单核细胞增生李斯特菌二硫键形成蛋白DsbA调控胞间迁移的机制

The disulfide bond formation protein DsbA in Listeria monocytogenes regulates cell-to-cell spread

摘要:

【目的】 实验室前期研究发现单核细胞增生李斯特菌(单增李斯特菌，Listeria monocytogenes)二硫键形成蛋白DsbA缺失后，细菌胞间迁移能力增强，本研究旨在深入解析DsbA介导该生物学过程的具体机制。【方法】 通过荧光定量PCR和蛋白质免疫印迹(Western blotting)方法比较单增李斯特菌野生株和dsbA缺失株毒力基因的转录和表达水平差异；利用免疫荧光共定位方法观察DsbA缺失后对单增李斯特菌胞间迁移相关毒力因子ActA招募宿主肌动蛋白能力的影响(分析ActA与肌动蛋白共定位在细菌一侧形成“彗星状尾巴”的长短和数量)；通过等温滴定量热法(isothermal titration calorimetry, ITC)研究DsbA与ActA互作情况。【结果】 dsbA缺失后，毒力基因转录水平无显著差异，但毒力因子InlA、InlB、PlcA和PlcB的分泌均显著降低，而ActA、溶血素O (listeriolysin O, LLO)分泌量显著升高。缺失株形成的彗星尾巴数量显著高于野生株且平均长度也较野生株增加，说明dsbA缺失导致细菌招募肌动蛋白能力明显增强。ITC试验发现DsbA与ActA结合存在吸热反应，说明二者互作。【结论】 本研究证实单增李斯特菌DsbA通过调控毒力蛋白减弱了对肌动蛋白募集，进而影响细菌胞间迁移。研究结果有助于系统理解单增李斯特菌在宿主感染过程中的毒力调控机制，对人兽共患胞内病原菌的污染控制具有重要公共卫生学意义。

关键词:单增李斯特菌;二硫键形成蛋白DsbA;胞间迁移;肌动蛋白募集

Abstract:

[Objective] In view of the enhanced cell-to-cell spread ability of the dsbA-deleted strain (ΔdsbA) of Listeria monocytogenes, this study aims to elucidate the mechanism that how the disulfide bond formation protein DsbA mediates this biological process. [Methods] The mRNA and protein levels of virulence factors in the wild type and ΔdsbA were compared by RT-qPCR and Western blotting, respectively. The immunofluorescence co-localization analysis method was employed to observe the impact of DsbA deficiency on the actin recruitment by the virulence factor ActA in the cell-to-cell spread of L. monocytogenes (analyzing the length and quantity of the comet tails formed on one side of the bacteria by co-localization of ActA and actin). The presence or absence of interaction between DsbA and ActA was determined by isothermal titration calorimetry (ITC). [Results] Compared with the wild type, ΔdsbA showed no significant changes in the mRNA levels of virulence factors, downregulated protein levels of InlA, InlB, PlcA, and PlcB, and upregulated protein levels of ActA and LLO. In addition, ΔdsbA showed increased number and average length of comet tails, which indicated that the actin recruitment of ΔdsbA was enhanced. The ITC results revealed that DsbA bound to ActA, which gradually showed endothermic reactions, suggesting the presence of interaction between DsbA and ActA. [Conclusion] This study proved for that DsbA attenuated the recruitment ability of actin by regulating virulence proteins, thus affecting the cell-to-cell spread of L. monocytogenes. The findings help to further dissect the virulence regulatory mechanisms of L. monocytogenes during host infection, which is of great importance for controlling the contamination of zoonotic intracellular pathogens threatening public health.

Key words:Listeria monocytogenes;disulfide bond formation protein DsbA;cell-to-cell spread;actin recruitment