Cysteine-Scanning Mutagenesis Supports the Importance of Clostridium perfringens Enterotoxin Amino Acids 80 to 106 for Membrane Insertion and Pore Formation

Abstract

Clostridium perfringens enterotoxin (CPE) causes the gastrointestinal symptoms of the second most common bacterial foodborne illness. Previous studies suggested that a region named TM1, which has amphipathic characteristics and spans from amino acids 81 to 106 of the native CPE protein, forms a ␤-hairpin involved in ␤-barrel pore formation. To further explore the potential role of TM1 in pore formation, the single Cys naturally present in CPE at residue 186 was first altered to alanine by mutagenesis; the resultant rCPE variant, named C186A, was shown to retain cytotoxic properties. Cys-scanning mutagenesis was then performed in which individual Cys mutations were introduced into each TM1 residue of the C186A variant. When those Cys variants were characterized, three variants were identified that exhibit reduced cytotoxicity despite possessing binding and oligomerization abilities similar to those of the C186A variant from which they were derived. Pronase challenge experiments suggested that the reduced cytotoxicity of those two Cys variants, i.e., the F91C and F95C variants, which model to the tip of the ␤-hairpin, was attributable to a lessened ability of these variants to insert into membranes after oligomerization. In contrast, another Cys variant, i.e., the G103C variant, with impaired cytotoxicity apparently inserted into membranes after oligomerization but could not form a pore with a fully functional channel. Collectively, these results support the TM1 region forming a ␤-hairpin as an important step in CPE insertion and pore formation. Furthermore, this work identifies the first amino acid residues specifically involved in those two steps in CPE action.

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.…”

“…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). The CH-1 prepore complex, which forms in both cultured Caco-2 cells and the small intestine, then inserts into membranes by using a ␤-hairpin formed by CPE amino acids 81 to 106 (59,61). This process results in formation of a cation-selective CPE pore that is initially permeable to molecules of Ͻ200 Da (1,(75)(76)(77).…”

Identification and Characterization of Clostridium perfringens Beta Toxin Variants with Differing Trypsin Sensitivity and In Vitro Cytotoxicity Activity

“…The protease sensitivity of oligomerized CPB complex present on the surface of HUVEC cells was assayed using a modification of previously described methods (27). Briefly, HUVEC cells were cultured in 6-well plates until confluent monolayers were achieved.…”

“…The formation of a functional CPB pore was assessed by measuring 86 Rb release from host cells, as described previously (27). Briefly, 24-well plates were seeded with HUVEC cells and grown to confluent monolayers.…”

“…Since the alteration of a single amino acid can affect the function of clostridial pore-forming toxins (21,23,(26)(27)(28), our current work identified natural CPB sequence variations and assessed their effects on the stability of these CPB variants in the presence of trypsin and on their CPB cytotoxicity in vitro.…”