title: Viral infection modulation and neutralization by camelid nanobodies.
abstract: Lactococcal phages belong to a large family of Siphoviridae and infect Lactococcus lactis, a gram-positive bacterium used in commercial dairy fermentations. These phages are believed to recognize and bind specifically to pellicle polysaccharides covering the entire bacterium. The phage TP901-1 baseplate, located at the tip of the tail, harbors 18 trimeric receptor binding proteins (RBPs) promoting adhesion to a specific lactococcal strain. Phage TP901-1 adhesion does not require major conformational changes or Ca(2+), which contrasts other lactococcal phages. Here, we produced and characterized llama nanobodies raised against the purified baseplate and the Tal protein of phage TP901-1 as tools to dissect the molecular determinants of phage TP901-1 infection. Using a set of complementary techniques, surface plasmon resonance, EM, and X-ray crystallography in a hybrid approach, we identified binders to the three components of the baseplate, analyzed their affinity for their targets, and determined their epitopes as well as their functional impact on TP901-1 phage infectivity. We determined the X-ray structures of three nanobodies in complex with the RBP. Two of them bind to the saccharide binding site of the RBP and are able to fully neutralize TP901-1 phage infectivity, even after 15 passages. These results provide clear evidence for a practical use of nanobodies in circumventing lactococcal phages viral infection in dairy fermentation.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, Unité Mixte de Recherche 7257 Centre National de la Recherche Scientifique and Aix-Marseille University, 13288 Marseille Cedex 09, France.
authors.forename: Aline
authors.initials: A
authors.lastname: Desmyter
authors.forename: Carine
authors.initials: C
authors.lastname: Farenc
authors.forename: Jennifer
authors.initials: J
authors.lastname: Mahony
authors.forename: Silvia
authors.initials: S
authors.lastname: Spinelli
authors.forename: Cecilia
authors.initials: C
authors.lastname: Bebeacua
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Blangy
authors.forename: David
authors.initials: D
authors.lastname: Veesler
authors.forename: Douwe
authors.initials: D
authors.lastname: van Sinderen
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D001665:Binding Sites; D002161:Camelids, New World; D018360:Crystallography, X-Ray; D000939:Epitopes / Q000737:chemistry; D005285:Fermentation; D013294:Lactococcus lactis / Q000821:virology*; D008854:Microscopy, Electron; D008958:Models, Molecular; D008968:Molecular Conformation; D036103:Nanotechnology; D017434:Protein Structure, Tertiary; D061905:Single-Domain Antibodies / Q000737:chemistry*; D017906:Siphoviridae / Q000502:physiology*; D020349:Surface Plasmon Resonance; D017376:Viral Tail Proteins / Q000737:chemistry*
chemical_list: D000939:Epitopes; D061905:Single-Domain Antibodies; D017376:Viral Tail Proteins