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.
Highlights
Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids
S. mutans is a highly acidogenic organism that accumulates in dental plaque where it clings to tooth surfaces in a tenacious biofilm encased in an extracellular matrix (ECM) composed of glucan and fructan polymers, proteins, and extracellular DNA
The protease resistant amyloid-like material harvested from the ECM of S. mutans biofilms exists in an apparent complex with eDNA8
Summary
Streptococcus mutans is an etiologic agent of human dental caries that forms dental plaque biofilms containing functional amyloids. Multiple bacterial functional amyloids have been identified that can contribute to adhesion, biofilm development, genetic competence, cell density regulation, host interactions, and/or aerial hyphae formation[9,10,11,12]. Smu_63c, a third amyloidogenic protein identified in S. mutans, is a secreted protein that appears to serve as a negative regulator of genetic competence and biofilm cell density[26,31]. All three of these S. mutans proteins influence biofilm development, which is inhibited by known inhibitors of amyloid fibrillization such as tannic acid and epigallocatechin gallate[26]. The new information described will facilitate the design of future experiments to evaluate environmental conditions that influence amyloid formation of S. mutans proteins in vitro and within biofilms
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