Periodic Reporting for period 1 - PSMNano (Probing the fibrillation of Staphylococcal amyloids and their interactions with Membranes at the Nanoscale)
Berichtszeitraum: 2022-07-01 bis 2024-06-30
Staphylococcus aureus is a human commensal of the microbiota and the human epithelia, which can turn into an opportunistic pathogen, eventually causing life-threatening diseases. Due to its key role in nosocomial infections and its resistance to many antibiotics, S. aureus constitutes major political and clinical issues worldwide. To elicit less antibioresistance, current drug development strategies focus on targeting virulence determinants of S. aureus, and require, first and foremost, a comprehensive understanding of the molecular mechanisms S. aureus has developed to passively or actively evade host elimination. In this context, S. aureus virulence has been shown to critically depend on the production of the phenol-soluble modulins α3 (PSMα3) peptides, that have recently drawn much attention due to their key roles in invasion and infection. Overproduced by multiresistant strains, those amphipathic peptides not only exhibit cytolytic activities towards human cells but also eventually trigger pro-inflammation processes in a receptor (FPR2)-dependent manner. Such diverse functions rely on specific mechanisms of interaction, either recognition and/or perturbation, with cell membranes. Interestingly, PSMα3 can self-assemble into insoluble fibrils, characterized by the unique cross-α structure, reminiscent of the cross-β scaffold of pathogenic amyloids studied in light of neurodegenerative disorders. Based on recent controversial data, the cross-α fibrillation would be required for PSMα3 to exert its functions. Yet, most of the current knowledge arises from ensemble techniques, only providing average behavior of all PSMα3 entities formed along the fibrillation, and thus partially ignoring the complex interplay between the structural polymorphism of PSMα3, and their interactions with cells. This project thus aims at providing insights into the structure-function relationship of the virulent factors PSMα3, by investigating, at the cellular and molecular levels, their underestimated dynamic interactions with cell membranes, the first key steps towards their physiological functions in S. aureus. To reach this objective, Combination of in vitro and in cellulo experiments, based on Atomic Force Microscopy (AFM)-related techniques, combined with complementary biophysical approaches, will be performed to: (1) unravel the fibrillation kinetics and structures of PSMα3 interacting with membranes, (2) decipher the mechanisms governing the interaction of PSMα3 and membranes, and (3) unveil the receptor-mediated processes turning PSMα3 into proinflammatory agents.
During this MSCA fellowship, we have demonstrated in vitro that the charge provided by the N-terminal capping of PSMα3 alters its local interactions with model membranes of controlled lipid composition, without compromising its intrinsic ability to form amyloid fibrils, yet with eventual distinct kinetics as probed by Nuclear Magnetic Resonance (NMR) and fluorescence spectroscopy. Atomic Force Microscopy (AFM) studies have revealed that the N-ter capping indeed eventually dictates PSMα3-membrane binding via electrostatic interactions with the lipid head groups: while N-formylated and N-acetylated peptides only bind membranes containing zwitterionic lipids (e.g. DOPC), N-deformylated peptides also deposit on negatively charged membranes (e.g. DOPG/DOPE). Furthermore, PSMα3 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 when present. Strikingly, real-time AFM imaging has provided compelling evidence supporting the role of intermediate protofibrillar entities, formed along PSMα3 self-assembly, in its formylated and acetylated forms, in the loss of membrane integrity. Those entities tend to accumulate and insert within the lipid membranes, subsequently leading to membrane thinning in a peptide concentration and lipid-dependent manner. Polarized Attenuated Total Reflectance (ATR) FTIR spectroscopy has further allowed structural characterization of those entities, that, promoted at the membrane interface, induced lipid depletion in specific membranes. They are enriched in α-helical content compared to their monomeric forms, thus suggesting their transition to cross-α (proto)fibrils, as previously demonstrated for PSMα3 on its own. Interestingly, nanoinfrared spectroscopy, a coupling between AFM and IR that enables structural investigation on individual nano-objects, has additionally emphasized that those protofibrils formed at the lipid membrane interface differ in their secondary structures from the mature fibrils of PSMα3. To get closer to physiological relevance, in cellulo experiments were also performed to explore this “oligomeric hypothesis”, i.e. to investigate if intermediate entities could be responsible for cytotoxic activities, as supported by associated lipid membrane damage in vitro. We first revealed that PSMα3 can only fibrillate in a cellular medium deprived of serum, suggesting lipoproteins eventually inhibit the formation of amyloid structures. In such minimal medium, PSMα3 induces a concentration and time-dependent cytotoxicity towards HEK cells, that is higher for the formylated form compared to the acetylated one, likely due to their distinct kinetics of self-assembly. Importantly, cells treated with the monomeric and fibrillary forms of the peptides remained mostly viable over the same timescales, emphasizing the role of fibrillation, and intermediate entities, in mediating lytic activities. Overall, this multiscale and multimodal approach has shed new light on the key roles of N-terminal capping and intermediate self-assembling entities in dictating deleterious interactions of PSMα3 with membrane lipids and living cells, likely underscoring its ultimate cellular toxicity in vivo, and in turn S. aureus pathogenesis.
While the remarkable cross-a fibrils of PSMa3 have been associated to its toxicity towards human cells in vivo, the molecular mechanisms underlying such functions have so far remained elusive. Here, combining spectroscopic and highly-resolved imaging techniques, we have bridged the in vitro behaviour of PSMa3 at the lipid membrane level, to its toxic activities in cellulo. We have demonstrated, for the first time, the key roles of N-terminal capping and intermediate oligomeric entities, rather than mature fibrils, in driving lipid-specific membrane damages, and in turn cytotoxic activities. Real-time imaging, at the nanoscale, importantly revealed dynamic and mutual interactions between PSMa3 and specific lipids, both via electrostatic and hydrophobic interactions, fibrillation being promoted at the membrane interface subsequently leading to disruption. Further, the serum-dependent cytotoxic function of PSMa3 was first revealed and associated to the role of lipoproteins as inhibitors of amyloid fibrillation, thus preventing the formation of toxic intermediate entities. Such findings interestingly fuel the increasing debate on the amyloid cascade hypothesis, even in the context of functional amyloids. For the future design of efficient therapeutics to S. aureus infections, e.g. able to specifically target those intermediate entities, one would need further molecular insights into PSMa3-membrane interactions. A structure-based peptide inhibitor could constitute an innovative strategy, thus requiring deeper characterization of the specific structures of toxic entities, and the mechanisms underlying their formation when interacting with cell membranes in cellulo. Such an investigation has been partially achieved in this project and would be pursued thanks to new fundings.