Trends in Microbiology
ReviewStructure and mode of action of clostridial glucosylating toxins: the ABCD model
Introduction
Antibiotic-associated diarrhea and pseudomembranous colitis induced by Clostridium difficile have emerged as important nosocomial infections. During the past decade, these diseases seem to have become more serious and more frequently refractory to therapy [1]. The occurrence of hypervirulent strains of C. difficile such as PCR ribotype 027/PFGE type NAP1 [2] with attributable mortality rates of up to 16.7% is concerning [3]. The major virulence factors of C. difficile are two protein toxins, named toxin A and toxin B (also designated TcdA and TcdB), which have been recognized for their important role in disease for the past 30 years. In addition, some strains of C. difficile produce a binary ADP-ribosylating toxin (C. difficile transferase, CDT) that modifies G-actin [4]. Notably, hypervirulent strains are characterized by production of 10- to 20-fold larger amounts of toxins A and B, the resistance to fluoroquinolones and production of the actin-ADP-ribosylating toxin [2].
Section snippets
Structure–function relationships of clostridial glucosylating toxins
C. difficile toxin A and toxin B are the prototypes of the family of clostridial glucosylating toxins 5, 6. Other members of this toxin family are the hemorrhagic and the lethal toxins from Clostridium sordellii and the α-toxin from Clostridium novyi. Moreover, several isoforms of toxin A and B have been described, adding to this family of cytotoxins 7, 8, 9. All of these toxins share sequence identities ranging from 36% to 90% and have molecular masses between 250 and 308 kDa 10, 11. Because
Toxins with multimodular structure
Toxin A consists of 2710 amino acid residues with a molecular mass of 308 kDa and toxin B comprises 2366 residues with a mass of 269.6 kDa. These toxins are therefore also known as large clostridial cytotoxins [10]. On the basis of their amino acid sequences, a tripartite structure for the toxins had been suggested 10, 12, 13, with a biologically active N-terminal domain, a middle translocation section characterized by a small hydrophobic stretch (prediction of transmembrane regions), and a
The C terminus binds to target cell membranes
The receptor-binding domain located at the C terminus of the clostridial glucosylating toxins consists of repeating polypeptide units 12, 15, 16, 17. Recently, two C-terminal fragments (terminal 127 and 255 residues) of toxin A (toxinotype VI) were crystallized 18, 19. These studies gave new insights into the overall structure of the C terminus of the toxin (Figure 1). Toxin A folds in a solenoid-like structure with 32 small repeats consisting of 15–21 residues and seven large repeats of 30
Toxin uptake: a question of cutting and delivery
Following receptor binding, the clostridial glucosylating toxins are endocytosed [25] (Figure 2) – the precise endocytosis pathway is not known. After endocytosis, the toxins translocate through the early endosomal membrane into the cytosol. This process depends on the acidification of endosomes by vesicular H+-ATPase. Bafilomycin, which blocks the H+-ATPase, inhibits cytosolic entry of the toxin and intoxication of cells [26]. Therefore, C. difficile toxins A and B belong to the short trip
The N-terminal enzyme domain
The N terminus harbors the glucosyltransferase activity of the toxins and is the biological activity domain [37]. The 543 amino acid residues, which are delivered into the cytosol of host cells, form the glucosyltransferase domain. Recently, the 3D-structure of the catalytic domain (residues 1–543) of toxin B has been solved [38]. The catalytic core is formed by a mixed α/β-fold with mostly parallel β-strands (Figure 3a). The overall structure of the catalytic core is similar to other bacterial
Interaction of toxin B with Rho GTPases
Glycosyltransferases are region-selective enzymes. In the case of the clostridial glucosylating toxins a specific threonine residue in the substrate proteins (small GTP-binding proteins) is monoglucosylated 54, 58. This threonine senses the integrity of the nucleotide GTP, which is bound to the GTPase in the active conformation and to the hydrolyzed GDP form in the inactive conformation. Sensing occurs via binding to a Mg2+ ion, which is also complexed with the phosphates of the nucleotide.
Concluding remarks
Studies from recent years have yielded important progress in our knowledge of the structure and mode of action of clostridial glucosylating toxins. These results indicate the need for a revision of the current AB-model of clostridial glucosylating toxins. Characterization of clostridial glucosylating toxins according to this model seems to be a scientific Procrustes’ bed. Thus, we suggest that an ABCD model is more appropriate for describing the structure and function of these toxins. However,
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