The cytotoxic Staphylococcus aureus PSMα3 reveals a cross-α amyloid-like fibril

Abstract

Amyloids are ordered protein aggregates, found in all kingdoms of life, and are involved in aggregation diseases as well as in physiological activities. In microbes, functional amyloids are often key virulence determinants, yet the structural basis for their activity remains elusive. We determined the fibril structure and function of the highly toxic, 22-residue phenol-soluble modulin α3 (PSMα3) peptide secreted by Staphylococcus aureus PSMα3 formed elongated fibrils that shared the morphological and tinctorial characteristics of canonical cross-β eukaryotic amyloids. However, the crystal structure of full-length PSMα3, solved de novo at 1.45 angstrom resolution, revealed a distinctive "cross-α" amyloid-like architecture, in which amphipathic α helices stacked perpendicular to the fibril axis into tight self-associating sheets. The cross-α fibrillation of PSMα3 facilitated cytotoxicity, suggesting that this assembly mode underlies function in S. aureus.

Fig. 1.. The cross-α amyloid-like fibril of the full-length PSMα3.

(A) An electron micrograph of PSMα3 fibrils. (B) Fluorescence microscopy images of Thioflavin T stained PSMα3 fibrils. (C) The sequence of S. aureus PSMα3 (UniProt accession number is indicated in brackets). (D and E) The crystal structure of PSMα3 at 1.45 Å resolution, colored according to hydrophobicity (a colored scale bar is shown). (D) A view down the fibril axis. PSMα3 forms parallel α-helical stacks, viewed as ribbons along with a semitransparent surface representation. Facing helical sheets are oriented head to tail. (E) A view perpendicular to the fibril axis. The helices, shown in surface representation, run horizontally. Eight layers of α-helices forming the cross-α structure are depicted. Theoretically, fibrils can contain tens of thousands of layers. The α-helical sheets interact via their hydrophobic face, creating a tight interface. The higher order packing of the crystal structure shows continuous rows of sheets that generate alternating hydrophobic and hydrophilic interfaces (fig. S6A). Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; F, Phe; G, Gly; K, Lys; L, Leu; M, Met; N, Asn; and V, Val.

Fig. 2.. PSMα3 cross-α fibril is reminiscent of amyloid cross-β structure.

(A) The crystal structure of PSMα3. Two mating α-helical sheets are shown. (B) The steric zipper structure of the NNQQNY N, Asn; Q, Gln; Y,Tyr) segment from yeast prion Sup35 (4) (PDB code 1YJO) forms the cross-β spine of amyloid-like fibrils. The two mating β-sheets are composed of parallel β-strands. In both PSMα3 (A) and NNQQNY (B) structures, side-chains protruding from the two sheets intermesh to form a dry, tightly self-complementing interface. The two sheets, in purple and gray, are shown as ribbons, with side chain as sticks. Heteroatoms are colored by atom type (nitrogen in blue, oxygen in red, and sulfur in yellow). In the left panels, the view looks down the fibril axis, and in the right panels, the view is roughly perpendicular to the fibril axis. The α-helices (A) and β-strands (B) run horizontally. Distances between mating sheets and between strands along the sheet are displayed (table S3).

Fig. 3.. PSMα3 toxicity against human T-cells is dependent on its ability to form fibrils.

(A) PSMα3 is toxic to human T-cells in a dose dependent manner. The F3A mutant and the K9P/F11P double mutant, which do not form fibrils (figs. S8 and S9), exhibited much lower levels of cytotoxicity compared to wild-type PSMα3. G16A, a mutant that is helical and which forms fibrils that bind Thioflavin T, served as a control mutant and proved cytotoxic (figs. S8 and S9). (B) Cytotoxicity of PSMα3 was significantly reduced with the addition of Tween 80, a biocompatible surfactant that diminishes fibrillation (figs. S8 and S10). In both panels, error bars represent the SEM of three replicates. The experiment was performed at least three times on different days. * P<0.05 and ** P<0.001 compared to 7.5 µM wild type PSMα3.