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

Abstract

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

CLC Number:

S852.61

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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Mechanism of Cytolethal Distending Toxin CdtB of Glaesserella parasuis Serotype 5 Destroys the Integrity of Swine Tracheal Epithelial Barrier

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