Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin CDT

Sci Rep. 2015 Jan 19:5:7850. doi: 10.1038/srep07850.

Abstract

The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / immunology
  • ADP Ribose Transferases / metabolism*
  • ADP Ribose Transferases / toxicity
  • Actins / metabolism
  • Amino Acid Sequence
  • Animals
  • Antibodies, Neutralizing / biosynthesis
  • Antibodies, Neutralizing / genetics
  • Antibodies, Neutralizing / immunology
  • Bacterial Proteins / immunology
  • Bacterial Proteins / metabolism*
  • Bacterial Proteins / toxicity
  • Cell Survival / drug effects
  • Clostridioides difficile / metabolism*
  • Clostridioides difficile / pathogenicity
  • Dogs
  • Epitope Mapping
  • Epitopes / immunology
  • HT29 Cells
  • Humans
  • Madin Darby Canine Kidney Cells
  • Microscopy, Interference
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • Protein Subunits / genetics
  • Protein Subunits / immunology
  • Protein Subunits / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / immunology
  • Recombinant Proteins / toxicity
  • Single-Domain Antibodies / biosynthesis
  • Single-Domain Antibodies / genetics
  • Single-Domain Antibodies / immunology*

Substances

  • Actins
  • Antibodies, Neutralizing
  • Bacterial Proteins
  • Epitopes
  • Protein Subunits
  • Recombinant Proteins
  • Single-Domain Antibodies
  • ADP Ribose Transferases
  • actin-specific ADP-ribosyltransferase, Clostridium