Enhanced purification coupled with biophysical analyses shows cross-β structure as a core building block for Streptococcus mutans functional amyloids

Abstract

Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Three amyloidogenic proteins, P1, WapA, and Smu_63c were previously identified. C123 and AgA are naturally occurring amyloid-forming fragments of P1 and WapA, respectively. We determined that four amyloidophilic dyes, ThT, CDy11, BD-oligo, and MK-H4, differentiate C123, AgA, and Smu_63c amyloid from monomers, but non-specific binding to bacterial cells in the absence of amyloid precludes their utility for identifying amyloid in biofilms. Congo red-induced birefringence is a more specific indicator of amyloid formation and differentiates biofilms formed by wild-type S. mutans from a triple ΔP1/WapA/Smu_63c mutant with reduced biofilm forming capabilities. Amyloid accumulation is a late event, appearing in older S. mutans biofilms after 60 hours of growth. Amyloid derived from pure preparations of all three proteins is visualized by electron microscopy as mat-like structures. Typical amyloid fibers become evident following protease digestion to eliminate non-specific aggregates and monomers. Amyloid mats, similar in appearance to those reported in S. mutans biofilm extracellular matrices, are reconstituted by co-incubation of monomers and amyloid fibers. X-ray fiber diffraction of amyloid mats and fibers from all three proteins demonstrate patterns reflective of a cross-β amyloid structure.

Figure 1

Transmission electron microscopy (TEM) of amyloid mats and purified fibers (A) TEM images of S. mutans amyloid before and after removal of residual monomer. a, c and e) Induced amyloid produced from purified C123, AgA, and Smu_63c, respectively. b, d and f). Amyloid material produced from C123, AgA, and Smu_63c following proteinase K digestion. (B) TEM of purified C123 amyloid fibers incubated without stirring with and without added monomers. a, c, and e) Purified C123 fibers incubated for 2 weeks alone (a) or with added C123 monomer at final concentrations of 1% (c) or 10% (e). e, d, and f) Purified C123 fibers incubated for 4 weeks alone (b) or with added C123 monomer at final concentrations of 1% (d) or 10% (f).

Figure 2

Amyloidophilic dye uptake assays. Ten micrograms of purified recombinant C123 (red bars), AgA (blue bars), or Smu_63c (green bars) were reacted with ThT, CDy11, BD-oligo, and MK-H4. Empty bars correspond to the purified monomeric proteins, solid bars to the samples after amyloid induction, and diagonal patterned bars to the purified amyloid fibers after treatment of the amyloid material to remove residual monomers. C123 and AgA polypeptides correspond to naturally occurring amyloidogenic derivatives of P1 and WapA, respectively. Arbitrary units (a.u.) ThT (Excitation = 440, Emission = 485), CDy11 and BD-Oligo (Ex = 530, Em = 580), and MK-H4 (Ex = 475, Em = 567). Protein concentration 0.1 mg/mL. Error bars represent standard deviations.

Figure 3

Congo red-induced birefringence of amyloid material before and after treatment with proteinase K (PK). Purified recombinant Smu_63c, and recombinant C123 and AgA polypeptides, which correspond to naturally occurring amyloidogenic derivatives of P1 and WapA respectively, were evaluated. Samples were visualized by bright-field microscopy and under crossed polarizing light filters. Scale Bar 50 µm.

Figure 4

Schematic representation of the solved or predicted tertiary structures of P1-C123 (a), WapA-AgA (b), and Smu_63c (c). The illustration of C123 is based on the solved crystal structure (PDB id:3QE5). The predicted structures of AgA and Smu_63C were determined using I-TASSER. Blue to red rainbow coloring indicates progression from N- to C-termini.

Figure 5

X-ray fiber diffraction patterns of amyloid mats and purified amyloid fibers derived from P1-C123, WapA-AgA and, Smu_63c. Black arrows indicate meridonal diffraction at ~4.8 Å. White arrows indicate equatorial diffraction at ~10.5 Å.

Figure 6

Assessment of amyloid formation during S. mutans biofilm growth in a microfluidics system. (a) Phase contrast and red fluorescence images following staining of 72 h biofilms of S. mutans wild-type and a triple mutant (Δ3) lacking P1 (encoded by spaP), WapA, and Smu_63c with CDy11. Similar patterns of dye binding to both strains suggests CDy11 reactivity with non-amyloid material. (b) CR-induced birefringence of 72 h biofilms of S. mutans wild-type (left) and the Δ3 triple mutant (right). Insets indicate pixel frequency of the brightest visual locations (red boxes) in each image. In contrast to staining with CDy11, biofilms of the WT and mutant strains could be differentiated on the basis of CR-induced birefringence.

Figure 7

Time course of amyloid formation in S. mutans biofilms. Extracellular birefringent rope-like structures were visualized in biofilm material harvested after 60 h of growth as static cultures. Images were captured by bright-field microscopy and under cross-polarizing light filters to visualize Congo Red-induced birefringence at the indicated time points. Scale Bar 50 µm.