Lactobacillli expressing llama VHH fragments neutralise Lactococcus phages

Background: Bacteriophages infecting lactic acid bacteria (LAB) are widely acknowledged as the main cause of milk fermentation failures. In this study, we describe the surface-expression as well as the secretion of two functional llama heavy-chain antibody fragments, one binding to the major capsid protein (MCP) and the other to the receptor-binding proteins (RBP) of the lactococcal bacteriophage p2, by lactobacilli in order to neutralise lactococcal phages. Results: The antibody fragment VHH5 that is directed against the RBP, was fused to a c-myc tag and expressed in a secreted form by a Lactobacillus strain. The fragment VHH2 that is binding to the MCP, was fused to an E-tag and anchored on the surface of the lactobacilli. Surface expression of VHH2 was confirmed by flow cytometry using an anti-E-tag antibody. Efficient binding of both the VHH2 and the secreted VHH5 fragment to the phage antigens was shown in ELISA. Scanning electron microscopy showed that lactobacilli expressing VHH2 anchored at their surface were able to bind lactococcal phages. A neutralisation assay also confirmed that the secreted VHH5 and the anchored VHH2 fragments prevented the adsorption of lactococcal phages to their host cells. Conclusion: Lactobacilli were able to express functional VHH fragments in both a secreted and a cell surface form and reduced phage infection of lactococcal cells. Lactobacilli expressing llama heavy-chain antibody fragments represent a novel way to limit phage infection.