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Proc Natl Acad Sci U S A. 2013 Apr 9;110(15):E1371-9. doi: 10.1073/pnas.1301336110. Epub 2013 Mar 25.

Viral infection modulation and neutralization by camelid nanobodies.

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  • 1Architecture 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.

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.

PMID:
23530214
PMCID:
PMC3625315
DOI:
10.1073/pnas.1301336110
[PubMed - indexed for MEDLINE]
Free PMC Article
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