title: Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility.
abstract: Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6.
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.affiliation: AbCelex Technologies, Inc., Toronto, ON, Canada L4V 1T4.
authors.forename: Ali
authors.initials: A
authors.lastname: Riazi
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6.
authors.forename: Shannon
authors.initials: S
authors.lastname: Ryan
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6.
authors.forename: Henk
authors.initials: H
authors.lastname: van Faassen
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 School of Environmental Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Roger
authors.initials: R
authors.lastname: MacKenzie
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 School of Environmental Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada K1N 6N5.
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
authors.affiliation: Human Health Therapeutics Portfolio, National Research Council Canada, Ottawa, ON, Canada K1A 0R6 School of Environmental Sciences, University of Guelph, Guelph, ON, Canada N1G 2W1 Department of Biology, Carleton University, Ottawa, ON, Canada K1S 5B6 mehdi.arbabi@nrc-cnrc.gc.ca.
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology*; D016123:Campylobacter jejuni / Q000187:drug effects / Q000502:physiology*; D002918:Chymotrypsin / Q000737:chemistry; D019020:Directed Molecular Evolution; D005407:Flagella / Q000276:immunology; D005408:Flagellin / Q000276:immunology; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000737:chemistry; D011485:Protein Binding; D011489:Protein Denaturation; D059748:Proteolysis; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D014357:Trypsin / Q000737:chemistry
chemical_list: D000900:Anti-Bacterial Agents; D057127:Single-Chain Antibodies; D005408:Flagellin; D002918:Chymotrypsin; D014357:Trypsin; D010434:Pepsin A
keywords: C. jejuni; VHH; motility; protease resistance; protein stability