F199E substitution reduced toxicity of Clostridium perfringens epsilon toxin by depriving the receptor binding capability

ABSTRACT

Epsilon toxin (ETX), a potent toxin, is produced by types B and D strains of Clostridium perfringens, which could cause severe diseases in humans and domestic animals. Mutant rETX^F199E was previously demonstrated to be a good vaccine candidate. However, the mechanism concerned remains unknown. To clarify how F199E substitution reduced ETX toxicity, we performed a series of experiments. The results showed that the cell-binding and pore-forming ability of rETX^F199E was almost abolished. We speculated that F199E substitution reduced toxicity by depriving the receptor binding capability of ETX, which contributed to the hypothesis that domain I of ETX is responsible for cell binding. In addition, our data suggested that ETX could cause Ca²⁺ release from intracellular Ca²⁺ stores, which may underlie an alternate pathway leading to cell death. Furthermore, ETX induced crenation of the MDCK cells was observed, with sags and crests first appearing on the surface of condensed MDCK cells, according to scanning electron microscopy. The data also demonstrated the safety and potentiality of rETX^F199E as a vaccine candidate for humans. In summary, findings of this work potentially contribute to a better understanding of the pathogenic mechanism of ETX and the development of vaccine against diseases caused by ETX, using mutant proteins.

KEYWORDS:

• Clostridium perfringens
• Epsilon toxin (ETX)
• Mutant
• mechanism
• Cell-binding
• Pore-forming

Abbreviations

CD	=	Circular dichroism
CDC	=	Centers for Disease Control and Prevention
ETX	=	Clostridium perfringens epsilon toxin
FBS	=	fetal bovine serum
MDCK	=	the Madin Darby Canine Kidney cell line
PBS	=	phosphate-buffered saline

Disclosure of potential conflicts of interest

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Acknowledgments

We greatly thank Prof. Zhihu Zhao for providing the pTIG-trx vector. We greatly acknowledge the help of Yating Zhang for confocal microscopy and scanning electron microscopy experiments.

Funding

This work was supported by project of State Key Laboratory of Pathogen and Biosecurity (SKLPBS1413) and National Natural Science Foundation of China (No. 31500108).

Author contributions

Wenwen Xin, Baohua Zhao and Jinglin Wang conceived and designed the experiments; Jingjing Kang performed the experiments; Jie Gao and Wenwu Yao analyzed the data; Lin Kang, Shan Gao, Hao Yang, Bin Ji, Ping Li, Jing Liu and Jiahao Yao contributed reagents/materials/analysis tools; Jingjing Kang and Wenwen Xin wrote the paper.