Cationic Lys53-C5aa endolysin modification boosts anti-Vibrio activity, disrupts membranes, and enhances seafood safety

Endolysins are phage-derived enzymes that specifically degrade the peptidoglycan layer of bacterial cell walls, offering a promising alternative to conventional antibiotics. However, their application against Gram-negative bacteria is severely limited by the outer membrane (OM) barrier, which prevents access to the cell wall. This structural challenge necessitates modifications to enable endolysins to penetrate the OM and exert their lytic activity. Addressing this gap, researchers are exploring cationic peptide fusions to enhance endolysin efficacy against problematic Gram-negative pathogens like Vibrio parahaemolyticus, a major cause of seafood-borne illness.

Researchers modified the endolysin Lys53 from phage vB_VpaS_1601 by fusing cationic peptides to its C-terminus, creating the engineered protein Lys53-C5aa. They evaluated its lytic activity against Vibrio parahaemolyticus at a concentration of 6.4 μM over 30 min, assessing bacterial reduction (log CFU/mL). The study also investigated its lysis spectrum, optimal temperature and pH ranges, and mechanisms of action through OM permeability assays, nucleic acid leakage measurements, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Furthermore, Lys53-C5aa's effects on V. parahaemolyticus biofilm formation and its ability to reduce bacterial counts in contaminated oysters and Pacific white shrimp at 4 °C and 25 °C were tested.

The engineered endolysin Lys53-C5aa demonstrated significantly enhanced anti-Vibrio parahaemolyticus activity, achieving a 3.51 log CFU/mL reduction at 6.4 μM within 30 min, alongside a broader lysis spectrum compared to its native form. It maintained optimal lytic activity across a wide temperature range of 4 to 65 °C and a pH range of 5 to 10. Mechanistically, Lys53-C5aa significantly enhanced the OM permeability of V. parahaemolyticus, leading to substantial leakage of intracellular nucleic acids, which peaked at 110.5 ng/μL after 75 min. SEM and AFM analyses confirmed that Lys53-C5aa directly disrupted the structural integrity of the bacterial cells. Beyond planktonic bacteria, Lys53-C5aa exhibited strong anti-biofilm effects, showing 41.83% removal and 61.01% inhibition of V. parahaemolyticus biofilms. > In practical applications, Lys53-C5aa effectively reduced V. parahaemolyticus counts in oysters by 2.14 log CFU/g (4 °C) and 2.40 log CFU/g (25 °C), and in Pacific white shrimp by 2.21 log CFU/g (4 °C) and 2.69 log CFU/g (25 °C).