Clostridium difficile, the causative agent of nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis, possesses two main virulence factors: the large clostridial cytotoxins A and B. It has been proposed that toxin B is cleaved by a cytosolic factor of the eukaryotic target cell during its cellular uptake. Here we report that cleavage of not only toxin B, but also all other large clostridial cytotoxins, is an autocatalytic process dependent on host cytosolic inositolphosphate cofactors. A covalent inhibitor of aspartate proteases, 1,2-epoxy-3-(p-nitrophenoxy)propane, completely blocked toxin B function on cultured cells and was used to identify its catalytically active protease site. To our knowledge this is the first report on a bacterial toxin that uses eukaryotic signals for induced autoproteolysis to deliver its toxic domain into the cytosol of target cells. On the basis of our data, we present an integrated model for the uptake and inositolphosphate-induced activation of toxin B.

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Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III. Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.Recently, the genetic element CdISt1 was identified in the genome of Clostridium difficile strain C34 (1, 5) and was shown to combine features of group I introns and insertion sequence (IS) elements. To distinguish between individual CdISt1 variants from different strains, the strain C34 element is herein designated CdISt1-C34, since the designation CdISt1 includes all variants combining features of the group I intron and the IS elements of the IS605 type. The 434 bp at the 5Ј end of CdISt1-C34 show the typical structures and key features of group I introns, while the 3Ј part harbors two open reading frames (ORFs) coding for a truncated (tlpA) and a complete (tlpB) protein. The putative proteins TlpA and TlpB show high homology to transposases, which are characteristic products of composite IS elements of the IS605 type ( Fig. 1) (4). In contrast to classical group I introns, CdISt1-C34 is found in proteinencoding genes of a bacterial chromosome (1, 2). Sequence analysis of CdISt1-C34, several of its variants (CdISt1a-C34 through CdISt1j-C34), and their integration sites revealed that these genetic elements show all features characteristic of an IS605-like mobility mechanism (1, 3). Therefore, we assumed that the IS element component mediates the spread of CdISt1 while the intron component is responsible for efficient splicing of CdISt1 from precursor mRNA. Due to their chimeric nature, we called these elements IStrons.Previously, two types of IStrons (CdISt1-I and CdISt1-II) were identified in C. difficile, and these types differ in the IS element component (Fig. 1) (1). We have grouped all characterized CdISt1 IStron variants into four types (CdISt1-0 to CdISt1-III) based on their structural features. Within each type, the variants differ in their genomic integration sites and the strains in which they are located. Compared to CdISt1-I IStrons, CdISt1-II IStrons show a deletion of about 100 bp covering nearly the entire tlpA gene (Fig. 1). To verify the proposal that CdISt1 was generated by fusion of a group I intron and an IS element from the IS605 family, we searched for additional CdISt1 types without deletion of the IS element component. C. difficile isolates were screened for CdISt1-related sequences by using two internal primers (P1, CGACAA CCTCAAAAATGATAAA, and P2, TCTTAATCCTTCTTT TAATATATTT) that amplify a major part of the CdISt1 sequence. PCR conditions were as follows: 30 cycles of...

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