Structural Tuning of HEWL Amyloid Polymorphs Enhances Antibacterial Activity Against Gram-Positive and Gram-Negative Pathogens

Amyloid fibrils are traditionally associated with protein misfolding disorders. However, increasing evidence indicates that they can also perform beneficial biological functions, including antimicrobial defense. Here, we investigated whether structurally distinct amyloid polymorphs of hen egg white lysozyme (HEWL) exhibit enhanced antibacterial activity compared with the native protein. HEWL was converted into two amyloid polymorphs, flexible fibrils and rigid fibrils, and their antibacterial activities were evaluated against the Gram positive bacterium Staphylococcus aureus and the Gram negative bacteria Escherichia coli (Top10) and Salmonella typhimurium. Fibril formation was confirmed by circular dichroism (CD) spectroscopy, thioflavin T (ThT) fluorescence, and transmission electron microscopy (TEM), demonstrating morphologically distinct amyloid assemblies with different secondary-structure organizations. Fluorescence-based bacterial growth assays showed that native HEWL exhibited only moderate antibacterial activity, whereas both amyloid polymorphs produced potent, concentration-dependent bacterial growth inhibition. Flexible fibrils and rigid fibrils consistently displayed greater antibacterial efficacy than native HEWL across all tested strains, with flexible fibrils exhibiting slightly stronger activity against S. typhimurium. At concentrations of 600 to 800 micro molar, flexible fibrils achieved >90% growth inhibition for all bacterial species examined. Cytotoxicity studies using SH SY5Y human neuroblastoma cells demonstrated minimal toxicity for native HEWL, modest effects for flexible fibrils, and substantially greater toxicity for rigid fibrils, indicating that amyloid polymorphism influences both antimicrobial activity and mammalian cell compatibility. Collectively, these findings establish a direct relationship between amyloid structure, antibacterial efficacy, and cytotoxicity. The combination of potent antibacterial activity and relatively low cytotoxicity identifies flexible fibrils as a promising functional amyloid biomaterial for the development of next-generation antimicrobial materials.

Source: Structural Tuning of HEWL Amyloid Polymorphs Enhances Antibacterial Activity Against Gram-Positive and Gram-Negative Pathogens