Genetic rearrangements in the pathogenicity locus of Clostridium difficile strain 8864 – implications for transcription, expression and enzymatic activity of toxins A and B

Abstract

The pathogenicity locus (PaLoc) of Clostridium difficile isolate 8864 was investigated to locate genetic rearrangements that would explain the exceptional pathogenicity of this particular isolate. Two major changes were defined: an insertion of 1.1 kb between the two genes tcdA and tcdE, coding for the enterotoxin and an accessory protein of unknown function, respectively, and a deletion of 5.9 kb encompassing the 3′ ends of tcdA and tcdC. Transcription of the tcdA–E genes is severely affected by both rearrangements, explaining the demonstrated complete lack of TcdA polypeptide. We present a model of coordinate, growth-related transcription of the tcdA–E genes that confirms our previous findings in strain 10463. Recombinant TcdA-8864 had UDP-glucose-glucosyltransferase activity, proving that the N-terminal 698 amino acids of the polypeptide represent the catalytic domain. However, this truncated TcdA molecule lacks a ligand and translocation domain. To assess the catalytic domain of TcdB-8864, the sequence of the 5′ end of its gene was determined. TcdB-8864 shows high homology to TcdB-1470 but lower homology to TcdB-10463 within this domain. This fits well with the altered glucosylation specificity of TcdB-8864 (Rac1, Rap2 and Ral). Having defined the variations of transcription, expression and enzymatic activity of toxins A and B, implications for the pathogenic potential of strain 8864 are discussed.