撒坝猪源E. coli高致病性毒力岛通过NLRP3/ASC/Caspase-1途径诱导IPEC-J2细胞焦亡

doi:10.11843/j.issn.0366-6964.2023.12.031

Abstract

Abstract: To study the mechanism of pyroptosis induced by highly pathogenic virulence island (HPI) of E. coli from Saba pig in IPEC-J2 cells, The E. coli HPI screened in the laboratory and the E. coli ΔHPI constructed by CRISPR/Cas9 gene knockout technology were used to infect IPEC-J2 cells to observe cell damage, LPS+ATP was used as a positive control. RT-qPCR was used to determine the mRNA transcription levels of key factors in the pyroptosis pathway, including NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, Gasdermin D (GSDMD), and inflammatory factors interleukin-18 (IL-18) and interleukin-1β (IL-1β) at 0.5, 3, 6, 9, and 12 h after treatment in each group. Laser confocal microscopy was used to observe the assembly and expression of NLRP3/Caspase-1 inflammasome complex. Western blot was used to determine the expression levels of GSDMD and its activated form GSDMD-N protein. PI staining was used to detect membrane integrity. The results showed that LPS+ATP treatment significantly promoted the occurrence of pyroptosis. The cells in the negative control group grew normally with clear contours. The cells in the E. coli infection group and LPS+ATP group showed obvious cytopathic effects (CPE) such as deformation, detachment, and blurred boundaries. HE staining showed that E. coli HPI infection caused more severe cell damage than E. coli ΔHPI infection. The mRNA levels of key factors and inflammatory factors in the pyroptosis pathway in the E.coli HPI group were significantly (P<0.05) or extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group at 3-9 h. NLRP3/Caspase-1 inflammasome complex was assembled in the cytoplasm, and the protein fluorescence intensity of NLRP3/Caspase-1, the expression levels of GSDMD and GSDMD-N protein, and the positive rate of PI staining in the E. coli HPI group were extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group. The CPE, HE staining, mRNA levels of key factors and inflammatory factors in the pyroptosis pathway, expression levels of GSDMD and GSDMD-N protein, and co-localization of NLRP3/Caspase-1 in the E.coliHPI group were similar to those in the LPS+ATP group. The results suggest that E. coli HPI from Saba pig induces pyroptosis in IPEC-J2 cells by upregulating the mRNA levels of key factors in the NLRP3/ASC/Caspase-1 signaling pathway, inducing the assembly of NLRP3/Caspase-1 inflammasome complex, promoting the expression of pyroptosis marker proteins GSDMD and GSDMD-N, and punching holes in the cell membrane of IPEC-J2 cells.

Key words: Escherichia coli, highly pathogenic virulence island, pyroptosis, porcine small intestinal epithelial cells, NLRP3/ASC/Caspase-1 pathway

CLC Number:

S852.3

XIAO Jinlong, WANG Hao, WAN Quan, SHEN Jue, ZHANG Bo, ZHAO Weiwei, DENG Jing, WANG Xi, ZHAO Ru, XIAO Peng, GAO Hong. Induction of IPEC-J2 Pyroptosis by E.coli HPI from Saba Pig via NLRP3/ASC/Caspase-1 Pathway[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5218-5227.

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Induction of IPEC-J2 Pyroptosis by E.coli HPI from Saba Pig via NLRP3/ASC/Caspase-1 Pathway

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