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Clostridium difficile toxin A, the chief pathogenicity factor of the antibiotic-associated pseudomembranous colitis, is an intracellular acting cytotoxin that reaches its targets, the Rho GTPases, after receptor-mediated endocytosis. The C-terminal part, constructed of repetitive peptide elements, is thought to bind to a lot of carbohydrate containing receptor molecules to induce clustering and endocytosis. To study which part of the receptor-binding domain is in charge of addressing toxin A into the target cells, we studied the functional, i.e., endocytosis-inducing, binding of toxin A. By a competition assay between various receptor-binding fragments of toxin A and the holotoxin A we found that the complete receptor-binding domain, encompassing the entire repetitive elements, but not parts of it, is necessary for binding-induced endocytosis. The receptor binding domain itself shows weaker competition with holotoxin A than the fragment consisting of receptor-binding domain plus intermediary part of the toxin. All toxin A fragments that compete with holotoxin A are capable of inducing their own endocytosis. Thus, the entire receptor-binding domain, covering the C-terminal third of the toxin A molecule, is responsible for cell uptake of toxin A and the intermediary part contributes to the correct folding and assembly of the repetitive domains.

Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B. We used the Bacillus megaterium expression system for expression of recombinant toxin A. The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector. The three parts were sequentially fused to the complete gene. The holotoxin gene was ligated into the expression vector pWH1520. This vector was modified to generate a toxin with a C-terminally located His-tag. Gene expression in the B. megaterium system resulted in an approximate 300 kDa protein, which was identified by specific antibody as toxin A. Recombinant, His-tagged toxin A was purified by Ni 2+ as well as thyroglobulin affinity chromatography. Characterization of the recombinant toxin A showed identical cytotoxicity and in vitro-glucosyltransferase activity as the native toxin A from C. difficile .

Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-γ-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-γ-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho–PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.