Lactobacillli expressing llama VHH fragments neutralise Lactococcus phages

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Bacteriophages are a major cause of fermentation failures in lactic acid bacteria (LAB).
• Controlling bacteriophage infection is crucial for successful dairy fermentation.
• Llama heavy-chain antibody fragments (VHHs) offer potential for phage neutralization.

Purpose of the Study:

• To engineer lactobacilli to express functional VHH fragments for neutralizing lactococcal phages.
• To investigate both secreted and cell-surface anchored VHH fragment expression.

Main Methods:

• Genetic engineering of Lactobacillus strains to express VHH fragments targeting major capsid protein (MCP) and receptor-binding proteins (RBP) of lactococcal bacteriophage p2.
• Utilizing c-myc and E-tags for VHH fragment detection and localization.
• Employing flow cytometry, ELISA, and scanning electron microscopy to confirm expression and binding.
• Conducting neutralization assays to assess phage adsorption inhibition.

Main Results:

• Functional VHH fragments (VHH2 targeting MCP, VHH5 targeting RBP) were successfully expressed by lactobacilli in both secreted and cell-surface anchored forms.
• Surface-expressed VHH2 and secreted VHH5 demonstrated efficient binding to phage antigens.
• Lactobacilli expressing VHH2 showed phage binding, and both VHH2 and VHH5 inhibited phage adsorption to host cells.

Conclusions:

• Lactobacilli can express functional VHH fragments, reducing phage infection.
• This approach offers a novel strategy for controlling bacteriophage infections in lactococcal cultures.
• Engineered lactobacilli represent a promising tool for preventing milk fermentation failures.