Salt-Inducing Assembly Polymorphism Strategy for Cytotoxicity Differentiation of Phenol-Soluble Modulin α3 Assemblies

Abstract

Phenol-soluble modulin α3 (PSMα3) can self-assemble into fibrous assemblies with a unique "cross-α" sheet structure, which serves as a key virulence factor in the infection of Staphylococcus aureus. However, the structure−cytotoxicity relationships of PSMα3 still remain elusive. Herein, we utilized the strategy of salt-inducing assembly polymorphism to controllably prepare three PSMα3 assemblies with morphological and structural distinctions, including amorphous aggregates (AAs), rigid fibrils (RFs), and oligomers/curvilinear fibrils (OCFs), which provided a convincing method to facilitate the structure−cytotoxicity investigation of PSMα3 assemblies. Our results affirmed that amyloid fibrillation was essential for the enhancement of PSMα3 cytotoxicity, which was proved based on the evidence that RFs and OCFs both triggered more obvious cytotoxicity than AAs. Furthermore, our study also demonstrated that the cytotoxicity was severely dependent on the size and structure of PSMα3 fibrils. In detail, smaller OCFs rich in α-helices exhibited stronger virulence than RFs with larger sizes and low α-helical contents. The cytotoxicity caused by such fibrils was achieved via a membrane-disrupting mechanism, in which RFs and OCFs might be prone to membrane thinning and perforation, respectively. This strategy of salt-inducing PSMα3 assembly polymorphism facilitated the comprehension of the relationship between the characteristics of PSMα3 assemblies and their cytotoxicity and was also helpful to understanding the intrinsic assembly mechanism of the PSMα3.

The Molecular Mechanism of PSMα3 Aggregation: A New View

“…However, interchain salt bridges operate only at lower-order aggregates, and peptides in the structurally similar oligomeric and fibrillar states are primarily held by van der Waals forces which is further exemplified by the aggregability of a sequence having no salt bridge forming ability. The fibril–oligomer structural similarity hints at the theory that the peptides initially assemble to an oligomeric state, which is supposed to be the toxic species and which drives further aggregation . The observed structural similarity originates due to the distorted curvilinear conformations acquired by the peptides, which appear as orthogonal relative orientation, which is believed to possess cytotoxic character .…”

“…A consensus on the origin of cytotoxicity via PSMα3 is yet to be achieved as a myriad of preconditions has been put forward to date. 14,15,17,21,23,27 The present work deconvolutes the complexity of the aggregation paradigm to identify the operative facts. The aggregation propensity of the wild-type PSMα3 increases at a faster rate as the local concentration of the peptide increases 11 and the monotonic trend breaks at the fibrillar concentration hinting toward the existence of a critical concentration of the peptide that can effectively self-assemble and remain aggregated, which indirectly proves the requirement of a "seed" for the faster formation of aggregates at higher concentrations of the sequence.…”

“…21 The observed structural similarity originates due to the distorted curvilinear conformations acquired by the peptides, which appear as orthogonal relative orientation, which is believed to possess cytotoxic character. 21 The fibrillar state, whether wild-type or mutated, undergoes conformational polymorphism, 14,24 and thus, it is an intrinsic property of the PSMα3 fibril 5 owing to the chameleon behavior. 14 Finally, the operative dominating forces vary in an aggregation-phase-dependent fashion considering the dynamic fluidity in the identity of the most toxic species (monomer, dimer, oligomer, and fibril).…”

“…The expected regular cross-β fibrillar form produced by other PSMs (structural) and PSMα3’s multifaceted pathogenicity on the human body (functional) drives the motivation behind the recent curiosity about this bacterial functional amyloid. Limited, yet seminal, works have bubbled up to rationalize the observed functionalities of PSMα3 based on its structure, e.g., characterization of the monomeric state, factors stabilizing the mature fibril, − the kinetics of aggregation, − nature/location-, − length-, − structure-, , and phase-specific ,,, roles of the constituting residues in diverse functionalities of the peptide, and membrane interaction with PSMα3. , However, the nascent phase of the research is filled with contradictory observations producing inconclusive inferences on the structure of the fibrillar state, ,,,, the factors required for cytotoxicity, − ,,,− sequence/structure-encoded functional specificity, and salt concentration dependence of the structure–toxicity relationship. , The lack of a comprehensive description of the deleterious aggregation process has motivated the present study, which focuses on the switching of specific roles of the charged and the hydrophobic residues at different stages of the aggregation pathway by a comparative kinetic–thermodynamic analysis between the wild-type (WT) PSMα3 sequence and three designed variants with isoleucine to model operatively increased hydrophobicity (WT → IMT, F/V/L < I), glutamine to model lack of charge with comparable hydrophilicity (WT → QMT, D/E/K ≈ Q), and proline to model moderately less hydrophilicity with one less charged residue (WT → PMT, D > P) mutations (see Supporting Information, justification behind the modeling of noncanonical mutants, Figures S1 and S2) at a low salt concentration (see Supporting Information, rationalization behind the choice of low salt concentration).…”

Amyloid‐Based Albumin Hydrogels

“…Another deciding factor during protein denaturation and amyloid fibril assembly is the presence of salts. [39,40] In preliminary experiments, we tested the effect of increasing ionic strength, by adding 150 mm NaCl in the precursor mixtures, and observed quick aggregation as evidenced by clouding of the solution and precipitation. Therefore, we prepared all subsequent samples in water.…”