THE ENZYMATIC DOMAIN OF CLOSTRIDIUM-DIFFICILE TOXIN-A IS LOCATED WITHIN ITS N-TERMINAL REGION

Clostridium difficile, an anaerobic pathogen encountered in human ente ric disease, produces two major virulence factors, toxins A and B, whi ch are members of a clostridial family of large cytotoxins. These are glucosyltransferases, which use a UDP-sugar as co-substrate to glucosy late and inactivate small GTPases of the Rho or Ras families, culminat ing in cytotoxicity. Clinically, toxin A is perhaps the most important family member, because it causes major tissue damage in the course of disease, leading to a potentially lethal, pseudomembranous colitis. T he location of the enzymatic domain of toxin A and mechanistic details of its action are not yet known, so we wished to localize this domain using gene deletion constructions from the full-length gene and by mo nitoring glucosylation activity of encoded protein products. Toxin A d eletions were obtained by successively truncating the C-terminal codin g region. These were transformed into E. coil, cell lysates were prepa red and they were assayed for their ability to glucosylate Rho A prote in, using an in vitro enzymatic assay. We report that the UDP-glucose binding site, the catalytic site for glucose transfer and the Rho A in teraction site occur within the first 659 N-terminal amino acids of to xin A, i.e., less than 25% of the length of holotoxin A. Localization of the enzymatic domain of toxin A to these 659 N-terminal amino acids should greatly simplify studies on mechanistic details of this clinic ally important toxin. (C) 1998 Academic Press.