副猪格拉瑟菌5型细胞致死性膨胀毒素CdtB破坏猪呼吸道上皮屏障的机制

doi:10.11843/j.issn.0366-6964.2023.01.028

摘要/Abstract

摘要： 旨在探究副猪格拉瑟菌(Glaesserella parasuis，GPS)突破猪呼吸道上皮屏障引起系统性感染的机制。通过超速离心和密度梯度离心提取副猪格拉瑟菌外膜囊泡(outer membrane vesicles,OMVs)，OMVs经SDS-PAGE显示，蛋白质条带分布在55~100ku，经透射电子显微镜(TEM)观察，OMVs的粒径在100~200nm，纳米粒子直径分析(NTA)结果显示，样品在100~200nm处的粒子数目最多。进而用制备的HbpA及OmpP2多克隆抗体对OMVs及不含OMVs的细菌上清进行Western blot验证，证实所提取的样品为外膜囊泡，且进一步结果证明细胞致死性膨胀毒素(CDT)在GPS培养物中主要以OMVs的形式存在。用OMVs或CdtB处理猪气管上皮细胞(swine tracheal epithelial cells,STEC)36h，检测STEC中cleaved-caspase3、ZO-1和Occludin的蛋白表达水平，并用FITC-葡聚糖(FD-4)检测STEC单层细胞的细胞旁通透性。结果发现，OMVs与CdtB处理后凋亡相关蛋白cleaved-caspase3表达水平升高，ZO-1和Occludin的表达水平降低，FD-4渗透率升高，同时，乳酸脱氢酶(LDH)细胞毒性检测试验发现，当CdtB与STEC单层细胞共孵育24h、OMVs与STEC单层细胞共孵育36h后，STEC存活率显著降低。而Pifithrin-α预处理STEC可抑制OMVs和CdtB引起的细胞凋亡和屏障通透性的增加。另外，用免疫磁珠捕获的方法去除OMVs中的CdtB(OMVs-△CdtB)，OMVs-△CdtB处理STEC 36h后cleaved-caspase3、ZO-1、Occludin表达水平与对照组相比无显著差异。综上，成功提取并纯化GPS OMVs，且试验证明GPS可通过OMVs转运CdtB诱导STEC凋亡和屏障损伤，为副猪格拉瑟菌突破呼吸道上皮屏障的机制提供新思路。

关键词: 副猪格拉瑟菌, 外膜囊泡, 细胞致死性膨胀毒素CdtB, 猪呼吸道上皮细胞

Abstract: To investigate the mechanism of systemic infection caused by Glaesserella parasuis (GPS) breaking through the epithelial barrier of swine respiratory tract, outer membrane vesicles (OMVs) were extracted by hypercentrifugation and density gradient centrifugation in this study.The protein bands of OMVs were detected by SDS-PAGE and distributed between 55 to 100 ku. The particle size of OMVs was found to be 100-200 nm observed by transmission electron microscope (TEM) and nanoparticle diameter analysis(NTA).The prepared HbpA and OmpP2 polyclonal antibodies were used to perform Western blot verification on OMVs and bacterial supernatants without OMVs. The results proved that the extracted samples were outer membrane vesicles, and further results proved that the cytolethal distending toxin (CDT) mainly exists in the form of OMVs in G. parasuis. Then, swine tracheal epithelial cells (STEC) were treated with OMVs or CdtB for 36 h, and the protein expression levels of cleaved-caspase3, ZO-1 and Occludin in STEC were detected, and FITC-dextran (FD-4) was used to detect the paracellular permeability. The results showed that after treated with OMVs or CdtB, the expression level of apoptosis-related protein cleaved-caspase3 was increased and the expression levels of ZO-1 and Occludin were decreased compared with the control group, and the permeability of FD-4 was increased. At the same time, the lactate dehydrogenase (LDH)cytotoxicity assay showed that the cell viability was significantly decreased after 24h of CdtB co-culture and 36h of OMVs co-culture. STEC pretreated with inhibitor of the apoptosis p53 pathway (pifithrin-α) could inhibit the cell apoptosis and barrier permeability increase induced by OMVs or CdtB. In addition, when CdtB in OMVs was removed by immunomagnetic bead capture (OMVs-△CdtB), the expression levels of cleaved-caspase3, ZO-1, and Occludin were not different after OMVs-△CdtB treated STEC for 36h compared with the control group. In conclusion, GPS OMVs were successfully extracted and purified, and the experiment proved that OMVs induced p53-dependent apoptosis and disrupted tight junctions in STEC by transporting CdtB, and finally increased the permeability of the tracheal epithelial barrier, providing a new idea for the mechanism of G. parasuis breaking through the respiratory epithelial barrier.

Key words: Glaesserella parasuis, outer membrane vesicles, cytolethal distending toxin CdtB, swine tracheal epithelial cells

中图分类号:

S852.61

陈敏, 刘明星, 张鹏云, 蔺辉星, 范红结. 副猪格拉瑟菌5型细胞致死性膨胀毒素CdtB破坏猪呼吸道上皮屏障的机制[J]. 畜牧兽医学报, 2023, 54(1): 304-316.

CHEN Min, LIU Mingxing, ZHANG Pengyun, LIN Huixing, FAN Hongjie. Mechanism of Cytolethal Distending Toxin CdtB of Glaesserella parasuis Serotype 5 Destroys the Integrity of Swine Tracheal Epithelial Barrier[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 304-316.

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