Binding of Clostridium difficile toxins to human milk oligosaccharides

Glycobiology. 2011 Sep;21(9):1217-27. doi: 10.1093/glycob/cwr055. Epub 2011 May 24.


The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(β1-4)Glc, Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Fuc(α1-2)Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc, Gal(β1-3)[Fuc(α1-4)]GlcNAc(β1-3)Gal(β1-4)Glc and Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-3)Gal(β1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3 )M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Sugars / metabolism
  • Animals
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / pharmacology
  • Bacterial Toxins / chemistry
  • Bacterial Toxins / metabolism*
  • Bacterial Toxins / pharmacology
  • Binding Sites
  • Carbohydrate Sequence
  • Cell Survival / drug effects
  • Chlorocebus aethiops
  • Clostridioides difficile / chemistry*
  • Enterotoxins / chemistry
  • Enterotoxins / metabolism*
  • Enterotoxins / pharmacology
  • Humans
  • Kinetics
  • Milk, Human / chemistry*
  • Molecular Dynamics Simulation
  • Molecular Sequence Data
  • Oligosaccharides* / analysis
  • Oligosaccharides* / chemistry
  • Oligosaccharides* / metabolism
  • Peptide Fragments / chemistry
  • Peptide Fragments / metabolism*
  • Peptide Fragments / pharmacology
  • Protein Binding
  • Spectrometry, Mass, Electrospray Ionization
  • Vero Cells


  • Amino Sugars
  • Bacterial Proteins
  • Bacterial Toxins
  • Enterotoxins
  • Oligosaccharides
  • Peptide Fragments
  • tcdA protein, Clostridium difficile
  • toxB protein, Clostridium difficile
  • lactosamine