA sequencing confirmed the successful construction of Δlmo2300 and CΔlmo2300. Deletion of lmo2300 did not affect the bacterial growth, motility, antibiotic susceptibility, adhesion, invasion or intracellular proliferation. However, the Lmo2300 protein possesses reductase activity, and the transcriptional levels of the thioredoxin gene lmo1903 and grx are significantly upregulated in the Δlmo2300 mutant. And markedly improved resistance to oxidative substances, including H₂O₂, diamide, CdCl₂, and MnSO₄ compared with the wild type. The Δlmo2300 mutation significantly enhanced intercellular migration ability, accompanied by an approximately 8-fold upregulation of the actA gene transcription, which is consistent with the enhanced intercellular migration ability.Conclusion This study demonstrates that lmo2300 plays a critical role in modulating the oxidative stress resistance and intercellular migration of L. monocytogenes, providing novel insights into the infection biology and adaptive responses of L. monocytogenes to host-derived oxidative challenges.