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.

Amyloid aggregation of Streptococcus mutans Cnm influences its collagen-binding activity

“…TEM visualization of amyloid material induced by mechanical agitation of purified proteins, followed by treatment with proteinase K, revealed a mat-like structure for glycosylated Cnm but a fibrillar morphology for non-glycosylated Cnm and CBD. Mat-like aggregates have been proposed as a more biologically germane form of S. mutans amyloids that represent a supramolecular structure composed of amyloid fibers, monomers, and oligomer intermediates (48) where pure fibers, achieved by proteolytic digestion of S. mutans amyloid-containing mats, are likely only seen in laboratory settings. It has also been shown that the addition of monomeric protein to purified fibrils shifts the morphology of fibrils back to mats (48).…”

“…Because S. mutans amyloid mats have been confirmed to exhibit the same characteristic X-ray fiber diffraction pattern as isolated amyloid fibers (48), and because glycosylation of Cnm has been shown to protect this protein from proteolytic degradation (41), the visualization of amyloid mats rather than fibers by TEM confirms the formation of amyloid by native glycosylated Cnm.…”

“…A defining feature of amyloid aggregates is protease resistance (45)(46)(47). In S. mutans, protein amyloids form mat-like composite aggregates in which typical amyloid fibrillar structures are only revealed following protease digestion of protein monomers or protease-sensitive oligomeric intermediates (48). To obtain direct evidence that Cnm forms amyloids, stirred samples of full-length glycosylated Cnm, non-glycosylated Cnm (rCnm) and rCBD were treated with proteinase K and visualized by transmission electron microscopy (TEM) (Figure 4).…”

The Multifaceted Nature of Streptococcal Antigen I/II Proteins in Colonization and Disease Pathogenesis

“…AgI/II fragments comprised of the C-domain are able to form the functional amyloid fibers in vitro (Tang et al, 2016;Barran-Berdon et al, 2020). Furthermore, AgI/II functional amyloids contribute to stabilization of S. mutans biofilms (Oli et al, 2012;Tang et al, 2016;Barran-Berdon et al, 2020). Together, this information suggests a critical role of AgI/II protein interactions for the stabilization of streptococcal communities, as well as in the formation of a structural foundation in biofilms that may be taken advantage of by other species.…”

“…These released fragments can interact with surface anchored AgI/II proteins, particularly through the V-domain of the surface anchored protein with either the V-or C-domains of the fragmented protein (Heim et al, 2015;Rego et al, 2016;Tang et al, 2016;Rivière et al, 2020). AgI/II fragments comprised of the C-domain are able to form the functional amyloid fibers in vitro (Tang et al, 2016;Barran-Berdon et al, 2020). Furthermore, AgI/II functional amyloids contribute to stabilization of S. mutans biofilms (Oli et al, 2012;Tang et al, 2016;Barran-Berdon et al, 2020).…”