Cytophysiologic effects and molecular inhibition of a functional actin-specific ADP-ribosyltransferase CDT from Clostridium difficile

Abstract

The emerging clinical importance of Clostridium difficile has prompted the application of targeted strategy to rapidly identify virulence determinants. Putative virulence-encoding gene fragments were identified and a complete binary ADP-ribosyltransferase cdt isolated. Transcriptional studies suggest bicistronic conformation of cdt operon while analysis of toxin activities revealed catalytically essential amino acid residues. CDTb was important in translocation while CDTa remodelled actin, triggering pro-apoptotic and stress effectors via integrin-cAMP-stress-related MAPK-ATF2 but not the MEK2-ERK1/2-AKT-Stat-3 route. CDTa antagonists were found including heterocyclic peptide antibiotics polyxin B and b-lactam cephalosporins which neutralized CDT's transferase and glycohydrolase activities. The strongest inhibitors were actin-binding proteins having extensive interfaces with G-actin, adjoining the CDT-ADP-ribose+ acceptor site and nucleotide cleft. Overall CDT is a potent agent that may enhance C. difficile pathogenicity by complementing deleterious effects of large clostridial toxins, partly explaining the increased isolation of an otherwise attenuated non-toxin A-producing pathogenic C. difficile strain.