title: Selection of nanobodies that block the enzymatic and cytotoxic activities of the binary Clostridium difficile toxin CDT.
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.
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Mandy
authors.initials: M
authors.lastname: Unger
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Anna Marei
authors.initials: AM
authors.lastname: Eichhoff
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Lucas
authors.initials: L
authors.lastname: Schumacher
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Moritz
authors.initials: M
authors.lastname: Strysio
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Menzel
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schwan
authors.affiliation: Fundacion Instituto Leloir, C1405 Buenos Aires, Argentina.
authors.forename: Vanina
authors.initials: V
authors.lastname: Alzogaray
authors.affiliation: Inmunova, C1405 Buenos Aires, Argentina.
authors.forename: Vanesa
authors.initials: V
authors.lastname: Zylberman
authors.affiliation: Normandy University, Institute for Research and Innovation in Biomedicine, 76183 Rouen, France.
authors.forename: Michel
authors.initials: M
authors.lastname: Seman
authors.affiliation: Department of Dermatology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Johanna
authors.initials: J
authors.lastname: Brandner
authors.affiliation: Institute of Microbiology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Rohde
authors.affiliation: Schrodinger Inc., 120 West 45th Street, New York, New York 10036, United States.
authors.forename: Kai
authors.initials: K
authors.lastname: Zhu
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Haag
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Hans-Willi
authors.initials: HW
authors.lastname: Mittrücker
authors.affiliation: Fundacion Instituto Leloir, C1405 Buenos Aires, Argentina.
authors.forename: Fernando
authors.initials: F
authors.lastname: Goldbaum
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Freiburg, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Aktories
authors.affiliation: Institute of Immunology, University Medical Center Hamburg-Eppendorf, Germany.
authors.forename: Friedrich
authors.initials: F
authors.lastname: Koch-Nolte
mesh_terms: D036002:ADP Ribose Transferases / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D057134:Antibodies, Neutralizing / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D001426:Bacterial Proteins / Q000276:immunology / Q000378:metabolism* / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D016360:Clostridioides difficile / Q000378:metabolism* / Q000472:pathogenicity; D004285:Dogs; D018604:Epitope Mapping; D000939:Epitopes / Q000276:immunology; D019073:HT29 Cells; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008857:Microscopy, Interference; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology / Q000633:toxicity; D061905:Single-Domain Antibodies / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D000199:Actins; D057134:Antibodies, Neutralizing; D001426:Bacterial Proteins; D000939:Epitopes; D021122:Protein Subunits; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; D036002:ADP Ribose Transferases; C410164:actin-specific ADP-ribosyltransferase, Clostridium