Inositol hexakisphosphate-dependent processing of Clostridium sordellii lethal toxin and Clostridium novyi alpha-toxin

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.

FIGURE 1.

Comparative analysis of CGT fragments comprising the GD and the putative CPD. A, InsP₆-induced in vitro cleavage of CGT fragments comprising the GD and the adjacent CPD. The toxin fragments were expressed as recombinant proteins in B. megaterium and purified. Processing of CGT fragments (2 μg) was induced with increasing concentrations of InsP₆ as indicated and incubation for 1 h at 37 °C. B, alignment of primary sequences of the CGTs representing the putative autocatalytic cleavage site (↓) and the conserved catalytic cysteine residue of the cysteine protease homology region (shown in boldface). C, the amino acids highlighted in B were substituted in each CGT fragment with alanine and analyzed for autocatalytic processing in the presence or absence of 100 μm InsP₆ and incubated for 1 h at 37 °C. D, in vitro glucosylation of Rac1 with CGT fragments and mutants shown in C in the presence of radiolabeled UDP-glucose or UDP-GlcNAc (α-toxin), respectively. Samples were subjected to SDS-PAGE, and modified Rac1 was visualized by autoradiography.

FIGURE 2.

Binding affinity of InsP₆ for the CGT CPDs determined by isothermal calorimetry. The respective CPDs of the CGTs were expressed as recombinant proteins in B. megaterium and purified prior to analysis of the binding of InsP₆ at pH 7.4 by isothermal calorimetry. A, toxin B-(544–807); B, lethal toxin-(544–807); C, α-toxin-(549–813).

FIGURE 3.

Functional characterization of recombinant CGTs. A, C. difficile toxin B, C. sordellii lethal toxin, and C. novyi α-toxin were purified from B. megaterium and subjected to SDS-PAGE, and separated proteins were stained with Coomassie Blue. The bands indicated by arrowheads represent the respective purified toxins. B, in vitro glucosylation of Rac1, Ha-Ras, and RhoA with recombinant CGTs. C, intoxication of Vero cells with recombinant CGTs. Images were obtained after overnight (control (mock)), 1-h (100 pm toxin B), 6-h (1 nm lethal toxin), and overnight (1 nm α-toxin) treatment. D, InsP₆-induced in vitro cleavage of recombinant CGTs. Processing of CGTs (2 μg) was induced with increasing concentrations of InsP₆ as indicated and incubation for 1 h at 37 °C. E, processing of CGTs (2 μg) was induced with 100 μm InsP₆, followed by an incubation period as indicated at 37 °C.

FIGURE 4.

Intoxication of cultured Vero cells with CPD-inactive toxin mutants. A, cultured Vero cells were incubated with wild-type toxins (100 pm toxin B, 1 nm lethal toxin, and 100 pm α-toxin) or CPD-inactive versions (100 pm C698A-toxin B, 1 nm C698A-lethal toxin, and 100 pm C707A-α-toxin), and images were obtained after 1 h (toxin B/C698A-toxin B) and 6 h (lethal toxin/C698A-lethal toxin and α-toxin/C707A-α-toxin). B, in vitro cleavage assay. The wild-type and mutant toxins (2 μg each) shown in A were incubated for 1 h at 37 °C with 100 μm (toxin B/C698A-toxin B and lethal toxin/C698A-lethal toxin) or 1 mm (α-toxin/C707A-α-toxin) InsP₆, followed by SDS-PAGE and visualization of proteins by Coomassie Blue staining. FL, full-length. C, time-dependent in vitro glucosylation of Rac1. 10 nm lethal toxin/C698A-lethal toxin or 10 nm α-toxin/C707A-α-toxin was incubated with 5 μm Rac1 in the presence of radiolabeled UDP-glucose or UDP-GlcNAc (α-toxin), respectively. Samples were taken at the indicated time points and subjected to SDS-PAGE, and modified Rac1 was visualized by autoradiography.

FIGURE 5.

Influence of pH in binding of InsP₆ to lethal toxin for induction of autocatalytic processing. A, filter binding assay. 1 μm recombinant CGT protein was incubated with [³H]InsP₆ for 10 min at 4 °C. Thereafter, the mixture was loaded onto nitrocellulose filters, and after extensive washing, bound radioactivity was measured by liquid scintillation counting. The GD of toxin B (toxin B-(1–546)) was included in the assay as a negative control. B, InsP₆-induced in vitro cleavage of lethal toxin at low pH. Processing of full-length toxin and GD-CPD fragments of lethal toxin was induced at pH 5 with increasing concentrations of InsP₆ as indicated and incubation for 1 h at 37 °C. C, the filter binding assay (in the presence of [³H]InsP₆) shown in A was repeated with lethal toxin and toxin B-(1–546) (negative control) at pH 5. D, the filter binding assay shown in A was performed with full-length toxin B and full-length lethal toxin under various pH conditions as indicated. The highest InsP₆-binding values for each toxin were set to 100%. E, filter binding assay in the presence of [³H]InsP₆ with lethal toxin and toxin B at pH 7.4 after lowering the pH of the mixture to pH 5 and after incubation of the toxins at pH 5 for 10 min, followed by readjustment from pH 5 to pH 7.4. Bound radioactivity is given as a percent of maximal binding (100%). F, isothermal calorimetry of the CPD of lethal toxin and InsP₆ at pH 5. Error bars in A and C–E represent S.D. (n ≥ 3).