Functional characterization of an extended binding component of the actin-ADP-ribosylating C2 toxin detected in Clostridium botulinum strain (C) 2300

Abstract

Clostridium botulinum C2 toxin consists of the binding component C2II and the enzyme component C2I, which ADP-ribosylates G-actin of eukaryotic cells. Trypsin-activated C2II (C2IIa) forms heptamers that mediate cell binding and translocation of C2I from acidic endosomes into the cytosol of target cells. By genome sequencing of C. botulinum strain (C) 2300, we found that C2II from this strain carries a C-terminal extension of 129 amino acids, unlike its homologous counterparts from strains (C) 203U28, (C) 468, and (D) 1873. This extension shows a high similarity to the C-terminal receptor-binding domain of C2II and is presumably the result of a duplication of this domain. The C2II extension facilitates the binding to cell surface receptors, which leads to an increased intoxication efficiency compared to that of C2II proteins from other C. botulinum strains.

FIG. 1.

Alignment of amino acid sequences of C2II from Clostridium botulinum strains (C) 2300, (C) 203U28, (C) 468, and (D) 1873. Three characters are used to mark conserved positions: an asterisk indicates positions which have a single, fully conserved residue; a colon indicates that one of the “strong” STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW groups is fully conserved; a period indicates that one of the “weaker” CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY groups is fully conserved. These are all the positively scoring groups that occur in the Gonnet Pam 250 matrix. The strong and weak groups are defined by a score of >0.5 and a score of <0.5, respectively. The alignment was done by ClustalX (21).

FIG. 2.

Nucleotide sequences of C2II genes near the prior stop codon (∼position 2166) of Clostridium botulinum strains (C) 2300, (C) 92-13, (C) 203U28, and (C) 468. The additional nucleotides are marked by gray boxes. Stop codons are marked by a black box.

FIG. 3.

Alignment of three amino acid sequences of C2II from Clostridium botulinum strain (C) 2300 and from Clostridium botulinum strain (C) 203U28. The amino acid sequence designated 2300ex from C2II of Clostridium botulinum strain (C) 2300 (amino acids 716 to 847) represents the C-terminal extension of C2II. The amino acid sequence designated 2300 is derived from the same strain and covers the amino acids 586 to 720. The amino acid sequence designated 203U28 is the C-terminal part of C2II of Clostridium botulinum (C) strain 203U28. This alignment was done with ClustalX (21). Three characters are used to mark conserved positions: an asterisk indicates positions which have a single, fully conserved residue; a colon indicates that one of the “strong” STA, NEQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, and FYW groups is fully conserved; a period indicates that one of the “weaker” CSA, ATV, SAG, STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, and HFY groups is fully conserved. These are all the positively scoring groups that occur in the Gonnet Pam 250 matrix. The strong and weak groups are defined by a score of >0.5 and a score of <0.5, respectively.

FIG. 4.

Time course of the intoxication of HeLa cells with C2 toxin possessing the long (C2IIa) or short (ΔC2IIa) version of the binding component. HeLa cells were treated with 430 pM C2I and 1 nM C2IIa or ΔC2IIa. As a control, HeLa cells were treated with 430 pM C2I alone. Cells were fixed after the indicated times, and the actin cytoskeleton was stained by TRITC-phalloidin (red). The nuclei were stained by DAPI (blue). Bars, 30 μm.

FIG. 5.

Cell intoxication by C2 toxin possessing the long (C2IIa) or short (ΔC2IIa) version of the binding component. (A) Human colon carcinoma (CaCo-2) cell monolayers were treated with 215 pM C2I and 500 pM C2IIa (▪) or ΔC2IIa (▵). Reduction of transepithelial resistance (TER) due to cell rounding over time (min) served as a parameter for intoxication. Results are mean values (±SD) of results for at least three independent experiments and are shown as percentages of starting TER. Controls are buffer (⋄) or C2I alone (⧫). Asterisks indicate significant differences (**, P < 0.02; ***, P < 0.001) calculated by analysis of variance (ANOVA). (B) CHO-K1 (▪, ▴) and CHO-RK14 (□, ▵) cells were treated with C2IIa (▪, □) or ΔC2IIa (▴, ▵) at the indicated concentrations. C2I was added to C2IIa or ΔC2IIa at a ratio of 3:7. Cells were further incubated at 37°C, and after 3 h, pictures were taken. The percentage of round cells per field (∼300 cells/field) is given as the mean ± SD (n = 3).

FIG. 6.

Comparison of pore formation by C2IIa and ΔC2IIa in HT-29 cells. Cell monolayers in 24-well culture plates were preloaded with ⁸⁶Rb⁺ (1 μCi/ml) for 48 h and, thereafter, washed twice with PBS. Subsequently, C2IIa (▪) or ΔC2IIa (▵) was allowed to bind at 4°C for 45 min. As a control, 20 nM C2I (⋄) was used. Afterwards, cells were exposed for 5 min to an acidic shift (pH 4.8) at 37°C to initiate membrane insertion of C2II and ΔC2IIa. After an additional incubation of 40 min at 4°C, the radioactivity in the medium was measured by using liquid scintillation counting. Asterisks indicate significant differences (**, P < 0.02; ***, P < 0.001) calculated by ANOVA.