Functional amyloids in Streptococcus mutans, their use as targets of biofilm inhibition and initial characterization of SMU_63c

Abstract

Amyloids have been identified as functional components of the extracellular matrix of bacterial biofilms. Streptococcus mutans is an established aetiologic agent of dental caries and a biofilm dweller. In addition to the previously identified amyloidogenic adhesin P1 (also known as AgI/II, PAc), we show that the naturally occurring antigen A derivative of S. mutans wall-associated protein A (WapA) and the secreted protein SMU_63c can also form amyloid fibrils. P1, WapA and SMU_63c were found to significantly influence biofilm development and architecture, and all three proteins were shown by immunogold electron microscopy to reside within the fibrillar extracellular matrix of the biofilms. We also showed that SMU_63c functions as a negative regulator of biofilm cell density and genetic competence. In addition, the naturally occurring C-terminal cleavage product of P1, C123 (also known as AgII), was shown to represent the amyloidogenic moiety of this protein. Thus, P1 and WapA both represent sortase substrates that are processed to amyloidogenic truncation derivatives. Our current results suggest a novel mechanism by which certain cell surface adhesins are processed and contribute to the amyloidogenic capability of S. mutans. We further demonstrate that the polyphenolic small molecules tannic acid and epigallocatechin-3-gallate, and the benzoquinone derivative AA-861, which all inhibit amyloid fibrillization of C123 and antigen A in vitro, also inhibit S. mutans biofilm formation via P1-and WapA-dependent mechanisms, indicating that these proteins serve as therapeutic targets of anti-amyloid compounds.

Anti-Biofilm Activity of a Self-Aggregating Peptide against Streptococcus mutans

“…In S. mutans , a major constituent of the S. mutans biofilm matrix was shown to be an amyloid-forming protein (Oli et al, 2012). This Ala-rich protein, known as “antigen I/II, PAc, or P1” (not to be confused with the synthetic peptide P1 used in this study), along with other cell-surface-localized amyloidogenic proteins, are important for biofilm stability (Oli et al, 2012; Besingi et al, 2017). The Ala-rich, β-sheet forming properties of the synthetic P1 peptide may interfere with native amyloid-forming proteins.…”

Characterization of an intermolecular quaternary interaction between discrete segments of the Streptococcus mutans adhesin P1 by NMR spectroscopy

“…Previous work had demonstrated the ability of the C123 truncation fragment (aka AgII) of S. mutans P1 to interact with covalently attached full‐length P1 (aka Ag I/II) on the bacterial cell surface , to adhere to salivary agglutinin , and to form amyloid fibrils within biofilms . However, previous experiments lacked sufficient resolution to characterize the molecular nature of the C123 interaction with other P1 polypeptides.…”

“…C123 has also been identified as the amyloid forming moiety of S. mutans P1 . S. mutans mutants lacking the spaP gene that encodes P1 lose sensitivity to biofilm inhibition by known inhibitors of amyloid fibrillization suggesting a specific contributory role for P1 amyloid formation during biofilm development.…”

“…It interacts with the salivary agglutinin glycoprotein complex, composed predominantly of the scavenger receptor gp340/DMBT1, host cell matrix proteins and other bacteria to adhere the pathogen to tooth surfaces . More recent work also highlights the role P1 plays in adhesive interactions and biofilm development, via specific quaternary structure assembly and amyloid formation , further contributing to S. mutans virulence properties. Characterizing P1’s quaternary interactions at the molecular level is essential to a more complete understanding of S. mutans pathogenesis, and the rational development of novel therapeutic interventions.…”

“…As part of that study, surface plasmon resonance experiments identified a specific interaction between recombinant purified A3VP1 and C123 polypeptides. It is also now known that functional amyloid formation by S. mutans contributes to biofilm development . P1 is one of the three S. mutans proteins identified thus far that forms amyloid fibrils, with C123 representing the amyloid forming moiety.…”