Toxins A and B from Clostridium difficile are single-chain protein toxins, which are the major pathogenicity factors of the antibiotic-associated pseudomembranous colitis. Both toxins act identically as enzymes which mono-glucosylate intracellular small GTPases of the Rho subfamily. Rho-GTPases are the master regulators of the actin cytoskeleton. Whereas the cytotoxic activity of these toxins can be fully explained by their inherent enzyme activity, the associated clinical features of pseudomembranous coliltis cannot: secretory diarrhoea and pronounced inflammation of the colonic mucosa.

Up to now, we have provided evidence that both toxins are capable to cause a small spectrum activation of pro-inflammatory signal pathways in the colonic cell line Caco-2 as well as in the human mast cell line HMC--1. Preferentially, p38 MAP kinase is activated. Mast cells are directly stimulated to release inflammatory mediators. Toxin A-caused alteration of gene expression has been checked by DNA micro arrays of Caco-2 showing a change of only few genes. Strongest change observed was the 15-fold up-regulation of the RhoB-GTPase, which was activated simultaneously. Based on these data the paradigm is disproved that toxin A is an exclusive inhibitor of Rho-GTPases. In the meantime we have established the generation of fully functional recombinant holo-toxin A which is the basis for the creation of mutant holo-toxin A.

Based on our current findings the following issues are to be addressed:

1. Which mechanism do the toxins use to stimulate the stress-activated signal cascades (p38 MAPK) and what is the functional implication for the target cell?

2. What is the functional relevance of the immediate–early gene product RhoB strongly up-regulated as response to the toxins? Especially the aspect of cellular defense has to be considered covering RhoB function in apoptosis and inhibition of NfkappaB. We assume that the toxins exhibit rather an anti- than a pro-inflammatory activity at the level of colonocytes.

3. The relevance of our in-vitro findings is to be checked in a mouse model. Firstly, the effects of toxin A on primary colonocytes prepared from mouse gut with respect to gene expression and alteration of signal pathways has to be analysed. Then, these issues are addressed to the intact (mouse)colon by intra-gut application of toxin A to induce pseudomembranous colitis; enzyme-deficient toxin A is compared with wild type one. The general goal is to understand the mechanism how toxin A induces inflammation of the colon.