Elsevier

Gene

Volume 759, 30 October 2020, 144999
Gene

Research paper
Clostridium perfringens beta2 toxin induced in vitro oxidative damage and its toxic assessment in porcine small intestinal epithelial cell lines

https://doi.org/10.1016/j.gene.2020.144999Get rights and content

Highlights

  • CPB2 can initiate inflammatory responses in IPEC-J2 cells.

  • CPB2 may cause intestinal epithelial barrier dysfunction.

  • CPB2 toxin can induce apoptosis of IPEC-J2 cells.

Abstract

Clostridium perfringens beta2 (CPB2), a key virulence factor, is produced by C. perfringens type C that is the main pathogenic microorganism causing diarrhea in piglets. However, little is known concerning the toxic damage effect of CPB2 on intestinal cells of piglets. In present study, CPB2 toxin obtained by genetic recombination technology was evaluated for its cytotoxicity property using the intestinal porcine epithelial (IPEC-J2) cells, which aims to attempt to understand and explain its mechanism of action in porcine small intestinal epithelial cells. IPEC-J2 cells were treated with different concentrations of CPB2 toxin (5, 10, 20, 30, 40, and 50 μg/mL), and MTT assay results showed that the cell viability of CPB2-treated IPEC-J2 cells decreased in a dose-dependent manner. Lactate dehydrogenase (LDH) assay results revealed that CPB2 significantly increased the LDH release, relative to the control. The expression of tumor necrosis factor α (TNF-α) and interleukin 8 (IL-8) gradually increased, while the expression of interleukin 10 (IL-10) gradually decreased in IPEC-J2 cells with increasing concentration of CPB2 (10–30 μg/mL), as analyzed by quantitative real-time PCR (RT-qPCR). Also, CPB2 increased the content of intracellular reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) of IPEC-J2 cells. Western blot and immunofluorescence results demonstrate that CPB2 decreased the expression of zonula occludens (ZO-1), claudin12 (CLDN12) and occludin (OCLN) in IPEC-J2 cells. In addition, CPB2 increased Bax expression, and inhibited Bcl-2 and Bcl-xL expression, as measured by Western blot. Considering all of these findings, it was concluded that CPB2 toxin shows significant cytotoxicity, cell growth inhibition and increase in cell permeability in IPEC-J2 cells in a concentration-dependent manner, thus leading to abnormal cell apoptosis and functions in porcine small intestinal epithelial cells.

Introduction

Diarrhea is one of the important causes of piglet death, causing huge economic losses to the world pig industry (Chan et al., 2012). In recent years, Clostridium perfringens (C. perfringens) is considered to be a major pathogen causing diarrhea in piglets and has become a major problem hindering the healthy development of the pig industry (Kich et al., 2014). C. perfringens is a bacterium widely found in nature that can cause a range of intestinal diseases in humans and livestock (Petit et al., 1999, Songer and Glenn, 1996). According to its ability to produce four major types of alpha, beta, epsilon and iota toxins, C. perfringens can be further divided into five different toxin types, namely A, B, C, D and E (Hassan et al., 2015).

Previous studies have shown that C. perfringens type C is one of the main pathogenic microorganism causing diarrhea in piglets, which can produce alpha and beta (1, 2) toxins (Michael et al., 2003, Sayeed et al., 2010). Pathologically, beta1 (CPB1) and beta2 (CPB2) are considered to be important toxic factors of necrotizing enteritis in humans and animals, especially in piglets (Gibert et al., 1997, van Asten et al., 2010). The molecular weight (MW) of CPB2 is 28 kDa, which is first identified in C. perfringens strains isolated from piglets died of necrotizing enterocolitis (Gibert et al., 1997). Subsequent studies report that CPB2 is not only isolated from human (Fisher et al., 2005) and domestic animals such as pigs (Klaasen et al., 1999), horses (Bacciarini et al., 2003), cattles (Lebrun et al., 2007), sheep (Gkiourtzidis et al., 2001), dogs (Thiede et al., 2001), and chickens (Engström et al., 2003). Most of the animals infected with CPB2 are characterized by diarrhea and necrotizing enteritis. At present, the research on CPB2 is mainly focused on the isolation and identification of toxins, while few reports regarding the specific mode of action of CPB2 are available. Additionally, although the toxic effect of CPB2 in some cell lines such as human embryonic intestinal epithelial cells (I407) (Gibert et al., 1997), human colonic epithelial cell lines (Caco2) (Smedley et al., 2004) and human colon epithelial cells (NCM460) (Zeng et al., 2016), has been studied, there are no reports on the effect of CPB2 produced by C. perfringens type C on intestine damage of piglets and the potential underlying mechanisms. In this study, we obtained the CPB2 recombinant protein by using prokaryotic expression technology, which is active against pig intestinal epithelial cells (IPEC-J2). By evaluating the toxicity of CPB2 to IPEC-J2 cells and investigating the toxicological mechanism of CPB2-induced apoptosis of IPEC-J2 cells, it provides a reference for further exploring the immune response mechanism of piglet diarrhea caused by C. perfringens type C infection.

Section snippets

Preparation of CPB2 recombinant protein

Based on the cpb2 DNA sequence in GenBank (GenBank: L77965.1), we synthesized a gene fragment that removes the signal peptide and added restriction sites Nde I and XholI at the 5′ and 3′ ends, respectively. Cloned upstream of the His tag of the pET-28a bacterial expression vector (Bioss, Beijing, China). After sequencing, the vector was successfully constructed and named pET-28a-CPB2. It was transformed into Escherichia coli (E. coli) strain Trans BL21 (DE3) pLysS (TransGen Biotech, Beijing,

Expression and purification of recombinant CPB2 toxin protein

BL21 (DE3) pLysS containing pET-28a-CPB2 vector was induced by IPTG, and it was found that the expected molecular weight ~28 kDa band was observed in SDS-PAGE after induction (lanes 1–2 in Fig. 1A). Observing SDS-PAGE, we found that a brighter protein band was found in the eluate with a concentration of 500 mm imidazole (lanes 5–8 in Fig. 1A). WB analysis further showed that the purified protein reacted specifically with His-tagged antibodies (Fig. 1B). This shows that we have the right size

Discussion

When the sensitive cells were treated with CPB2, they would show cell rounding, vesicle formation and finally dissociation from the cell culture matrix (Gibert et al., 1997). Studies have shown that CPB2 toxin has certain toxicity to CHO and I407 (Gibert et al., 1997). I407 cells were rounded with 20 μg/mL CPB2 toxin for 18 h (Gibert et al., 1997). Analogously, in this study, we observed the obvious morphological changes, such as cell rounding, loss of cell-cell adhesion in IPEC-J2 cells

Conclusions

In conclusion, our results indicate that CPB2 toxin can cause a dose-dependent inhibition of the growth of IPEC-J2 cells and result in cell inflammation. CPB2 induced IPEC-J2 cells can reduce the expression of tight junction protein, leading to barrier dysfunction of the intestinal epithelium. This study also showed that CPB2 can increase the accumulation of ROS, reduce ΔΨm, increase the ratio of Bax/Bcl-2, and thus lead to apoptosis of IPEC-J2 cells.

Author contributions

SG and RL conceived and designed the experiments; RL, XG, WW, KX and PW performed the experiments and analyzed the data; RL wrote the paper; SG, QY, XH and ZY revised the manuscript. All authors read and approved the final manuscript.

CRediT authorship contribution statement

Ruirui Luo: Conceptualization, Investigation, Visualization, Writing - original draft. Qiaoli Yang: Writing - review & editing. Xiaoyu Huang: Writing - review & editing. Zunqiang Yan: Writing - review & editing. Xiaoli Gao: Formal analysis. Wei Wang: Visualization. Kaihui Xie: Investigation. Pengfei Wang: Data curation. Shuangbao Gun: Writing - review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by the Scientific Research Start-Up Funds for Openly-Recruited Doctors of Gansu Agricultural University (GAU-KYQD-2019-07) and the National Natural Science Foundation of China (grant number 31660646 and 31960646).

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