N-formylation modifies membrane damage associated to PSMα3 interfacial fibrillation

The virulence of Staphylococcus aureus, a multi-drug resistant pathogen, notably depends on the expression of the phenol soluble modulins 3 (PSM3) peptides, able to self-assemble into amyloid-like cross- fibrils. Despite remarkable advances evidencing the crucial, yet insufficient, role of fibrils in PSM3 cytotoxic activities towards host cells, the relationship between its molecular structures, assembly propensities, and modes of action remains an open intriguing problem. In this study, combining Atomic Force Microscopy (AFM) imaging and infrared spectroscopy, we first demonstrated in vitro that the charge provided by the N-terminal capping of PSM3 alters its interactions with model membranes of controlled lipid composition, without compromising its fibrillation kinetics or morphology. N-formylation eventually dictates PSM3 - membrane binding via electrostatic interactions with the lipid head groups. Furthermore, PSM3 insertion within the lipid bilayer is favoured by hydrophobic interactions with the lipid acyl chains, only in the fluid-phase of membranes, and not in the gel-like ordered domains. Strikingly, our real-time AFM imaging emphasizes how intermediate protofibrillar entities, formed along PSM3 self-assembly and promoted at the membrane interface, likely disrupt membrane integrity via peptide accumulation, and subsequent membrane thinning in a peptide concentration and lipid-dependent manner. Overall, our multiscale and multimodal approach sheds new light on the key roles of N-formylation and intermediate self-assembling entities, rather than mature fibrils, in dictating deleterious interactions of PSM3 with specific membrane lipids, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.