Structural and functional analysis of Clostridium difficile toxins A and B

Abstract

The pathogenesis of Clostridium difficile is dependent on two large homologous toxins, TcdA and TcdB. These toxins contain glucosyltransferase domains that inactivate host Rho proteins by glucosylation. Delivery of the glucosyltransferase domain into the target cell is achieved by binding to the target cell, pH-dependent translocation of the glucosyltransferase domain across the membrane, and release of the domain by autoproteolysis. These steps in delivery are mediated by receptor-binding, pore-forming, and autoprotease domains. Structures existed for a portion of the TcdA receptor-binding domain and the TcdB glucosyltransferase domain, but there had previously been no structural information for the protease and pore-forming domains or for the holotoxins. I have visualized the TcdA and TcdB holotoxins by negative stain electron microscopy, determined the TcdA three-dimensional structure by random conical tilt, and mapped the TcdA functional domains within this structure. A second structure, obtained at acidic pH, revealed significant structural changes involved in delivery of the glucosyltransferase domain across the membrane. In addition, I have elucidated the structure of the TcdA autoprotease domain and used complementary functional assays to uncover key aspects of its function. Finally, I have determined the structure of the TcdA glucosyltransferase domain. The activity of the TcdA glucosyltransferase domain was compared with that of TcdB and shown to be a potent enzyme with a broader range of substrates than TcdB. These data provide a structural framework for understanding the molecular mechanisms by which TcdA and TcdB deliver their cytotoxic cargo into the target cell and disrupt host processes.