Inositol Hexakisphosphate-dependent Processing of Clostridium sordellii Lethal Toxin and Clostridium novyi α-Toxin

Abstract

Clostridium sordellii lethal toxin and Clostridium novyi ␣-toxin, which are virulence factors involved in the toxic shock and gas gangrene syndromes, are members of the family of clostridial glucosylating toxins. The toxins inactivate Rho/Ras proteins by glucosylation or attachment of GlcNAc (␣-toxin). Here, we studied the activation of the autoproteolytic processing of the toxins by inositol hexakisphosphate (InsP 6 ) and compared it with the processing of Clostridium difficile toxin B. In the presence of low concentrations of InsP 6 (<1 M), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNActransferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi ␣-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and ␣-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of ␣-toxin) were identified. Affinity of the CPDs for binding InsP 6 was determined by isothermal titration calorimetry. In contrast to fulllength toxin B and ␣-toxin, autocatalytic cleavage and InsP 6 binding of full-length lethal toxin depended on low pH (pH 5) conditions. The data indicate that C. sordellii lethal toxin and C. novyi ␣-toxin are InsP 6 -dependently processed. However, full-length lethal toxin, but not its short toxin fragments consisting of the glucosyltransferase domain and the CPD, requires a pH-sensitive conformational change to allow binding of InsP 6 and subsequent processing of the toxin.

Molecular Characteristics of Clostridium perfringens TpeL Toxin and Consequences of Mono-O-GlcNAcylation of Ras in Living Cells

“…Our study highlights that TpeL also undergoes InsP 6 -dependent autocatalytic processing and that a cysteine protease domain-like region (TpeL543-805), located next to the glucosyltransferase domain, is sufficient for cleavage. Accordingly, the K D value for the interaction of InsP 6 with TpeL543-805, determined by iso- thermal titration calorimetry, was ϳ2.5 M. This sensitivity characteristics are very similar to those of C. difficile toxin B, C. sordellii lethal toxin, and C. novyi ␣-toxin (30).…”

“…4D). Based on the obtained K D values (2.1 and 2.7 M), InsP 6 bound with a similar affinity to the CDP of TpeL when compared with the CPDs of other clostridial glucosylating toxins (30).…”

Identification of the Cellular Receptor of Clostridium spiroforme Toxin

“…The methodology was adapted from protocols that were previously established for the recombinant production of various other clostridial toxins (11,17,20,22). The B. megaterium expression system often offers an advantage over conventional bacterial expression systems (e.g., Escherichia coli) in producing clostridial toxins, obviously due to a similar codon usage of the related genera Bacillus and Clostridium.…”

“…This system was extremely instrumental for expression of members of the family of large glucosylating clostridial toxins, which possess a mass of 250 to 308 kDa and are notoriously poorly expressed in Escherichia coli (22). We previously employed this procedure for recombinant production of the clostridial toxins C. sordellii lethal toxin, C. novyi alpha-toxin, and C. difficile transferase (11,17,20). The current study describes the usefulness of the B. megaterium expression system for recombinant production of CSTa and CSTb.…”

Development of a cyclic voltammetry method for the detection of Clostridium novyi in black disease

“…It may be present on skin surfaces and is a potential source of wound infections (Plomp et al, 2007). The lethal and necrotizing toxins (primarily α toxin) damage hepatic parenchyma, thereby permitting the bacteria to multiply and produce a lethal amount of toxin (Guttenberg et al, 2011). Most of characteristic lesions are the grayish yellow, necrotic foci in the liver that often follow the migratory tracks of the young flukes.…”