The enterotoxin from Clostridium difficile (ToxA) monoglucosylates the Rho proteins

Area of Science:

• Microbiology
• Cell Biology
• Toxicology

Background:

• Clostridium difficile enterotoxin (ToxA) causes antibiotic-associated pseudomembranous colitis.
• ToxA induces cytotoxic effects by altering the actin cytoskeleton in cultured cells.
• Previous studies linked ToxA's effects to ADP-ribosylation of Rho proteins.

Purpose of the Study:

• To identify the specific modification induced by ToxA on Rho proteins.
• To elucidate the molecular mechanism underlying ToxA's cytotoxic activity.

Main Methods:

• Electrospray mass spectrometry to determine the mass of the modification.
• Tandem mass spectrometry to identify the acceptor amino acid.
• Site-directed mutagenesis to confirm the target residue.
• Enzymatic assays using UDP-glucose and various Rho family proteins.

Main Results:

• ToxA-induced modification of Rho proteins has a mass of 162 Da, consistent with hexose incorporation.
• UDP-glucose was identified as the cosubstrate for ToxA-catalyzed modification.
• Threonine-37 (Thr-37) was identified as the specific acceptor amino acid for glucosylation.
• Mutation of Thr-37 to alanine abolished glucosylation, confirming its role.
• Rho family proteins (RhoA, Rac1, Cdc42Hs) were substrates, while H-Ras, Rab5, and Arf1 were not.
• ToxA-pretreated cells showed reduced [14C]glucose incorporation, indicating prior glucosylation.

Conclusions:

• ToxA catalyzes the glucosylation of Rho subtype proteins at Thr-37.
• This glucosylation is the molecular mechanism by which C. difficile ToxA exerts its cytotoxic effects.
• The modification disrupts the actin cytoskeleton, leading to cell damage and disease.