DegU调控单核细胞增生李斯特菌感染宿主细胞及适应高温的机制研究

The orphan response regulator DegU regulates host cell infection and high-temperature adaptation of Listeria monocytogenes

【目的】本研究旨在探究孤儿调节因子DegU在介导单核细胞增生李斯特菌(Listeria monocytogenes)宿主感染和高温环境适应性方面的调控机制。【方法】本研究以单增李斯特菌参考菌株EGD-e、degU基因缺失菌株ΔdegU和回补菌株CΔdegU为研究材料,通过细胞模型、实时荧光定量聚合酶链式反应和凝胶阻滞试验等方法探究DegU对单增李斯特菌感染宿主细胞和适应高温的调控机制。【结果】研究结果表明:缺失degU后,单增李斯特菌在Caco-2上的黏附和侵袭能力显著降低,在RAW264.7中的增殖能力显著降低,在L929中的空斑形成能力也显著降低;进一步通过实时荧光定量聚合酶链式反应检测degU基因缺失后引起的单增李斯特菌毒力因子转录水平变化,发现多个重要毒力因子转录水平均显著下调;该试验结果还发现与毒力相关的热应激基因clpE(受CtsR抑制的ATP依赖蛋白水解酶编码基因)转录水平显著升高,而在43℃高温条件下,clpE转录水平显著降低;进一步通过凝胶阻滞试验结果表明DegU能够与clpE的启动子直接结合。【结论】综上所述,degU基因缺失能够降低单增李斯特菌在宿主感染过程中的细胞黏附、侵袭、胞内增殖和胞间迁移能力;DegU能够与clpE启动子结合,直接调控clpE基因的转录水平来适应高温环境。该研究为进一步深入解析单增李斯特菌的宿主感染和环境适应性机制奠定了试验基础。

[Objective]Listeria monocytogenes is a food-borne bacterial pathogen that is widely distributed in the environment.This study aims to investigate the regulatory role of the orphan response regulator DegU in the infection and high-temperature adaptation of L.monocytogenes.[Methods]The regulatory roles of DegU in three strains including wild-type EGD-e,ΔdegU,and CΔdegU of L.monocytogenes were studied.The infection models established with human epithelial cell line Caco-2,mouse RAW264.7 macrophages,and murine fibroblast L929,real time quantitative polymerase chain reaction(RT-qPCR),and electrophoretic mobility shift assay(EMSA)were employed to investigate the roles of DegU in the infection and high-temperature adaptation.[Results]Compared with wild-type EGD-e,ΔdegU showed weakened abilities to adhere,invade,proliferate,and migrate between host cells.The RT-qPCR results revealed that the transcription levels of the multiple virulence factors were down-regulated while that of clpE was up-regulated inΔdegU.However,the transcription level of clpE was down-regulated at 43℃.Moreover,EMSA demonstrated that DegU bound directly with the promoter of clpE.[Conclusion]DegU plays a vital role in adhesion,invasion,proliferation,and migration of L.monocytogenes.Additionally,DegU directly binds with the promoter and regulates the transcriptional level of clpE to adapt to the high-temperature environment.The findings provide a basis for probing into the infection and environmental adaptation mechanisms of L.monocytogenes.