Cleavage of a C-terminal Peptide Is Essential for Heptamerization of Clostridium perfringens ε-Toxin in the Synaptosomal Membrane

Abstract

Activation of Clostridium perfringens ⑀-protoxin by tryptic digestion is accompanied by removal of the 13 N-terminal and 22 C-terminal amino acid residues. In this study, we examined the toxicity of four constructs: an ⑀-protoxin derivative (PD), in which a factor Xa cleavage site was generated at the C-terminal trypsin-sensitive site; PD without the 13 N-terminal residues (⌬N-PD); PD without the 23 C-terminal residues (⌬C-PD); and PD without either the N-or C-terminal residues (⌬NC-PD). A mouse lethality test showed that ⌬N-PD was inactive, as is PD, whereas ⌬C-PD and ⌬NC-PD were equally active. ⌬C-PD and ⌬NC-PD, but not the other constructs formed a large SDS-resistant complex in rat synaptosomal membranes as demonstrated by SDSpolyacrylamide gel electrophoresis. When ⌬NC-PD and ⌬C-PD, both labeled with 32 P and mixed in various ratios, were incubated with membranes, eight distinct high molecular weight bands corresponding to six heteropolymers and two homopolymers were detected on a SDS-polyacrylamide gel, indicating the active toxin forms a heptameric complex. These results indicate that C-terminal processing is responsible for activation of the toxin and that it is essential for its heptamerization within the membrane. ⑀-Toxin produced by Clostridium perfringens types B and D is the third most potent clostridial toxin after botulinum and tetanus toxins, and is responsible for the pathogenesis of fatal enterotoxemia in domestic animals caused by the organisms (1). This toxin exhibits toxicity toward neuronal cells via the glutamatergic system (2, 3) or extravasation in the brain (4) after infection of experimental animals. It has been suggested to be a pore-forming toxin based on the following observations. (i) ⑀-Toxin can form a large complex in the membrane of MDCK 1 cells, and it permeabilizes them (5, 6); (ii) the large complex formed by ⑀-toxin is not dissociated by SDS treatment (6), which is a common feature of pore-forming toxins such as aerolysin (7), Clostridium septicum ␣-toxin (8), and Pseudomonas aeruginosa cytotoxin (9); and (iii) the CD spectrum of ⑀-toxin shows it mainly consists of ␤-sheets (10), as is characteristically observed for pore-forming ␤-barrel toxins.The structures of many bacterial pore-forming toxins or toxin components such as perfringolysin O (11), Bacillus thuringiensis ␦-toxin (12), aerolysin (13), staphylococcal ␣-toxin (14), and protective antigen of anthrax toxin (15) have been determined. These pore-forming toxins are believed to undergo a drastic conformational change upon interaction with a membrane. Since these toxins are inserted into the cytoplasmic membrane without the aid of other proteins such as chaperones or the translocation machinery, characterization of their metamorphosis has been regarded as a novel means for studying membrane-protein interactions (16). A characteristic feature of ⑀-toxin is its potent neurotoxicity, which is not seen for the structurally well defined pore-forming toxins. Thus, it could serve as a useful tool for extending our knowledge o...

Large scale purification of Clostridium perfringens toxins: a review

“…It has been suggested that is a pore-forming toxin based on the following observations: (i) ε-toxin can form a large complex in the membrane of Madin-Darby canine kidney cells, and permeabilizes them (Petit et al 1997;Nagahama, Ochi, Sakurai, 1998); (ii) the large complex formed by ε-toxin is not dissociated by SDS-treatment, which is a common feature of pore-forming toxins (Petit et al 1997); and (iii) the CD spectrum of ε-toxin shows that it mainly consists of β-sheets (Habeeb, Lee, Atassi, 1973), as observed for pore-forming β-barrel toxins. A characteristic feature of ε-toxin is its potent neurotoxicity, which is not observed for other structurally well-defined pore-forming toxins (Miyata et al, 2001). Another characteristic of ε-toxin is the activation of the inactive precursor (ε-protoxin) by proteases such as trypsin, chymotrypsin (Hunter et al, 1992), and λ-protease produced by C. perfringens.…”

Clostridium perfringens ε-Toxin Forms a Heptameric Pore within the Detergent-insoluble Microdomains of Madin-Darby Canine Kidney Cells and Rat Synaptosomes

“…, that encodes ProET (20) was digested with EcoRI and XhoI to abolish the HindIII site in the non-coding region, blunt-ended, and then religated. Two synthetic oligonucleotides, 5Ј-TATGGGCCATCATCATCATCATC-ACAGCAGCGGCATCGAAGGTCGTATGAGACGTGCGTCTGTTAA-3Ј (sense; the His 6 tag, factor Xa, and protein kinase A recognition sequences are underlined, doubly underlined, and italicized, respectively) and 5Ј-AGCTTTAACAGACGCACGTCTCATACGACCTTCGATGCCG-CTGCTGTGATGATGATGATGATGGCCCA-3Ј (antisense), were annealed and cloned into the above plasmid, which had been digested with NdeI and HindIII.…”

“…This toxin accumulates mainly in the brain and kidneys when intravenously injected into rats (14), causing injury to neuronal cells (15)(16)(17) and cerebral blood vessels (18). One characteristic feature of the toxin is its extraordinarily high potency; 20 ng of ET kills a mouse within 1 h. 2 We have previously shown that proteolytic activation of ⑀-protoxin (ProET) is due to the removal of a C-terminal peptide (19,20) and that activated ET forms a heptamer in rat synaptosomal membranes (20). Similar ET oligomerization has been reported for Madin-Darby canine kidney (MDCK) cell membranes, where the potassium ion permeability is increased by the toxin (21).…”

Cinética dos anticorpos de origem colostral contra a toxina épsilon de Clostridium perfringens tipo D em cordeiros

“…A prototoxina épsilon utilizada como antígeno no ELISA Indireto, cedida pelo Laboratório de Enfermidades Infecciosas dos Animais da Faculdade de Medicina Veterinária de Araçatuba, UNESP Araçatuba, SP, produzida in house (Veschi 2006) teve sua toxigenicidade avaliada pela observação de sintomatologia de camundongos Balb-c (peso médio 17-20g) inoculados via endovenosa com diferentes diluições da prototoxina ativada com tripsina 0,05% (Miyata et al 2001), por eletroforese em gel de poliacrilamida 12 % (SDS--PAGE) para caracterização da fração protéica (Asubel et al 1994) e pela neutralização com antitoxina homóloga.…”