C. difficile toxin A (TcdA)

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'''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[1]. 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[1]. 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[1].
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'''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<ref name="Just 1">Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.</ref>. 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<ref name="Just 1"/>. 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<ref name="Just 1"/>.
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'''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[2]. 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 Lewis<sup>x</sup> and Lewis<sup>y</sup>, both of which are present in human intestinal epithelium[3]. 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 Lewis<sup>a</sup>; 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-.
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'''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<ref>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.
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</ref>. 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 Lewis<sup>x</sup> and Lewis<sup>y</sup>, both of which are present in human intestinal epithelium<ref>Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.</ref>. 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 Lewis<sup>a</sup>; 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 contributing to the understanding of this paradigm ==
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CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
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* CFG Participating Investigators (PIs) contributing to the understanding of TcdA include: Borden Lacy, Yashwant Mahida, Kenneth Ng
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Non-CFG PIs include: Brian Dieckgraefe
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* Non-CFG PIs include: Brian Dieckgraefe
== Progress toward understanding this GBP paradigm ==
== Progress toward understanding this GBP paradigm ==
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=== Glycan array ===
=== Glycan array ===
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The CFG glycan array has been used to examine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2693 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.  
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The CFG glycan array has been used to examine the [http://www.functionalglycomics.org/glycomics/HServlet?operation=view&sideMenu=no&psId=primscreen_2693 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.
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''== Related GBPs ==
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== Related GBPs ==
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C. difficile'' toxin B (TcdB), ''C. sordellii'' lethal toxin and hemorrhagic toxin, ''C. novyi'' alpha-toxin.
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''C. difficile'' toxin B (TcdB), ''C. sordellii'' lethal toxin and hemorrhagic toxin, ''C. novyi'' alpha-toxin.
== References ==
== References ==

Revision as of 18:47, 9 April 2010

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[1]. 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[1]. 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[1].

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[2]. 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[3]. 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-.

Contents

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

Carbohydrate ligands


Cellular expression


Structure


Biological roles of GBP-ligand interaction


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".

Glycan profiling


Glycogene microarray


Knockout mouse lines


Glycan array

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.

Related GBPs

C. difficile toxin B (TcdB), C. sordellii lethal toxin and hemorrhagic toxin, C. novyi alpha-toxin.

References

  1. 1.0 1.1 1.2 Just. I., Gerhard, R. (2004). Large clostridial cytotoxins. Rev. Physiol. Biochem. Pharmacol. 152:23-47.
  2. 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. Voth, D. E., and Ballard, J. D. (2005). Clostridium difficile toxins: mechanism of action and role in disease. Clin. Microbiol. Rev. 18, 247-263.

Acknowledgements

The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton

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