C. difficile toxin A (TcdA)
The large clostridial cytotoxins (LCT) are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B [also termed TcdA and TcdB, respectively), C. sordellii (lethal and hemorrhagic toxin) and C. novyi (alpha-toxin). The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes. The LCTs are single-chain protein toxins, comprising three domains: receptor-binding, translocation and catalysis, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Enzymatic activity involves transfer of a glucosyl moiety from UDP-glucose (or the N-acetyl-glucosaminyl moiety from UDP-GlcNAc in the case of alpha toxin) to a conserved threonine within the effector regions of the intracellular Rho and Ras GTPases. The C-terminal receptor-binding domain comprises up to one third of the LCT molecule, and contains repetitive peptide elements called combined repetitive oligopetides (CROPs). The repeating units binding regions of certain streptococcal glycosyltransferases.
TcdA is the better characterized of the two LCTs produced by C. difficile. Collectively, TcdA and TcdB are directly responsible for the increasingly common and serious human gastrointestinal disease caused when antibiotic treatment enables C. difficile to out-compete commensal gut microflora. Recently, the crystal structure of a C-terminal fragment of TcdA has been solved, revealing a solenoid-like structure, which consists of 32 short repeats with 15–21 residues and seven long repeats with 30 residues. Solenoid structures are often found in bacterial surface proteins, and their extended surface allows protein–protein or protein–carbohydrate interactions. TcdA is known to bind to Galα1-3Galβ1-4GlcNAc- glycans, although this structure is not present in humans. However, it is also known to recognize a variety of other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium. Clearly, a better understanding of the identity/structure of the human receptor(s) for these toxins will facilitate design of novel therapeutics capable of blocking toxin binding. Glycan array analysis conducted by the CFG showed that TcdA bound most strongly to glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa; other bound structures included Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-.
CFG Participating Investigators contributing to the understanding of this paradigm
- CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
- Non-CFG PIs include: Brian Dieckgraefe
Progress toward understanding this GBP paradigm
- Galα1-3Galβ1-4GlcNAc- glycans (not present in humans).
- Other structures with a Galβ[1→4]GlcNAcβ- backbone, including the fucosylated blood group antigens Lewisx and Lewisy, both of which are present in human intestinal epithelium.
- Strongest binding: glycans terminating in Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4-GlcNAc-, which mimics the blood group antigen Lewisa.
- Also binds: Galα1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ-, NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ-, and Fucαa1-2[GalNAcαa1-3]Galβ1-4GlcNAcβ1-3Galβb1-4GlcNAcβ-.
Cellular expression of GBP and ligands
TcdA is produced by Clostridium difficile. Structurally and functionally related exotoxins in the LCT family are produced by C. sordellii (lethal and hemorrhagic toxin) and C. novyi (alpha-toxin).
TcdA binds the fucosylated blood group antigens Lewisx and Lewisy, which are present in human intestinal epithelium (see above).
Biosynthesis of ligands
The highest affinity ligand, Lewisa is created by the action of
acting on type 1 chains.
On type 2 chains, the biosynthesis of the alternative Lewisx and Lewisy ligands requires the action of Fucosyltransferase 4 and Fucosyltransferase 9 for addition of the α1-3 fucose and in the latter case action of Fucosyltransferase 1 and Fucosyltransferase 2 for the addition of α1-2 fucose.
Members of the LCT family are single-chain protein toxins with receptor-binding, translocation and catalysis domains, which mediate cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and enzymatic cytotoxic activity, respectively. Up to one third of the LCT molecule is comprised of the C-terminal receptor-binding domain, which contains repetitive peptide elements called combined repetitive oligopeptides (CROPs).
X-ray crystallography has revealed the three-dimensional structures of several recombinant fragments of TcdA and related toxins. The N-terminal domain consists of a GT-A family glucosyltransferase that catalyzes the transfer of glucose from UDP-glucose to specific threonine residues of small GTPases regulating the structure and dynamics of the cytoskeleton. Although the three-dimensional structure of the GT domain of TcdA has not been solved by crystallography, the structures of closely related GT domains from TcdB and the letahal toxin from Clostridium sordellii have been determined.
The crystallographic structure of a cysteine protease domain bound to inositol hexakisphosphate has also been determined. This domain follows the N-terminal GT domain and is responsible for the autoproteolysis of TcdA that is important for releasing the GT domain into the cytoplasm.
Crystallographic structures of several recombinant fragments from the highly repetitive C-terminal domain have revealed an extended beta-solenoid structure that presents seven binding sites for complex oligosaccharide receptors. The bulk of this carbohydrate-binding domain is comprised of multiple copies of short repeats that interspersed with single copies of long repeats. Carbohydrate-binding sites appear to be formed at the junctions of long repeats and the preceding and following short repeats.
Crystallographic structures of recombinant fragments of TcdA have been determined with oligosaccharides, including the Gal-α(1,3)-LacNAc trisaccharide shown below. This trisaccharide was determined to be a likely native receptor for TcdA in the brush border membranes of intestinal epithelial cells from hamster, an organism which is susceptible to C. difficile infection (CDI) and is often used as a model for studying the disease.
Biological roles of GBP-ligand interaction
TcdA and TcdB, the other LCT produced by C. difficile, cause the serious human gastrointestinal disease that results when antibiotic treatment enables C. difficile to out-compete commensal gut microflora. The LCTs are major virulence factors, which in addition to their in vivo effects, are cytotoxic to cultured cell lines, causing reorganization of the cytoskeleton accompanied by morphological changes.
CFG resources used in investigations
The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for "difficile".
TcdA is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.
Knockout mouse lines
The CFG glycan array has been used to examine the glycan-binding specificity of TcdA. In addition, the CFG's glycan synthesis capability is being used to prepare synthetic oligosaccharides indentified in the glycan array screens (with or without biotin tags) to further probe TcdA-receptor interactions.
C. difficile toxin B (TcdB; CFG data), C. sordellii lethal toxin and hemorrhagic toxin, C. novyi alpha-toxin.
- ↑ 1.0 1.1 1.2 1.3 Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.
- ↑ 2.0 2.1 Thomas Jank, Torsten Giesemann and Klaus Aktories. (2007). Rho-glucosylating Clostridium difficile toxins A and B: new insights into structure and function. Glycobiology. 17: 15R–22R.
- ↑ 3.0 3.1 3.2 Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.
- ↑ Greco, A et al. (2006). Carbohydrate recognition by Clostridium difficile toxin A. Nat. Struct. Mol. Biol. 13:460-461.
- ↑ Krivan, HC et al (1986). Cell-surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Galα1,3-Galβ1,4-GlcNAc. Infect. Imm. 53:573-581.
The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, Kenneth Ng, James Paton