Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain

Area of Science:

• Microbiology
• Molecular Biology
• Toxicology

Background:

• Clostridium species produce various toxins affecting host cells.
• ADP-ribosyltransferases are enzymes that modify cellular proteins, impacting cell function.
• Clostridium difficile toxins A and B are well-known virulence factors.

Purpose of the Study:

• To screen Clostridium species for novel ADP-ribosyltransferase activities.
• To characterize a newly identified ADP-ribosyltransferase from Clostridium difficile.
• To investigate the relationship between this enzyme and known toxins.

Main Methods:

• Screening of culture supernatants from Clostridium species.
• Biochemical characterization of the novel ADP-ribosyltransferase (molecular weight, isoelectric point).
• Assays for cytotoxic and lethal activity.
• In vitro ADP-ribosylation assays using cell actin.
• Immunoblot analysis with antibodies against the novel enzyme.

Main Results:

• A novel ADP-ribosyltransferase (CDT) was identified in Clostridium difficile strain CD196.
• CDT (43 kDa, pI 7.8) covalently modifies cell actin.
• CDT showed no direct cytotoxic or lethal activity.
• CDT is distinct from C. difficile toxins A and B, and C. botulinum C2 toxin component I.
• Pre-treatment of Vero cells with C. difficile toxin B reduced actin's ADP-ribosylation by CDT.
• Antibodies to CDT recognized a similar protein in Clostridium perfringens type E supernatant.

Conclusions:

• Clostridium difficile produces a novel actin-modifying ADP-ribosyltransferase, CDT.
• CDT's activity is distinct from known C. difficile toxins but may be influenced by Toxin B.
• CDT shares antigenic similarities with the iota toxin component from Clostridium perfringens type E.