Clostridium perfringens Iota-Toxin Requires Activation of Both Binding and Enzymatic Components for Cytopathic Activity

Abstract

Iota-toxin is produced by Clostridium perfringens type E strains and consists of two independent components, the enzymatic and binding components, referred to as Ia and Ib, respectively. A recombinant C. perfringens strain, strain 667/pMRP147, produced processed Ia and partially processed Ib, while a recombinant C. perfringens type A strain, strain TS133/pMRP147, in which the VirR-VirS two-component system is inactivated, produced only precursor forms of Ia and Ib. This suggests that iota-toxin is processed by a VirR-VirSresponsive protease, although not completely in the recombinant type A strain. The precursor forms of Ia and Ib were purified from cultures of the latter strain, and their proteolytic activation was examined. Treatment with proteases cleaved off small peptides (9 to 13 amino acid residues) and a 20-kDa peptide from the N termini of the Ia and Ib precursors, respectively, leading to their active forms. They were activated efficiently by ␣-chymotrypsin, pepsin, proteinase K, subtilisin, and thermolysin but only weakly by trypsin, as demonstrated by the cell-rounding assay. -Protease from the C. perfringens type E strain, which was found to be a zinc-dependent protease related to thermolysin, activated iota-toxin as efficiently as did ␣-chymotrypsin. These results suggest that -protease is most responsible for the activation of iota-toxin in type E strains.Clostridium perfringens is a ubiquitous pathogen which causes food poisoning and gas gangrene in humans and digestive diseases in other animals. This organism is divided into five toxin types on the basis of the production of four major lethal toxins, alpha-, beta-, epsilon-, and iota-toxins (8). C. perfringens type E, which produces iota-toxin, has been implicated in the enterotoxemia of calves and lambs (22).Iota-toxin is a member of the actin ADP-ribosylating iotatoxin family, which includes immunologically related clostridial toxins such as Clostridium spiroforme toxin (7) and Clostridium difficile ADP-ribosyltransferase (CDT) (17). They are binary toxins consisting of two independent polypeptides, enzymatic and binding components. The binding component of iota-toxin (Ib; M r 80,000) is involved in the binding and internalization of the enzymatic component (Ia; M r 47,500) (4). Ia catalyzes the ADP-ribosylation of monomeric G-actin of the muscle and nonmuscle type at Arg-177, leading to disorganization of the actin filaments (1, 24). The components of the iota-toxin family are interchangeable: Ib can internalize the enzymatic components of C. spiroforme toxin and CDT. In contrast, Clostridium botulinum C2 toxin, which is structurally and functionally related but immunologically unrelated to iota-toxin, does not translocate the enzymatic component of the iota-toxin family (4,19).It has been shown that iota-toxin is produced as inactive precursor (23). The proteolytic activation of an Ib precursor is accompanied by removal of a N-terminal 20-kDa peptide, and it has been generally considered that only Ib requires the proteolytic activation to ex...

Interaction of Clostridium perfringensIota-Toxin with Lipid Bilayer Membranes

“…Ib and Ia components were purified as described previously (19). Ib (200 g/ml) was activated by incubation in 20 g/ml trypsin for 30 min at room temperature followed by the addition of 100 g/ml trypsin inhibitor (19). Rabbit anti-Ib antibodies were obtained as has been described previously by immunizing the animals with activated Ib (21).…”

“…Unprocessed Ib was produced from C. perfringens strain TS133 harboring the recombinant plasmid pMRP384, and Ia was produced from strain 667 harboring pMRP147. Ib and Ia components were purified as described previously (19). Ib (200 g/ml) was activated by incubation in 20 g/ml trypsin for 30 min at room temperature followed by the addition of 100 g/ml trypsin inhibitor (19).…”

“…Essential for the activity of the binding component Ib within the toxin translocation of Ia is the activation of the former by partial proteolysis (11,19). The active fragment has 664 amino acids (about 80 kDa) because 172 amino acids (about 20 kDa) are cleaved from the N terminus of the Ib precursor during the activation process.…”

Update on the toxins of Clostridium perfringens and their actions

“…Τα δυο συστατικά εκκρίνονται ως προτοξίνες και για την ενεργοποίηση τους απαιτείται η δράση της τοξίνης -λ ή της χυμοθρυψίνης (Gibert et al 2000). To lb συνδέεται με τον υποδοχέα επιφάνειας και συνεπώς μεσολαβεί στην εσωτερίκευση του ενζυμα-τικοΰ συστατικού.…”

“…Το la καταλύει την ADP-ριβοσυ-λίωση των μονομερών ακτίνης, με αποτέλεσμα τον αποπολυμερισμό της ακτίνης και την αναστολή των κυτταρικών λειτουργιών που βασίζονταν στον κυττα-ροσκελετό της ακτίνης, οδηγώντας στο θάνατο. Τέλος, αξίζει να σημειωθεί ότι η τοξίνη -ι θα μπορούσε να αποτελέσει χρήσιμο βιολογικό εργαλείο για τη μετα φορά πρωτεϊνών μέσα στα κύτταρα (Rood and Cole 1991, Songer 1996, Petit et al 1999, Gibert et al 2000, Johansson 2006). …”