The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality

• Published in: FEMS microbiology letters Vol. 155; no. 1; pp. 45 - 54
• Main Authors: Sauerborn, Markus, Leukel, Petra, von Eichel-Streiber, Christoph
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.1997; Oxford University Press
• Subjects: Affinity chromatography; Animals; Bacterial Toxins; Binding; Binding Sites; Biocompatibility; Carbohydrate Sequence; Carbohydrates; Cattle; Cell Line; Chemical compounds; Clostridium difficile; Clostridium difficile - genetics; Clostridium difficile - metabolism; Clostridium difficile - pathogenicity; Cytotoxicity; Enterotoxins - genetics; Enterotoxins - metabolism; Enterotoxins - toxicity; Female; Galactose; Immunization; Immunofluorescence; In Vitro Techniques; Intoxication; Lectins; Lectin‐like domain; Lethal dose; Lethality; Ligands; Mice; Mice, Inbred BALB C; Microbiology; Molecular Sequence Data; Monoclonal antibodies; Neutralization Tests; Pharmacology; Receptor binding; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Recombinant Proteins - toxicity; Teratocarcinoma; Teratocarcinoma cells; Thyroglobulin; Thyroglobulin - chemistry; Thyroglobulin - metabolism; Toxicity; Toxin A; Toxin B; Toxins; Vaccines; Lectin-like domain; Toxin A; Immunization; Receptor binding
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1111/j.1574-6968.1997.tb12684.x

Abstract We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Lex-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Lex as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.