Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen
| Autor: | Chanan Angsuthanasombat, Chompounoot Imtong, Wanpen Chaicumpa, Aijaz Ahmad Malik, Gerd Katzenmeier, Nitat Sookrung |
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| Rok vydání: | 2016 |
| Předmět: |
Models
Molecular 0301 basic medicine Bordetella pertussis Erythrocytes Phage display Health Toxicology and Mutagenesis CyaA-RTX Toxicology Article Epitope Epitopes Hemolysin Proteins 03 medical and health sciences Antigen intermolecular docking CyaA-hemolysin VH/VHH phage display Homology modeling Antigens Bacterial biology Single-Domain Antibodies cyaA biology.organism_classification Antibodies Neutralizing Molecular biology Bordetella 030104 developmental biology Adenylate Cyclase Toxin Linker |
| Zdroj: | Toxins Toxins; Volume 8; Issue 4; Pages: 99 |
| ISSN: | 2072-6651 |
| DOI: | 10.3390/toxins8040099 |
| Popis: | Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity. |
| Databáze: | OpenAIRE |
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