New method to generate enzymatically deficient Clostridium difficile toxinB as an antigen for immunization

The family of the large clostridial cytotoxins, encompassing Clostridium di fficile toxins A and B as well as the lethal and hemorrhagic toxins from Cl ostridium sordellii, monoglucosylate the Rho GTPases by transferring a gluc ose moiety from the cosubstrate UDP-glucose, Here we present a new detoxifi cation procedure to block the enzyme activity by treatment with the reactiv e UDP-2',3'-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alky lated toxin competes with the native toxin at the cell receptor, Alkylated toxins are good antigens resulting in antibodies recognizing only the C-ter minally located receptor binding domain, whereas formaldehyde treatment res ulted in antibodies recognizing both the receptor binding domain and the ca talytic domain, indicating that the catalytic domain is concealed under nat ive conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic do main inhibited enzyme activity. However, only antibodies against the recept or binding domain protected intact cells from the cytotoxic activity of tox in B, whereas antibodies against the catalytic domain mere protective only when inside the cell.