Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

Abstract

Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity.

F199E substitution reduced toxicity ofClostridium perfringensepsilon toxin by depriving the receptor binding capability

“…Previous studies have shown that mutant ETX proteins with amino acid substitutions near F199 are defective in binding MDCK cells, but are not defective in binding human ACHN cells. 13 Binding assay of toxins on human ACHN cells was also performed, and we found rETX was more likely to bind to ACHN cells. In contrast, rETX F199E was defective in binding to ACHN cells (Fig.…”

“…13 Our previous study also showed that C-terminal peptide reduced toxicity of ETX probably via covering receptorbinding region in domain III. 30 Therefore, there is still a possibility that the receptor binding region is located in domain III.…”

“…Domain I contains a group of key amino acid residues (Tyr196, Phe199, Tyr29, Tyr30 and Tyr36), suggesting a relationship of binding to receptor. 13,14 Domain II is a b-sandwich including a 2-stranded sheet and a 5-stranded sheet, and is likely to be involved in oligomerization. 15 Domain III is also a b-sandwich with one 3-stranded and one 4-stranded sheet, playing a role in mediating ETX insertion into membranes.…”

Red Blood Cell Susceptibility to Pneumolysin

“…L363A -For expression of eGFP-⌬6PLY L363A , recombinant plasmid was transferred into Escherichia coli Rosetta 2 (DE3) cells (Merck, Darmstadt, Germany), and expression of eGFP-⌬6PLY L363A was induced using the autoinduction system as described previously (51).…”

Structural relationships between small β-pore-forming toxins from Clostridium perfringens

“…However, ε-toxin has no equivalent to domain I of aerolysin, and the structural homology between the N-terminal ε-toxin domain and domain II in aerolysin is much less than that between the rest of ε-toxin and domains III and IV of aerolysin. A cluster of surface-exposed hydrophobic aromatic residues are present in this domain, which have been shown to be important for interactions with target cells [67]. Chassin et al [68] demonstrated that, unlike aerolysin, the ε-toxin receptor is not GPI-anchored, as PI-PLC treatment of target cells does not impair binding or complex formation.…”