Clostridium sordellii lethal toxin is maintained in a multimeric protein complex.
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.
Voth
Daniel E
DE
Department of Botany and Microbiology, The University of Oklahoma, Norman, Oklahoma 73019, USA.
Qa'Dan
Maen
M
Hamm
Elaine E
EE
Pelfrey
Joy M
JM
Ballard
Jimmy D
JD
D001427:Bacterial Toxins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity*; D003013:Clostridium / Q000472:pathogenicity*; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D016695:Glycosyltransferases / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight
D001427:Bacterial Toxins; C072292:lethal toxin LT, Clostridium sordellii; D016695:Glycosyltransferases; D010450:Endopeptidases