0000000000466380
AUTHOR
Maja Rupnik
Autocatalytic cleavage of Clostridium difficile toxin B.
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 prote…
Clostridium difficile IStron CdISt1: Discovery of a Variant Encoding Two Complete Transposase-Like Proteins
ABSTRACT Screening a Clostridium difficile strain collection for the chimeric element Cd ISt1 , we identified two additional variants, designated Cd ISt1 -0 and Cd ISt1 -III. In in vitro assays, we could prove the self-splicing ribozyme activity of these variants. Structural comparison of all known Cd ISt1 variants led us to define four types of IStrons that we designated Cd ISt1 -0 through Cd ISt1 -III. Since Cd ISt1 -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.
A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X.
Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene. To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470. Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots. Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed. In addition, this fragment showed no …
Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile.
We have used six independent polymerase chain reactions (A1–A3 and B1–B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains. With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA. Characteristic differences between strains were mainly focused to the 5′ third of tcdB (B1 fragment) and/or the 3′ third of tcdA (A3 fragment). The possible use of our approach for typing of C. difficile toxin genes is discussed.
A chimeric ribozyme in Clostridium difficile combines features of group I introns and insertion elements
CdlSt1, a DNA insertion of 1975 bp, was identified within tcdA-C34, the enterotoxin gene of the Clostridium difficile isolate C34. Located in the catalytic domain A1-C34, Cd/St1 combines features of two genetic elements. Within the first 434 nt structures characteristic for group I introns were found; encoding the two transposase-like proteins tlpA and tlpB nucleotides 435-1975 represent the remainder of a IS605-like insertion element. We show that the entire CdlSt1 is accurately spliced from tcdA-C34 primary transcripts and that purified TcdA-C34 toxin is of regular size and catalytic activity. A search for CdlSt1-related sequences demonstrates that the element is widespread in toxinogenic…
Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells
Clostridium difficiletoxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB10463between Leu543and Gly544and the naturally occurring variant TcdB8864between Leu544and Gly545. The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB10463) and 62 761 Da (TcdB8864)] harbour the cytotox…