Abstract
Some Clostridium difficile strains produce, in addition to toxins A and B, the binary toxin Clostridium difficile transferase (CDT), which ADP-ribosylates actin and may contribute to the hypervirulence of these strains. The separate binding and translocation component CDTb mediates transport of the enzyme component CDTa into mammalian target cells. CDTb binds to its receptor on the cell surface, CDTa assembles and CDTb/CDTa complexes are internalised. In acidic endosomes, CDTb mediates the delivery of CDTa into the cytosol, most likely by forming a translocation pore in endosomal membranes. We demonstrate that a seven-fold symmetrical positively charged β-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin, which was developed earlier as a potent inhibitor of the translocation pores of related binary toxins of Bacillus anthracis, Clostridium botulinum and Clostridium perfringens, protects cells from intoxication with CDT. The pore blocker did not interfere with the CDTa-catalyzed ADP-ribosylation of actin or toxin binding to Vero cells but inhibited the pH-dependent membrane translocation of CDTa into the cytosol. In conclusion, the cationic β-cyclodextrin could serve as the lead compound in a development of novel pharmacological strategies against the CDT-producing strains of C. difficile.
Highlights
Clostridium difficile (C. difficile) causes enteric diseases in patients treated with broad-spectrum antibiotics that range from diarrhea to severe, potentially life-threatening pseudomembranous colitis because disturbance of the gut flora enables spore germination and growth of this pathogen [1].The causative agents of C. difficile-associated diseases are the exotoxins A (TcdA, 308 kDa) andB (TcdB, 270 kDa), which catalyse the glucosylation of Rho, Rac and Cdc42 in the cytosol of cells thereby inhibiting signal transduction via these GTPases [2,3]
We have performed a series of experiments to demonstrate that the symmetrical positively charged β-cyclodextrin derivative, per-6-S-(3-aminomethyl)benzylthio-β-cyclodextrin (AMBnTβ-CD), efficiently protects cultured epithelial cells from intoxication with the binary toxin Clostridium difficile transferase (CDT) of C. difficile
The more detailed investigation of the underlying mechanism strongly suggests that this compound inhibited the pH-dependent translocation of the enzyme component CDTa across cell membranes, which is mediated by trans-membrane pores formed by the separate binding/translocation component
Summary
Clostridium difficile (C. difficile) causes enteric diseases in patients treated with broad-spectrum antibiotics that range from diarrhea to severe, potentially life-threatening pseudomembranous colitis because disturbance of the gut flora enables spore germination and growth of this pathogen [1]. The molecular and cellular consequences following toxin-catalysed mono-ADP-ribosylation of actin at arginine-177 were described in detail for the related C2 and iota toxins [14,30,31,32,33,34,35,36,37] Taken together, this modification inhibits actin polymerization [38] and causes cell-rounding. In addition to the pores, cytosolic host cell factors including chaperones and protein folding helper enzymes are involved in membrane translocation of the enzyme components of C2 toxin [57,58], iota toxin [28,59] and CDT [28] Due to their essential role in toxin uptake, the translocation pores represent attractive molecular drug targets [60] to protect cells from these binary toxins. AMBnTβ-CD inhibits translocation of CDTa and protects cells from intoxication with CDT
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