Closing in on the toxic domain through analysis of a variant Clostridium difficile cytotoxin B

Abstract

Strain 1470 is the standard typing strain for serogroup F of Clostridium difficile containing both toxin genes, toxA-1470 and toxB-1470. A polymerase chain reaction (PCR)-based approach to the sequencing of the total toxB-1470 gene identified an open reading frame (ORF) of 7104 nucleotides. In comparison with the previously sequenced toxB of C. difficile VP10463, 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 comparison with ToxB, ToxB-1470 differs primarily in the N-terminal region between positions 1 and 868 where 148 amino acids residues are changed. The C-terminal region between residues 869-2367 is highly conserved with only six amino acid alterations. Dot matrix comparison of ToxB-1470 with ToxA and ToxB reveals the highest homology between ToxB-1470 and ToxB. Thus ToxB-1470 did not originate from recombination between ToxA and ToxB. On cultured endothelial cells, from porcine pulmonary artery, purified ToxB-1470 is less potent than ToxB. The cytopathic effects of ToxB-1470 are indistinguishable from those caused by the lethal toxin (LT) of Clostridium sordellii, but are clearly different from the patterns observed after exposure of endothelial cells to ToxA and ToxB of C. difficile (VPI10463) or alpha-toxin (Tcn alpha) of Clostridium novyi. The LT-like action of ToxB-1470 was not due to altered internalization processes, as microinjection and addition to the medium induced identical effects on the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Clostridium difficile Testing: after 20 Years, Still Challenging

“…In A Ϫ B ϩ isolates, however, the substrate specificity of toxin B is broader, resulting in glycosylation of other G proteins. In this regard, toxin B from A Ϫ B ϩ isolates resembles toxin LT of C. sordellii, which crossreacts with toxin B but which is more lethal (3,19). Speculatively, the broader specificity may explain the increased toxicity of the aberrant toxin B, particularly its ability to damage the intestinal mucosa.…”

R-Ras Glucosylation and Transient RhoA Activation Determine the Cytopathic Effect Produced by Toxin B Variants from Toxin A-negative Strains of Clostridium difficile

“…In addition to TcdB and TcdA from the prototype strain VPI-10463, toxin variants of C. difficile toxin B, TcdB-1470 and TcdB-8864, have been isolated from TcdA-negative strains (8,9). These variants have been reported to produce a somehow different cytopathic effect (CPE) than the classical neurite-like morphology induced in fibroblasts by TcdB-10463 (9,10).…”

G13-dependent Activation of MAPK by Thyrotropin

“…Since a variant of this toxin, TcdB 1470, which exhibits no inhibitory activity toward Rho proteins (48), was also effective, we conclude that Rho is not involved in this pathway. This conclusion is supported by the fact that a fusion toxin with clostridial C3 activity, which acts as a more specific Rho inhibitor (49,50), did not prevent TSH-induced p44/42 MAPK activation in both cell lines.…”

“…3 and 8). Thus, we conclude that a reduced responsiveness of PHT cells to EGF is not responsible for the lack of receptor transactivation, suggesting that, despite the common role of G 13 observed in TSHpromoted MAPK activation in both cell types, divergent signaling pathways connect G 13 (48). Surprisingly, pretreatment of FTC-133 WT TSHR and PHT cells with both toxin variants inhibited TSH-dependent stimulation of MAPK (Fig.…”