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

Among the several potent toxins produced by the gram-positive, spore-forming anaerobe Clostridium perfringens is the C. perfringens enterotoxin (CPE). Despite being produced by less than 5% of global type A isolates, CPE remains biomedically relevant due to its role in enteric disease. CPE causes the symptoms of several human gastrointestinal diseases, one of which (C. perfringens type A food poisoning) is currently ranked as the third most prevalent food-borne illness in the United States (23). CPE is a single polypeptide chain of 319 amino acids with a molecular weight of 35,346 (4). As a pore-forming toxin (12), CPE permeabilizes plasma membranes of sensitive cells to molecules up to 200 Da within a short period of time (18). CPE first binds via protein receptors which are thought to be certain members of the claudin family of proteins involved in tight junction structure and function (6, 15, 37). Under physiologic conditions, the enterotoxin rapidly forms a large CPE-containing complex that is resistant to sodium dodecyl sulfate (SDS) and has an approximate molecular mass of 155 kDa (40). Changes in cellular membrane permeability typically accompany formation of the ∼155-kDa large complex, suggesting that this complex functions as the transmembrane pore (or a subunit thereof). Upon longer CPE treatment, intoxicated cells can also form a second SDS-resistant, CPE-containing large complex with an estimated molecular mass of 200 kDa (31). This second large complex also includes another tight junction protein, named occludin (30). Calcium influx through CPE pores drives cell death via either the apoptotic or oncotic pathways, depending on the CPE dose (1, 2). Previous studies using CPE variants blocked for one or more CPE functions have provided important insights into the action of this toxin (8, 10, 16, 17, 32). Binding activity has been mapped to the extreme C terminus, based upon the binding ability of recombinant CPE (rCPE) fragments containing C-terminal sequences (8-11) and the binding deficiencies of rCPE fragments lacking 5, 10, or 30 amino acids from the C terminus (17). In contrast, rCPE fragments lacking 36 or 44 amino acids at the N terminus were found to be approximately twofold more cytotoxic than full-length rCPE, indicating that sequences in the N terminus of CPE are mildly inhibitory for cytotoxicity (17). The same study also identified a region between amino acids 45 and 53 of rCPE that is essential for cytotoxicity, since rCPE fragments lacking these 9 amino acids were unable to form a large complex or induce pore formation. A subsequent site-directed mutagenesis study fine mapped this N-terminal CPE cytotoxicity region and demonstrated that the aspartic acid at position 48 is essential for large-complex formation and cytotoxicity (32). While previous structure-function studies have mainly focused on regions of CPE important for binding and large-complex formation, the region (if any) involved in pore formation/membrane insertion has not yet been investigated. Despite lacking primary sequence homology with other bacterial pore-forming toxins, CPE amino acids 81 to 106 show a marked alternating side chain hydrophobicity pattern (Fig. ​(Fig.1)1) closely resembling transmembrane domains (TMDs) found in members of the β-barrel pore-forming toxin (β-PFT) family. In this family, one or two β-hairpins from each monomer in a toxin oligomer collectively insert into the membrane to form the phospholipid bilayer-penetrating channel (25, 35, 36). In efforts to begin ascertaining whether amino acids 81 to 106 in CPE might be important for membrane insertion/pore formation, the present study deleted this region in both the wild-type and D48A point mutant rCPE backgrounds. Careful characterization of these variants provided qualitative, as well as temporal, insights into the molecular mechanism of CPE action. FIG. 1. Functional map of CPE. Previous analysis of CPE identified two major functional regions (10, 17). Within the 5-amino-acid core cytotoxicity sequence at CPE's N terminus, the aspartic acid residue at position 48 (inverted triangle) is essential for cytotoxicity ...