The findings show that bacterial virulence can be regulated through environmentally controlled transitions between protein assemblies, identifying membrane accumulation as a promising anti-virulence target.
Abstract
Phenol-soluble modulin α1 (PSMα1) is a cytolytic peptide secreted by Staphylococcus aureus that contributes to host-cell damage and biofilm stability, yet the relationship between its assembly behavior and function remains incompletely understood. Here, we combine cellular assays, molecular spectroscopy, and high-resolution structural approaches to elucidate how environmental conditions govern PSMα1 activity and supramolecular organization. Live-cell imaging and cytotoxicity assays show that PSMα1 accumulates at the plasma membrane of human cells prior to membrane permeabilization, linking membrane association to cytotoxic outcomes. This process is strongly attenuated by epigallocatechin gallate (EGCG). Cryogenic electron microscopy (cryo-EM) reveals two polymorphic canonical amyloid fibril architectures that share a conserved hydrophobic core and protofilament interface. In parallel, we identify pH as a key determinant of PSMα1 assembly pathways, driving a bifurcation between cross-β amyloid fibrils at extreme acidic and alkaline conditions and heterogeneous, long-lived, thermally stable α-helical nanotubular assemblies at acidic, near-neutral, and slightly alkaline conditions, which act as transient intermediates under highly acidic conditions. Together, these findings demonstrate that PSMα1 is not a single amyloid structure but a condition-dependent structural system in which environmental cues dictate assembly, membrane interaction, and cytotoxic function. This work provides a framework for understanding how polymorphic assembly of bacterial virulence peptides interfaces with host-cell interactions and suggests new avenues for targeting PSM-mediated pathogenicity. Statement of significance Staphylococcus aureus causes severe infections and uses the peptide PSMα1 to damage host cells and strengthen protective biofilms. Like many disease-associated proteins, PSMα1 self-assembles into amyloid fibrils, though their role in virulence remains unclear. We show that PSMα1 does not adopt a single architecture. Instead, environmental changes, such as those at infection sites, drive the peptide into distinct assemblies, including cross-β amyloid fibrils and unexpectedly stable nanotubes with α-helical features. Live-cell imaging shows PSMα1 accumulates at the plasma membrane before cell death, and that epigallocatechin gallate reduces membrane association and toxicity. These findings show that bacterial virulence can be regulated through environmentally controlled transitions between protein assemblies, identifying membrane accumulation as a promising anti-virulence target.
It is demonstrated for the first time that amyloid formation, similar to MV production, is regulated by environmental stresses, and a potential role for this complex in the environmental adaptation of S. aureus is suggested.
Zhuang Zhu, Jinxi Yue, Yalan Feng et al.· Microbial Pathogenesis· 0 citations
Early soluble intermediates in amyloid formation are difficult to characterize because they are transient and obscured by competing insoluble aggregation. Here, we use scE2C, an engineered covalently linked version of the HPV16 E2 DNA-binding dimer, as a tractable model to stabilize and characterize soluble amyloid-like assemblies that are poorly resolved in the native dimeric protein. Whereas native E2C progresses toward visible insoluble aggregation upon heating, scE2C undergoes an irreversible transition to a soluble β-rich state. This state displays amyloid-sensitive signatures, including enhanced thioflavin T fluorescence, a Congo Red spectral shift, and increased ANS binding. Dynamic light scattering and atomic force microscopy revealed soluble assemblies comprising small spherical particles, larger spherical particles, and worm-like protofibrillar structures, which were further visualized by negative-stain and cryo-electron microscopy. Quantitative comparison of DLS- and AFM-derived dimensions supports a hierarchical organization in which smaller spherical particles behave as building blocks for larger particles and necklace-like worms. Isothermal circular dichroism kinetics across concentration and temperature were consistent with an assembly-coupled structural transition, with an apparent low-order nucleus of approximately 2 ± 1 scE2C protomeric units and growth compatible with low-order addition. Specific DNA binding fully inhibited the thermal transition, indicating that access to this protofibrillar branch is constrained by the functional DNA-bound state. Together, these results support the view that covalent linkage of the E2C dimer can provide experimental access to soluble early amyloid-like assembly states under conditions where native E2C progresses toward insoluble aggregation.
Protein aggregation is increasingly recognized as a biologically relevant process in viral proteins, yet the molecular determinants governing such phenomena remain poorly understood. Intrinsically disordered proteins (IDPs) are ubiquitous in viral proteomes, where conformational plasticity not only enables functional diversity but also permits aberrant self‐assembly. Members of the Paramyxoviridae family, including henipaviruses such as the Nipah and Hendra viruses (two biosafety level‐4 pathogens), encode the P, V, and W proteins that share a common intrinsically disordered N‐terminal domain (NTD). V and W are virulence factors that undergo fibrillation. Here, we explored the aggregation landscape of seven homologous PNT1 subdomains from the NTD that harbors a conserved cryptic amyloidogenic region (CAR). By integrating Taylor dispersion analysis (TDA), Raman spectroscopy, and all‐atom molecular dynamics (MD) simulations, we unraveled both early assembly kinetics and structural outcomes. Despite the conserved CAR motif, the PNT1 homologs exhibit strikingly divergent aggregation behaviors. TDA revealed distinct oligomerization pathways with variations in nucleation, growth kinetics, and monomer recruitment, indicating virus‐specific pathways. Raman spectroscopy unveiled substantial variations in β‐sheet content, side‐chain packing, and aromatic residue environments, while MD simulations showed that conformational heterogeneity in the CAR flanking regions modulates hydrogen‐bond networks and structural stability. These findings indicate that the sequence context beyond a conserved amyloidogenic core governs the aggregation landscape of Henipavirus P, V, and W proteins. Evolutionary diversification modulates conformational heterogeneity and assembly pathways, giving rise to distinct structural outcomes. This work sheds light on the molecular grammar underlying viral protein fibrillation with potential implications for viral pathogenicity.
Harshita Sawdekar, Julien Mignon, Frank Gondelaud et al.· Protein Science· 0 citations
Polysaccharides remain the least understood biomacromolecules, particularly in terms of the relationship between their chemical structure and physical properties. On the other hand, polysaccharides often serve as the main structural components in biofilms: surface-attached aggregates of bacterial cells encased within a mechanically resilient extracellular matrix. The large chemical space explored by bacteria within biofilms provides excellent opportunities to establish the structure-function relationship for polysaccharides. In this paper, we systematically characterize various polymer properties of Vibrio polysaccharide (VPS), the major exopolysaccharide in biofilms formed by Vibrio cholerae, the causative agent of pandemic cholera. Using a combination of shear rheology, dynamic and static light scattering, and small-angle X-ray scattering, we measure the viscosity, molecular weight, persistence length, radius of gyration, and hydrodynamic radius of this chemically unique biopolymer. Combining all-atom and coarse-grained simulations, we show how the conformational flexibility of a single glycosidic linkage within each VPS monomer can lead to dramatic compaction of the entire polymer chain and nonclassical entanglement behavior. Our comprehensive quantification represents a rare endeavor for bacterial biofilms, whose matrix composition and physical properties remain largely nebulous; it also represents a significant step towards a detailed understanding of the molecular origins of biofilm mechanics.
Kee-Myoung Nam, Nathan Fowler, Rajan Kandel et al.· bioRxiv· 0 citations
The tumor suppressor protein p16INK4a plays a key role in cell cycle control and is frequently inactivated in human cancers. We previously discovered that oxidation of its single cysteine residue induces a structural transition from the functional monomeric form into inactive amyloid fibrils. Here, we investigate whether five physiologically relevant oxidants can trigger the conformational switch of the protein. Using time‐resolved SDS‐PAGE, thioflavin‐T fluorescence assays, mass spectrometry, and electron microscopy, we show that peroxymonocarbonate and hypothiocyanous acid (HOSCN) efficiently induce cysteine‐dependent dimerization with subsequent amyloid formation of recombinant p16. In contrast, hydrogen peroxide, hypochlorous acid, and taurine chloramine are less efficient and predominantly lead to the formation of amorphous aggregates. These findings demonstrate that physiological oxidants differ in their ability to induce amyloid transitions in p16, with potential implications for oxidative stress‐mediated inactivation of this cell‐cycle regulator protein.
Briana R. Smith, N. J. Magon, A. Sethi et al.· Protein Science· 0 citations
Findings provide direct structural evidence for amyloid evolution in vivo and support a chaperone-mediated mechanism of conformer selection within a polymorphic amyloid population.
Ziang Wang, Samantha L. Weetman, Barbara Altenhuber et al.· bioRxiv· 0 citations
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