Abstract
Clostridium sordellii lethal toxin and Clostridium novyi α-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 (α-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 μM), toxin fragments consisting of the N-terminal glucosyltransferase (or GlcNAc-transferase) domains and the cysteine protease domains (CPDs) of C. sordellii lethal toxin, C. novyi α-toxin, and C. difficile toxin B were autocatalytically processed. The cleavage sites of lethal toxin (Leu-543) and α-toxin (Leu-548) and the catalytic cysteine residues (Cys-698 of lethal toxin and Cys-707 of α-toxin) were identified. Affinity of the CPDs for binding InsP(6) was determined by isothermal titration calorimetry. In contrast to full-length toxin B and α-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 α-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.
Highlights
Various pathogens of the genus Clostridium produce highly potent glucosylating toxins, clostridial glucosylating toxins (CGTs),3 which act on host target cells by modification and inactivation of Rho and Ras GTPases (1–5)
cysteine protease domain (CPD) of C. sordellii Lethal Toxin and C. novyi ␣-Toxin Are Responsible for Autocatalytic Processing—Studies by Pruitt et al (21) delimited the CPD of toxin A to amino acids 543– 809
To study the autocatalytic cleavage of the toxins, we expressed toxin fragments consisting of the glucosyltransferase domain (GD) and the putative CPDs from various CGTs (toxin B-(1– 807), lethal toxin-(1– 807), and ␣-toxin-(1
Summary
Various pathogens of the genus Clostridium produce highly potent glucosylating toxins, clostridial glucosylating toxins (CGTs), which act on host target cells by modification and inactivation of Rho and Ras GTPases (1–5). This group of toxins, comprising Clostridium difficile toxins A and B, Clostridium novyi ␣-toxin, and Clostridium sordellii lethal and hemorrhagic toxins, are major virulence factors (6). 3 The abbreviations used are: CGT, clostridial glucosylating toxin; CPD, cysteine protease domain; InsP6, inositol hexakisphosphate; GD, glucosyltransferase domain. Previous studies revealed that autocatalytic processing of CGTs depends on inositol hexakisphosphate (InsP6) (17, 18) It was shown for C. difficile toxins A and B that InsP6 can bind to the intrinsic CPD (20, 21). Binding of InsP6 causes conformational changes in the catalytic center of the CPD, resulting in activation of the protease, cleavage of the toxin molecule between the glucosyltransferase domain and the CPD, and release of the glucosyltransferase into the cytosol (21–23)
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