RNA Selectively Modulates Activity of Virulent Amyloid PSMα3 and Host Defense LL-37 via Phase Separation and Aggregation Dynamics

Amyloids, classically associated with neurodegenerative disease, also play important roles in infection and immunity. Phenol-soluble modulins (PSMs) from Staphylococcus aureus are amyloid-forming virulence peptides that contribute to cytotoxicity, immune modulation, and biofilm stability. PSMα3 forms cross-α amyloid fibrils and shares sequence and α-helical self-assembly features with LL-37, a human host-defence peptide that forms non-amyloid α-helical assemblies. Here, we identify RNA as a context-dependent regulator of their assembly pathways and biological activity. RNA consistently reduces LL-37 cytotoxicity toward human cells without compromising its antibacterial function, suggesting a host-protective effect. In contrast, RNA preserves PSMα3 cytotoxic and antimicrobial activity over time by reshaping its assembly landscape, promoting liquid–liquid phase separation at low concentrations and stabilizing dynamic α-helical intermediates. At higher RNA concentrations, both peptides transition into distinct aggregated states, amorphous for LL-37 and fibrillar for PSMα3, correlating with divergent functional outcomes. The amyloid inhibitor EGCG abolishes the bioactivity of both peptides by redirecting assembly into non-functional aggregates, highlighting that activity depends on supramolecular architecture and reversibility rather than aggregation per se. Together, these findings establish RNA as an environmental regulator of α-helical peptide assemblies and reveal phase transitions as tuneable determinants of peptide function, with implications for microbial virulence, innate immunity, and therapeutic intervention in infectious and protein-aggregation–associated diseases.