CLOSING IN ON THE TOXIC DOMAIN THROUGH ANALYSIS OF A VARIANT CLOSTRIDIUM-DIFFICILE CYTOTOXIN-B

Strain 1470 is the standard typing strain for serogroup F of Clostridi um difficile containing both toxin genes, toxA-1470 and toxB-1470. A p olymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 n ucleotides. In comparison with the previously sequenced toxB of C. dif ficile VPI10463, the toxB-1470 gene has 16 additional nucleotides, 13 within the 5'-untranslated region and three within the coding region. The M(r) of ToxB-1470 is 269 262, with an isoelectric point (IP) of 4. 16. The equivalent values for ToxB are M(r) 269 709 and IP 4.13. In co mparison with ToxB, ToxB-1470 differs primarily in the N-terminal regi on between positions 1 and 868 where 148 amino acids residues are chan ged. The C-terminal region between residues 869-2367 is highly conserv ed with only six amino acid alterations. Dot matrix comparison of ToxB -1470 with ToxA and ToxB reveals the highest homology between ToxB-147 0 and ToxB. Thus ToxB-1470 did not originate from recombination betwee n ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic e ffects of ToxB-1470 are indistinguishable from those caused by the let hal toxin (LT) of Clostridium sordellii, but are clearly different fro m the patterns observed after exposure of endothelial cells to ToxA an d ToxB of C. difficile (VPI10463) or alpha-toxin (Tcn alpha) of Clostr idium novyi. The LT-like action of ToxB-1470 was not due to altered in ternalization processes, as microinjection and addition to the medium induced identical effects on the cells. Since the differences between ToxB and ToxB-1470 are clustered within the N-terminal third of the re spective proteins, we conclude that these domains carry the toxic dete rminants. A three-domain structure is proposed for the family of large clostridal cytotoxins.