Clostridium sordellii lethal toxin is maintained in a multimeric protein complex

Abstract

Clostridium sordellii lethal toxin (TcsL) is distinct among large clostridial toxins (LCTs), as it is markedly reduced in its rate of intoxication at pH 8.0 yet is cytotoxic at pH 4.0. Results from the present study suggest that TcsL's slow rate of intoxication at pH 8.0 is linked to formation of a high-molecular-weight complex containing dissociable pH 4.0-sensitive polypeptides. The cytosolic delivery of TcsL's enzymatic domain by using a surrogate cell entry system resulted in cytopathic effect rates similar to those of other LCTs at pH 8.0, further indicating that rate-limiting steps occurred at the point of cell entry. Since these rate-limiting steps could be overcome at pH 4.0, TcsL was examined across a range of pH values and was found to dissociate into distinct 45- to 55-kDa polypeptides between pH 4.0 and pH 5.0. The polypeptides reassociated when shifted back to pH 8.0. At pH 8.0, this complex was resistant to sodium dodecyl sulfate (SDS) and multiple proteases; however, following dissociation, the polypeptides became protease sensitive. Dissociation of TcsL, and cytotoxicity, could be blocked by preincubation with ethylene glycol bis(sulfosuccinimidylsuccinate), resulting in cross-linking of the polypeptides. TcsL was also examined at pH 8.0 by using SDS-agarose gel electrophoresis and transmission electron microscopy and was found to exist in a higher-molecular-weight complex which resolved at a size exceeding 750 kDa and also dissociated at pH 4.0. However, this complex did not reassemble following a shift back to pH 8.0. Collectively, these data suggest that TcsL is maintained in a protease-resistant, high-molecular-weight complex, which dissociates at pH 4.0, leading to cytotoxicity.

FIG. 1.

Time course of CPE in TcsL- and LFnTcsL_1-556-treated cells. HeLa cells were grown to confluence in a 96-well plate to ∼5 × 10⁴ cells/well. Cells were treated with either TcdB (100 fmol), TcsL (100 fmol), LFnTcsL_1-556 (1 pmol), or LFnTcdB_1-556 (1 pmol) in a volume of 100 μl. PA (30 pmol) was included with the fusion proteins. Control cells were treated with media, 20 mM Tris, heat-inactivated toxin, or PA plus LFn alone. Following treatment, CPE were observed for 10 h and calculated by visualizing the number of rounded cells from a field of at least 100 cells. Samples were analyzed in triplicate and error bars represent the standard deviation from the mean. ◊, TcdB; □, TcsL; ○, LFnTcdB_1-556; ×, LFnTcsL_1-556.

FIG. 2.

Acid pH-induced dissociation of TcsL. Samples of purified TcsL (10 μg) were incubated for 30 min with various amounts of 100 mM ammonium acetate buffer, pH 3.8, to attain the indicated pH. Proteins were separated via SDS-10% PAGE and observed by using Coomassie blue staining.

FIG. 3.

Impact of pH shifts and cross-linking on TcsL stability. TcsL samples (10 μg) were analyzed via SDS-10% PAGE and observed by using Coomassie blue staining following incubation under various pH conditions. In lanes 3 and 6, pH 4.0 was shifted back to pH 8.0 (4/8). +, TcsL with sulfo-EGS; − TcsL without sulfo-EGS.

FIG. 4.

Toxicity, cell binding, and enzymatic activity of cross-linked TcsL. HeLa cells were incubated with TcsL, TcsL-sulfo-EGS, or TcsL plus TcsL-sulfo-EGS (2 μg/ml) for up to 72 h and observed for morphological changes associated with TcsL-induced cytotoxicity. As a comparison, HeLa cells were also incubated with TcdB or TcdB-sulfo-EGS (2 μg/ml) and observed for morphological changes. TcsL or TcsL-sulfo-EGS was also incubated in a Ras glucosylation assay as described in Materials and Methods. (A) Control untreated cells. (B) Percent viability of HeLa cells treated with TcsL, TcsL-sulfo-EGS, and TcsL plus TcsL-sulfo-EGS. Samples were analyzed in triplicate and error bars indicate the standard deviation from the mean. Inset compares glucosyltransferase activity of TcsL (lane 1) and TcsL-sulfo-EGS (lane 2). (C) TcsL-treated cells. (D) TcsL-sulfo-EGS-treated cells. (E) TcdB-treated cells. (F) TcdB-sulfo-EGS-treated cells.

FIG. 5.

Protease treatment of TcsL. TcsL (10 μg) was incubated with an excess of each indicated protease (15 μg) overnight prior to SDS-PAGE analysis. The pH value is indicated above the gel and each pH section follows this order of protease treatment: Lanes 2 and 7, TcsL; lanes 3 and 8, TcsL plus trypsin; lanes 4 and 9, TcsL plus α-chymotrypsin; lanes 5 and 10, TcsL plus V8 protease; and lanes 6 and 11, TcsL plus proteinase K. Lane 1 shows the molecular mass markers.

FIG. 6.

SDS-AGE analysis of TcsL, TcdB, and Tcnα and TEM analysis of TcsL. (A to C) Each respective toxin was separated by SDS-1.5% AGE following incubation under the indicated pH conditions. Each lane represents 15 μg of protein. TcsL at pH 8.0 (D) and pH 4.0 (E) was stained with PTA and subsequently analyzed by TEM at a magnification of ×10⁵. Arrows indicate examples of oligomeric structures.

FIG. 7.

Electroelution of TcsL and TcsL complex proteins. Protein bands representing TcsL and the TcsL complex were subjected to electroelution in the presence of SDS for 2 h at room temperature. Following electroelution, proteins were separated via SDS-10% PAGE and observed by using Coomassie blue staining. Lane 1, molecular mass markers; lane 2, protein electroeluted from TcsL; lane 3, protein electroeluted from the oligomer.