F199E substitution reduced toxicity ofClostridium perfringensepsilon 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 Ca 2C release from intracellular Ca 2C 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.

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

“…The morphological effects of ETX-induced hemolysis in RBCs appeared to resemble those in MDCK cells. Similar sags and crests were observed on the surface of RBCs as those on ETX-treated MDCK cells [20]. A stable complex was detected in the ETX-treated membrane of both RBCs and MDCK cells (S3 Fig).…”

“…To verify that the observed hemolysis was induced by rETX and not contaminating proteins from the engineered E. coli strain, we tested the effect of the attenuated rETX mutant rETX F199E (the 199th amino acid residue of phenylalanine was replaced by a glutamic acid) [20] on the hemolysis of human erythrocytes. rETX F199E was expressed and purified using the same method as rETX.…”

“…The toxin protein used in this assay was freshly purified. MTS (3-(4, 5-dimethylthiazol-2-yl) -5(3-carboxymethoxyphenyl) -2-(4-sulfopheny)-2H-tetrazolium, inner salt) (Promega, United States) assay was performed as described previously [20]. …”

Clostridium perfringens epsilon toxin vaccine candidate lacking toxicity to cells expressing myelin and lymphocyte protein

“…These shortfalls have prompted work to devise improved vaccines, and a number of recombinant immunogens have been reported, including formaldehyde-treated epsilon toxin produced from Escherichia coli 17 and site-directed mutants (genetic toxoids) of epsilon toxin with reduced toxicity. 11,18–22 Site-directed mutants offer the advantage that chemical detoxification, a process that can result in batch-to-batch variation in immunogenicity, is avoided. However, the high potency of the toxin can make it difficult to completely abolish toxicity.…”

A Novel Panel of Rabbit Monoclonal Antibodies and Their Diverse Applications Including Inhibition of Clostridium perfringens Epsilon Toxin Oligomerization

“…Domain I contains numerous aromatic amino acids and the sole tryptophan residue, which contributes to receptor binding [3,23]. Single point mutations within this domain inhibit binding to susceptible cells [24,25,26,27,28,29,30,31,32,33]. Domain II is believed to play an important role in toxin oligomerization, stabilization, and insertion into the membrane [23,31,32].…”