Probing bacteriophage-induced membrane perturbation in Aeromonas salmonicida subsp. salmonicida by in-cell solid-state NMR.

Aeromonas salmonicida subsp. salmonicida is a major bacterial pathogen of salmonids in aquaculture, and bacteriophage therapy could provide an alternative to antibiotics for its control. Bacteriophage infection of this Gram-negative bacterium begins at the cell surface and involves interactions with outer membrane components including lipopolysaccharides and surface structures such as the A-layer. Although these interactions influence phage adsorption, their impact on bacterial membrane organization remains poorly understood. Here, we investigated membrane dynamics in three strains differing in envelope architecture and phage sensitivity: AS-R3 (A-layer-positive, phage-sensitive), AS-R5 (A-layer-negative, phage-sensitive), and BIM93 (A-layer-negative, phage-insensitive). We used in vivo2H solid-state nuclear magnetic resonance combined with phospholipid and fatty acid analyses. Exogenous perdeuterated palmitic acid enabled efficient membrane labelling in intact bacteria without significantly altering the overall lipid composition. While all strains shared similar phospholipid classes, BIM93 displayed a higher proportion of saturated fatty acids and increased membrane order than AS-R3 and AS-R5. Upon exposure to phage SW69-9, membrane order decreased markedly in the sensitive strains, with a pronounced loss of viability in AS-R5. In contrast, BIM93 maintained membrane organization and high viability, whereas AS-R3 showed an intermediate response with significant membrane perturbation despite limited cell lysis. These results indicate that phage-induced membrane perturbations depend on both phage sensitivity and bacterial envelope architecture. More broadly, they suggest that changes in membrane organization during phage exposure are influenced by surface-associated bacterial structures. This study highlights the role of bacterial surface structures and membrane physical properties in shaping phage-host interactions.