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