title: A non-haemagglutinating form of Clostridium difficile toxin A.
abstract: Analysis of crude culture filtrate of Clostridium difficile by Mono Q-anion exchange fast protein liquid chromatography (FPLC) demonstrated that toxin A had distinct peaks of activity for cytotoxicity and haemagglutination, as also did highly purified toxin A obtained by thyroglobulin affinity chromatography (TG) followed by two sequential anion-exchange chromatographic steps with Q-Sepharose FF and Mono Q. From TG unbound fractions a highly cytotoxic but weakly haemagglutinating variant (toxin A') of toxin A was obtained by Q-Sepharose FF and Mono Q chromatography. Analysis of toxins A and A' from cultures of C. difficile in a chemically defined medium, and of toxin A dialysed against brain heart infusion broth, indicated that A' was not merely toxin A coupled to a component of the growth medium. Polyacrylamide gel electrophoresis under non-denaturing conditions showed that toxins A and A' had the same Mr. Immunoblotting with mouse monospecific A antitoxin showed that five bands larger than the major 240-Kda band were more strongly developed in toxin A than in A' in denaturing but non-reducing conditions, and in reducing conditions eight bands (38-175 Kda) were seen in toxin A but not A'. Immunoblotting with a monoclonal antibody (PCG-4) showed that, in both reducing and non-reducing conditions, two bands of 160 and 155 Kda were more prominent in toxins A and A' respectively, and four bands (195, 180, 175 and 125 Kda) were detected only in toxin A'.
authors.affiliation: Microbial Pathogenicity Research Group, MRC Clinical Research Centre, Harrow, Middlesex.
authors.forename: S
authors.initials: S
authors.lastname: Kamiya
authors.forename: S P
authors.initials: SP
authors.lastname: Borriello
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D016360:Clostridioides difficile; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D006384:Hemagglutination / Q000187:drug effects*; D015151:Immunoblotting; D014709:Vero Cells
chemical_list: D001427:Bacterial Toxins; D004768:Enterotoxins; C041348:tcdA protein, Clostridium difficile