Effect of terminal modifications on aggregation properties and in vitro cytotoxicity of Staphylococcus aureus phenol soluble modulin β1 (PSMβ1)

We evaluated the effects of terminal modifications on the aggregation behaviour and in vitro cytotoxicity of Staphylococcus aureus phenol-soluble modulin β1 (PSMβ1). Using the wild-type peptide and its N-deformylated, C-amidated analogue (dfPSMβ1), we comparatively analysed fibrillation kinetics, morphology, and cytotoxicity. We demonstrated that terminal modifications markedly altered PSMβ1 aggregation behaviour. The wild-type peptide exhibited concentration-dependent amyloid fibrillation characterised by sigmoidal kinetics at lower concentrations and more complex, multiphasic aggregation behaviour at higher concentrations. In contrast, dfPSMβ1 generated only ~10% of the Thioflavin T (ThT) fluorescence observed for the wild-type peptide, lacked detectable signal at 15 μM, and exhibited attenuated elongation kinetics, consistent with the formation of structurally distinct fibrillar assemblies. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) analysis confirmed that both peptide variants exhibited pronounced structural rearrangements upon incubation, consistent with amyloid fibril formation driven by shifts from helix/random coil-dominated monomers toward a β-sheet-rich conformation. This transition is more pronounced for PSMβ1, where a dramatic increase in β-sheet content was observed, accompanied by a simultaneous significant decrease in helix/random coil structures. In contrast, dfPSMβ1 undergoes a less extensive structural rearrangement and remains more heterogeneous, retaining a relatively high helix/random coil content despite increased β-sheet formation. Cryogenic electron microscopy (cryo-EM) revealed distinct morphologies for two peptide variants. PSMβ1 formed long, thin, unbranched fibrils with regular helical twist, while dfPSMβ1 produced shorter, heterogeneous, and entangled fibrils alongside dense rod- or plate-like aggregates. Cytotoxicity assays in human umbilical vein endothelial cells (HUVECs) showed that dfPSMβ1 was markedly less toxic than the wild-type peptide.