Effects of Clostridium perfringens beta2 toxin on apoptosis, inflammation, and barrier function of intestinal porcine epithelial cells

Gao, Xiaoli; Yang, Qiaoli; Huang, Xiaoyu; Yan, Zunqiang; Zhang, Shengwei; Luo, Ruirui; Wang, Pengfei; Wang, Wei; Xie, Kaihui; Jiang, Tiantuan; Gun, Shuangbao

doi:10.1016/j.micpath.2020.104379

Cited by 28 publications

(32 citation statements)

References 45 publications

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“…13 However, the C. perfringens subtypes containing cpb2 gene have been found to play an important role in preterm necrotizing enterocolitis (NEC). 22,23…”

Section: Discussionmentioning

confidence: 99%

Prevalence and Genetic Diversity of Clostridium perfringens Isolates in Hospitalized Diarrheal Patients from Central China

Wang¹,

Dong²,

Ma³

et al. 2021

IDR

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“…13 However, the C. perfringens subtypes containing cpb2 gene have been found to play an important role in preterm necrotizing enterocolitis (NEC). 22,23…”

Section: Discussionmentioning

confidence: 99%

Prevalence and Genetic Diversity of Clostridium perfringens Isolates in Hospitalized Diarrheal Patients from Central China

Wang¹,

Dong²,

Ma³

et al. 2021

IDR

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“…IPEC-J2 cells were seeded in 6-well plates at a density of 1×10 5 cells/mL and cultured overnight (24 h). Then, cells in three wells were each treated with 20 μg/mL CPB2 (treatment group) while cells in the other three wells were not exposed to toxin (control group) ( Gao et al, 2020 ; Luo et al, 2020 ). Total RNA was extracted by using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, United States), and the m 6 A content was detected with an m 6 A RNA methylation quantification kit (Epigentek, Farmingdale, United States).…”

Section: Methodsmentioning

confidence: 99%

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing

Zhang

Yang

et al. 2021

Front. Genet.

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Piglet diarrhea is a swine disease responsible for serious economic impacts in the pig industry. Clostridium perfringens beta2 toxin (CPB2), which is a major toxin of C. perfringens type C, may cause intestinal diseases in many domestic animals. N6-methyladenosine (m6A) RNA methylation plays critical roles in many immune and inflammatory diseases in livestock and other animals. However, the role of m6A methylation in porcine intestinal epithelial (IPEC-J2) cells exposed to CPB2 has not been studied. To address this issue, we treated IPEC-J2 cells with CPB2 toxin and then quantified methylation-related enzyme expression by RT-qPCR and assessed the m6A methylation status of the samples by colorimetric N6-methyladenosine quantification. The results showed that the methylation enzymes changed to varying degrees while the m6A methylation level increased (p < 0.01). On this basis, we performed N6-methyladenosine sequencing (m6A-seq) and RNA sequencing (RNA-seq) to examine the detailed m6A modifications and gene expression of the IPEC-J2 cells following CPB2 toxin exposure. Our results indicated that 1,448 m6A modification sites, including 437 up-regulated and 1,011 down-regulated, differed significantly between CPB2 toxin exposed cells and non-exposed cells (p < 0.05). KEGG pathway analysis results showed that m6A peaks up-regulated genes (n = 394) were mainly enriched in cancer, Cushing syndrome and Wnt signaling pathways, while m6A peaks down-regulated genes (n = 920) were mainly associated with apoptosis, small cell lung cancer, and the herpes simplex virus 1 infection signaling pathway. Furthermore, gene expression (RNA-seq data) analysis identified 1,636 differentially expressed genes (DEGs), of which 1,094 were up-regulated and 542 were down-regulated in the toxin exposed group compared with the control group. In addition, the down-regulated genes were involved in the Hippo and Wnt signaling pathways. Interestingly, the combined results of m6A-seq and RNA-seq identified genes with up-regulated m6A peaks but with down-regulated expression, here referred to as “hyper-down” genes (n = 18), which were mainly enriched in the Wnt signaling pathway. Therefore, we speculate that the genes in the Wnt signaling pathway may be modified by m6A methylation in CPB2-induced IPEC-J2 cells. These findings provide new insights enabling further exploration of the mechanisms underlying piglet diarrhea caused by CPB2 toxin.

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“…We used the method described by Gao and Luo ( 17 , 18 ) to extract and purify the CPB2 toxin protein. Then, the endotoxin was removed using Endotoxin Erasol (Genscript, Nanjing, China).…”

Section: Methodsmentioning

confidence: 99%

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin

Yang

Zhang

et al. 2021

Front. Immunol.

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BackgroundThe n6-methyladenosine (m6A) modification is present widely in mRNAs and long non-coding RNAs (lncRNAs), and is related to the occurrence and development of certain diseases. However, the role of m6A methylation in Clostridium perfringens type C infectious diarrhea remains unclear.MethodsHere, we treated intestinal porcine jejunum epithelial cells (IPEC-J2 cells) with Clostridium perfringens beta2 (CPB2) toxin to construct an in vitro model of Clostridium perfringens type C (C. perfringens type C) infectious diarrhea, and then used methylated RNA immunoprecipitation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) to identify the methylation profiles of mRNAs and lncRNAs in IPEC-J2 cells.ResultsWe identified 6,413 peaks, representing 5,825 m6A-modified mRNAs and 433 modified lncRNAs, of which 4,356 m6A modified mRNAs and 221 m6A modified lncRNAs were significantly differential expressed between the control group and CPB2 group. The motif GGACU was enriched significantly in both the control group and the CPB2 group. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation analysis showed that the differentially methylated modified mRNAs were mainly enriched in Hippo signaling pathway and Wnt signaling pathway. In addition, the target genes of the differentially m6A modified lncRNAs were related to defense response to virus and immune response. For example, ENSSSCG00000042575, ENSSSCG00000048701 and ENSSSCG00000048785 might regulate the defense response to virus, immune and inflammatory response to resist the harmful effects of viruses on cells.ConclusionIn summary, this study established the m6A transcription profile of mRNAs and lncRNAs in IPEC-J2 cells treated by CPB2 toxin. Further analysis showed that m6A-modified RNAs were related to defense against viruses and immune response after CPB2 toxin treatment of the cells. Threem6A-modified lncRNAs, ENSSSCG00000042575, ENSSSCG00000048785 and ENSSSCG00000048701, were most likely to play a key role in CPB2 toxin-treated IPEC-J2 cells. The results provide a theoretical basis for further research on the role of m6A modification in piglet diarrhea.

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Effects of Clostridium perfringens beta2 toxin on apoptosis, inflammation, and barrier function of intestinal porcine epithelial cells

Cited by 28 publications

References 45 publications

Prevalence and Genetic Diversity of Clostridium perfringens Isolates in Hospitalized Diarrheal Patients from Central China

Prevalence and Genetic Diversity of Clostridium perfringens Isolates in Hospitalized Diarrheal Patients from Central China

Comprehensive Analysis of Transcriptome-wide m6A Methylome Upon Clostridium perfringens Beta2 Toxin Exposure in Porcine Intestinal Epithelial Cells by m6A Sequencing

N6-Methyladenosine Methylation Analysis of Long Noncoding RNAs and mRNAs in IPEC-J2 Cells Treated With Clostridium perfringens beta2 Toxin

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