Identification of a Prepore Large-Complex Stage in the Mechanism of Action of Clostridium perfringens Enterotoxin

Abstract

Clostridium perfringens enterotoxin (CPE) is the etiological agent of the third most common food-borne illness in the United States. The enteropathogenic effects of CPE result from formation of large CPEcontaining complexes in eukaryotic cell membranes. Formation of these ϳ155-and ϳ200-kDa complexes coincides with plasma membrane permeability changes in eukaryotic cells, causing a Ca 2؉ influx that drives cell death pathways. CPE contains a stretch of amino acids (residues 81 to 106) that alternates markedly in side chain polarity (a pattern shared by the transmembrane domains of the ␤-barrel pore-forming toxin family). The goal of this study, therefore, was to investigate whether this CPE region is involved in pore formation. Complete deletion of the CPE region from 81 to 106 produced a CPE variant that was noncytotoxic for Caco-2 cells and was unable to form CPE pores. However, this variant maintained the ability to form the ϳ155-kDa large complex. This large complex appears to be a prepore present on the plasma membrane surface since it showed greater susceptibility to proteases, increased complex instability, and a higher degree of dissociation from membranes compared to the large complex formed by recombinant CPE. When a D48A mutation was engineered into this prepore-forming CPE variant, the resultant variant was unable to form any prepore ϳ155-kDa large complex. Collectively these findings reveal a new step in CPE action, whereby receptor binding is followed by formation of a prepore large complex, which then inserts into membranes to form a pore.

Novel Clostridium perfringens enterotoxin suicide gene therapy for selective treatment of claudin-3- and -4-overexpressing tumors

“…[15][16][17] The targeted action of CPE is mediated by binding to a subset of claudins, mainly to claudin-3 and -4, which are frequently overexpressed in epithelial tumors. 22,24,25 This binding leads to disintegration of plasma membrane in association with rapid cytolysis of treated cells. 34 Several in vitro and in vivo studies demonstrated the antitumoral activity of recombinant CPE, limited by the need for frequent, repeated CPE application to achieve antitumoral effects.…”

“…These insert into the plasma membrane, leading to the loss of osmotic equilibrium and cell death. [20][21][22] CPE binds to a subset of the claudin family of tight junction proteins, such as claudin-3 and -4, initially defined as CPE receptors. 23 The C-terminus of CPE permits this binding, whereas the N-terminus of CPE is rather associated with cytotoxicity.…”

Structural Constraints for the Binding of Short Peptides to Claudin-4 Revealed by Surface Plasmon Resonance

“…Despite that fact that the C-Cpe peptide no longer contains direct cytotoxic activity, a recent report showed that a similar size truncated version of Cpe still retained the ability to form membrane "prepore complexes," suggesting that this peptide retained the ability to form structures distinct from its ability to simply bind the second extracellular domain of Cldn-4 (18,19). Some studies also showed binding by the smaller Cpe30 peptide, but our present studies show that Cpe peptides as small as Cpe17 also can bind with high affinity, and even shorter versions, such as a mutated version (MT2) only 12 amino acids long could still bind with high affinity.…”

Toxin Plasmids of Clostridium perfringens

“…Furthermore, those structural analyses, coupled with mutagenesis studies (56)(57)(58)(59)(60)(61), indicated that CPE contains a C-terminal domain that binds to claudin receptors on host cells and an N-terminal domain, consisting of two halves, that is critical for pore formation by mediating oligomerization and membrane insertion.…”

“…At 37°C, CPE in a small complex rapidly oligomerizes on the membrane surface to form a large (ϳ450-kDa) prepore complex named CPE hexamer 1 (CH-1) (59,66,74). In addition to six copies of the toxin, CH-1 contains both receptor and nonreceptor claudins (the presence of nonreceptor claudins in CH-1 likely reflects a propensity for claudin-claudin interactions) (66).…”