Clostridium sordelliiLethal 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.

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

“…More recently, it was proposed that lethal toxin forms high molecular complexes, which dissociate at low pH and reassociate after an increase in pH (31). We suggest that these studies performed with native lethal toxin purified from C. sordellii might be biased by pH-dependent impurities, which could affect complex formation.…”

The AlternativeTranslational Profile That Underlies the Immune-Evasive State of Persistence in Chlamydiaceae Exploits Differential Tryptophan Contents of the Protein Repertoire

“…Among the chlamydial organisms, the absolute number of Trp residues utilized (Trp burden) for cytotoxin input by C. muridarum is truly amazing, since the three adjacent paralogs require a total of 124 Trp residues. This enormous demand for Trp input might be amplified even further, to the extent that the cytotoxin might be multimeric, as has been reported for the large clostridial cytotoxin of Clostridium sordellii (131).…”

Bacillus anthracis oedema toxin as a cause of tissue necrosis and cell type-specific cytotoxicity

“…Both RAW 264.7 and IC-21 cells were pre-treated for 1 h with a pan-caspase inhibitor, ZVAD-FMK, before treatment with OT. As shown in , a fusion protein that consists of LFn (the PA-binding domain of LF) fused to the enzymatic domain of Clostridium sordellii lethal toxin and a potent inducer of apoptosis (Voth et al ., 2004), revealed activation of both caspase-3 and PARP. Finally, in line with these results, TUNEL staining for 3 ¢ nicked DNA did not reveal any notable chromosomal fragmentation in OT-treated macrophages (Fig.…”

“…Following overnight induction, the proteins were purified using nickel affinity chromatography according to the manufacturer's instructions (Novagen). As previously described (Voth et al, 2004), LFn/TcsL 1-556 was expressed from pMQ1 and also isolated by nickel affinity chromatography.…”