Extreme amyloid polymorphism in Staphylococcus aureus virulent PSMα peptides

Abstract

Members of the Staphylococcus aureus phenol-soluble modulin (PSM) peptide family are secreted as functional amyloids that serve diverse roles in pathogenicity and may be present as full-length peptides or as naturally occurring truncations. We recently showed that the activity of PSMα3, the most toxic member, stems from the formation of cross-α fibrils, which are at variance with the cross-β fibrils linked with eukaryotic amyloid pathologies. Here, we show that PSMα1 and PSMα4, involved in biofilm structuring, form canonical cross-β amyloid fibrils wherein β-sheets tightly mate through steric zipper interfaces, conferring high stability. Contrastingly, a truncated PSMα3 has antibacterial activity, forms reversible fibrils, and reveals two polymorphic and atypical β-rich fibril architectures. These architectures are radically different from both the cross-α fibrils formed by full-length PSMα3, and from the canonical cross-β fibrils. Our results point to structural plasticity being at the basis of the functional diversity exhibited by S. aureus PSMαs.

Fig. 1

PSMα1 and PSMα4 form cross-β amyloid fibrils. Electron micrographs of PSMα1 (a) and PSMα4 (b) show elongated fibrils; scale bars represent 100 nm. X-ray fibril diffraction of PSMα1 (c) and PSMα4 (d) shows major diffraction orthogonal arches at 4.6 Å and 12 Å spacings, indicative of the cross-β signature

Fig. 2

PSMα1 and PSMα4 spine segments form steric zipper fibrils. a Crystal structure of the PSMα1 spine segment IIKVIK determined at 1.1 Å resolution, showing the canonical steric-zipper architecture of tightly mated β-sheets composed of parallel β-strands. In the left panel, the view is perpendicular to the fibril axis and the β-strands run horizontally. In the right panel, the view is down the fibril axis. The segments are shown in ribbon representation, with the side chains shown as sticks. The carbons within each β-sheet are colored either gray or light blue, and nitrogen atoms in side chains are colored blue. Here, six layers of β-strands are presented. Theoretically, fibrils can contain thousands of layers. A similar structure of the PSMα4-IIKIIK and its crystal packing are shown in Supplementary Fig. 3. b Electron micrographs visualizing PSMα1-IIKVIK and PSMα4-IIKIIK fibrils; scale bars represent 100 nm. c X-ray fibril diffraction of PSMα1-IIKVIK and PSMα4-IIKIIK shows major diffraction orthogonal arches at 4.6 Å and 10 Å spacings, indicative of the cross-β signature

Fig. 3

Antibacterial activity of the fibril-forming LFKFFK from S. aureus PSMα3. Disc diffusion assay testing antibacterial activity against different bacteria. In this assay, the antibacterial agent diffuses into the agar and inhibits germination and growth of the test microorganism. a LFKFFK and KLFKFFK segments from PSMα3, but not the steric-zipper forming segments PSMα1-IIKVIK and PSMα4-IIKIIK, showed dose-dependent antibacterial activity against M. luteus and S. hominis. LFKFFK and KLFKFFK were not toxic to S. aureus, the bacterium which secretes PSMα3. Discs soaked with only DDW or Dimethyl-sulfoxide (DMSO) served as controls. The results of the disc diffusion assay coincide with the antibacterial activity seen in solution (Supplementary Fig. 4). b Electron micrographs visualizing fibrils and nano-crystals formed by LFKFFK; scale bar represents 400 nm, and straight and twisted crystalline fibrils of KLFKFFK; scale bar represents 200 nm

Fig. 4

Structural polymorphism of the LFKFFK segment from S. aureus PSMα3. a Crystal structure of polymorph I of the PSMα3 spine segment LFKFFK determined at 1.5 Å resolution. The structure reveals a unique amyloid-like hexameric architecture, which forms elongated cylindrical cavities along the fibril axis. The view is down the fibril axis. The segments are shown in ribbon representation, with side chains shown as sticks with gray carbons and blue nitrogen atoms. Water molecules (oxygen in red) and chloride ions (green) that counteract the charge of the lysine side chains, are shown as small spheres. b Crystal structure of LFKFFK polymorph II determined at 1.85 Å resolution, revealed a rare amyloid-like architecture of out-of-register β-sheets, in which each β-strand is at an angle of ~ 50° from the fibril axis, instead of the close to 90° angle found for in-register sheets. In both polymorphs, the β-sheets are composed of anti-parallel strands. In the left panel, the view is perpendicular to the fibril axis, and in the right panel, the view is down the fibril axis. The segments are shown in ribbon representation, with side chain shown as sticks. The carbons within each β-sheet are colored either gray or light blue, and nitrogen atoms in side chains are colored blue

Fig. 5

LFKFFK forms thermo-reversible fibrils compared to the stable IIKIIK fibrils. LFKFFK formed fibrils at room temperature, which dissolved upon heating to 50 °C and reformed after cooling, albeit with a different morphology; scale bars represent 500 nm for the sample at room temperature and 300 nm for the samples taken after heating to 50 °C and after re-cooling. In contrast, IIKIIK fibrils were thermo-stable; scale bars represent 100 nm for the sample at room temperature, 50 nm for the sample taken after heating to 50 °C, and 300 nm for the sample taken after re-cooling. Both peptides were incubated for 3 days at room temperature before fixation and heating-cooling cycles. The same sample was assayed following temperature change. Thermostability of the fibrils corresponded to the crystal structures; IIKIIK showed the highly stable steric-zipper architecture, while LFKFFK formed atypical amyloid-like structures