SummaryClostridium botulinum and Clostridium tetani , respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus. Neurotoxin synthesis is a regulated process in Clostridium

The translocation domain (T domain) of the diphtheria toxin contributes to the transfer of the catalytic domain from the cell endosome to the cytosol, where it blocks protein synthesis. Translocation is initiated when endosome acidification induces the interaction of the T domain with the membrane of the compartment. We found that the protonation of histidine side chains triggers the conformational changes required for membrane interaction. All histidines are involved in a concerted manner, but none is indispensable. However, the preponderance of each histidine varies according to the transition observed. The pair His 223 -His 257 and His 251 are the most sensitive triggers for the formation of the molten globule state in solution, whereas His 322 -His 323 and His 251 are the most sensitive triggers for membrane binding. Interestingly, the histidines are located at key positions throughout the structure of the protein, in hinges and at the interface between each of the three layers of helices forming the domain. Their protonation induces local destabilizations, disrupting the tertiary structure and favoring membrane interaction. We propose that the selection of histidine residues as triggers of membrane interaction enables the T domain to initiate translocation at the rather mild pH found in the endosome, contributing to toxin efficacy.

Production of botulinum neurotoxin A (BoNT/A) and associated non-toxic proteins (ANTPs), which include a non-toxic non-haemagglutinin (NTNH/A) as well as haemagglutinins (HAs), was found previously to be dependent upon an RNA polymerase alternative sigma factor (BotR/A). Expression of the botR/A, bont/A and antp genes, monitored by reverse transcription and real-time PCR analysis, occurred concomitantly at the transition between the exponential and stationary growth phases of Clostridium botulinum A. The botR/A expression level was about 100-fold less than those of the bont/A and antp genes. Therefore, BotR/A is an alternative sigma factor controlling the botulinum A locus genes during the transition phase. The highest toxin concentration was released into the culture supernatant 12 h after maximum expression of the botR/A, bont/A and antp genes, without any apparent bacterial lysis. Toxin levels were then stable over 5 days in cultures at 37 6C, whereas a dramatic decrease in lethal activity was observed between 24 and 48 h in cultures at 44 6C. High temperature did inhibit transcription, since expression levels of the botR/A, bont/A and antp genes were similar in cultures at 37 and 44 6C. However, incubation at 44 6C triggered a calcium-dependent protease that degraded BoNT/A and NTNH/A, but not HAs. In C. botulinum E, which contains no gene related to botR, the bont/E and p47 genes were also expressed during the transition phase, and no protease activation at 44 6C was evident.

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...