Autoproteolytic cleavage mediates cytotoxicity of Clostridium difficile toxin A

• Published in: Naunyn-Schmiedeberg's archives of pharmacology 2011-03, Vol.383 (3), p.253-262
• Main Authors: Kreimeyer, Isa, Euler, Friederike, Marckscheffel, Alexander, Tatge, Helma, Pich, Andreas, Olling, Alexandra, Schwarz, Janett, Just, Ingo, Gerhard, Ralf
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - physiology; Animals; Apoptosis - drug effects; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bacterial Proteins - pharmacology; Bacterial Toxins - chemistry; Bacterial Toxins - metabolism; Bacterial Toxins - pharmacology; Biomedical and Life Sciences; Biomedicine; Caspase 3 - metabolism; Caspase 8 - metabolism; cdc42 GTP-Binding Protein - metabolism; Cell Membrane - metabolism; Cell Shape - drug effects; Clostridium difficile; Cysteine Proteases - metabolism; Cytosol - metabolism; Cytotoxins - chemistry; Cytotoxins - metabolism; Cytotoxins - pharmacology; Dithiothreitol - chemistry; Dose-Response Relationship, Drug; Enterotoxins - chemistry; Enterotoxins - metabolism; Enterotoxins - pharmacology; Glucosyltransferases - metabolism; Glycosylation; HT29 Cells; Humans; Mice; Neurosciences; NIH 3T3 Cells; Original Article; Peptide Fragments - metabolism; Pharmacology/Toxicology; Phytic Acid - chemistry; Protein Structure, Tertiary - physiology; rac1 GTP-Binding Protein - metabolism; Recombinant Proteins - metabolism; rhoA GTP-Binding Protein - metabolism
• ISSN: 0028-1298; 1432-1912
• DOI: 10.1007/s00210-010-0574-x

Toxin A and toxin B from Clostridium difficile are the causative agents of the antibiotic-associated pseudomembranous colitis. They are of an A/B structure type and possess inositol hexakisphosphate-inducible autoproteolytic activity to release their glucosyltransferase domain to the cytoplasm of target cells. In this study, we investigated the effect of extracellular and intracellular autoproteolytic cleavage on the function of TcdA. Extracellular cleavage led to functional inactivation albeit TcdA was less susceptible to inositol hexakisphosphate-induced autoproteolysis than TcdB. A non-cleavable TcdA mutant (TcdA A541 G542 A543) was generated to investigate whether autoproteolysis is a prerequisite for intracellular function of TcdA. Although the EC 50 regarding cell rounding was about 75-fold reduced in short-term assay, non-cleavable TcdA was able to induce complete cell rounding and apoptosis after 36 h comparable to wildtype TcdA when continuously present. Studies with limited uptake of toxins revealed progressive Rac1 glucosylation and complete cell rounding for TcdA, whereas the effect induced by non-cleavable TcdA was reversible. These findings argue for cytosolic accumulation of the released glucosyltransferase domain of wild-type TcdA and rapid degradation of the non-cleavable TcdA. In summary, extracellular cleavage functionally inactivates TcdA (and TcdB), whereas intracellular autoproteolytic cleavage is not essential for function of TcdA but defines its potency.