Structural organization of the functional domains of Clostridium difficile toxins A and B

Abstract

Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a "pincher-like" head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.bacterial toxin | electron microscopy | pore formation

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

“…After a library screening, we identified two unique V H Hs, 7F and E3, that bind with high affinity to GTD yet inhibit distinct TcdB enzymatic activities located in different functional domains. Specifically, E3 interferes with the ability of TcdB to glucosylate Rac1 in cell-free assays, whereas 7F blocks toxin autocleavage by allosteric cofactor InsP 6 . The inhibitory action of 7F binding likely impedes toxin self-cleavage by interfering with catalytic processing of substrate in the CPD.…”

“…However, it is unknown whether CPD-mediated autoprocessing occurs before or after the translocation of the toxins. It is reasonable to assume that InsP 6 or other activators may accumulate in both cytosol and endosome to concentrations that are sufficient to cause activation of the cysteine protease leading to autoprocessing. Because the intrabodies are expressed in the cytosol and are unlikely reach into endosomes, our data thus provide experimental evidence indicating that autoprocessing occurs after toxin translocation from endosomes into the cytosol.…”

“…Evidence that GTD release into the cytoplasm is mediated by CPD activity is largely based on in vitro studies. This autoproteolytic activity in TcdA and TcdB is mediated by allosteric cofactors, inositol hexakis-and heptakisphosphate (InsP 6 and InsP 7 ) (24,28,29). We along with others have demonstrated using cysteine protease activity-deficient TcdB mutants, as well as a noncleavable TcdA or TcdB, that blocking the release of GTD into the host cell cytosol delays, but does not prevent, the cytopathic and cytotoxic activities of TcdA or TcdB (30,31).…”

“…Disease in patients with C. difficile infection is strongly associated with the two exotoxins, TcdA and TcdB (3). Both toxins are large, homologous single-chain proteins that contain at least four distinct domains (4)(5)(6): the N terminus glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a C terminus receptor binding domain (RBD; also known as combined repetitive oligopeptides, or CROPs). A recent study suggests that there might also be an additional receptor binding region besides the N-terminal CROP region (7) although the specific region has yet to be identified.…”

Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

“…The glucosyltransferase domain double point mutation (TcdA D285/287N and TcdB D286/ 288N) plasmids (pBL764 and pBL765, respectively) were made using the TcdA and TcdB parent plasmids (42) according to the QuikChange protocol (Stratagene). Recombinant TcdA, TcdA D285/287N, TcdB, and TcdB D286/288N proteins were expressed in Bacillus megaterium and purified as previously described (42).…”