鸭疫里氏杆菌抗氧化应激调节子OxyR的功能鉴定及调控机制

Functions and regulatory mechanisms of the antioxidative stress regulator OxyR in Riemerella anatipestifer

摘要:

背景 鸭疫里氏杆菌(Riemerella anatipestifer)是一种感染鸭和其他禽类的革兰氏阴性病原菌，其感染常常导致禽类的大量死亡，给我国养禽业造成巨大经济损失。该菌在外界环境或宿主体内会遭受氧化应激刺激，然而其应对氧化应激刺激的机制未完全被阐明。目的 以鸭疫里氏杆菌基因中的oxyR为研究对象，分析其功能以及抗氧化应激反应的调控机制。方法 构建RA CH-1株的oxyR基因无痕缺失株(ΔoxyR)并通过测定生长曲线、H₂O₂的敏感性、胞内活性氧(reactive oxygen species, ROS)、转录组、凝胶迁移试验(electrophoretic mobility shift assay, EMSA)、毒力及定殖等试验解析氧化应激调节子OxyR的功能以及其抗氧化应激反应的调控机制。结果 相较于亲本株，ΔoxyR在TSB培养基中的生长无显著差异，而在含H₂O₂的TSB培养基中的生长显著变缓；ΔoxyR在对数生长期对H₂O₂更敏感而在平台期无显著差异、oxyR缺失后胞内ROS含量显著增多；转录组结果表明，H₂O₂压力下OxyR激活71个基因的转录，占H₂O₂压力下亲本株上调基因总数的27%；H₂O₂压力下OxyR抑制74个基因的转录，占H₂O₂压力下亲本株下调基因总数的53%；凝胶迁移试验结果表明，重组OxyR蛋白能与被激活基因上游区域的DNA序列结合而不与该基因编码区序列结合；相较于亲本株，oxyR缺失后不影响该菌的毒力和定殖。结论 oxyR基因参与编码鸭疫里氏杆菌抗氧化反应调节子的功能，OxyR蛋白能与激活基因上游区域的DNA序列结合而发挥调控作用，OxyR不参与鸭疫里氏杆菌CH-1株在宿主体内的定殖。

Abstract:

Background Riemerella anatipestifer (RA) is a Gram-negative bacterium that infects ducks and other poultry, often causing large-scale deaths and resulting in serious economic losses to the poultry industry in China. This bacterium is subjected to oxidative stress in both the external environment and the host, while the mechanism by which it responds to oxidative stress has not been fully elucidated.Objective To analyze the functions and regulatory mechanisms of oxyR in the oxidative stress response.Methods We constructed an oxyR knockout strain (ΔoxyR) from RA CH-1 and analyzed the functions and regulatory mechanisms of OxyR in the antioxidative stress response through growth curve measurement, H₂O₂ sensitivity measurement, intracellular reactive oxygen species (ROS) measurement, transcriptomics, electrophoretic mobility shift assay (EMSA), and the virulence and colonization assays in ducklings.Results The growth of ΔoxyR in the TSB medium was not significantly different from that of the wildtype strain, while its growth in the TSB medium supplemented with H₂O₂ was significantly slower than that of the wildtype. Compared with the wildtype strain, ΔoxyR was more sensitive to H₂O₂ in the logarithmic growth phase but showed no significant difference in the stationary phase. The intracellular ROS content was significantly increased after oxyR deletion. Transcriptomics results indicated that under H₂O₂ stress, OxyR activated the transcription of 71 genes, which accounted for 27% of the total upregulated genes in the wildtype strain, and it inhibited the transcription of 74 genes, which accounted for 53% of the total downregulated genes in the wildtype strain. EMSA results indicated that the recombinant OxyR could bind to the upstream DNA sequence of the activated gene but not to the coding region sequence of the gene. Compared with wildtype strain, the deletion of oxyR did not affect the virulence or colonization of the bacterium in ducklings.Conclusion The oxyR gene was involved in encoding the regulator of the antioxidative response in RA, and OxyR can bind to the DNA sequences upstream of activated genes to exert its regulatory function. OxyR did not participate in the colonization of RA CH-1 within the host.