Small molecule targeting amyloid fibrils inhibits Streptococcus mutans biofilm formation

Abstract

Amyloid fibrils are important scaffold in bacterial biofilms. Streptococcus mutans is an established cariogenic bacteria dwelling within biofilms, and C123 segment of P1 protein is known to form amyloid fibrils in S. mutans biofilms, among which C3 segment could serve as a promising anti-amyloid target due to its critical role in C123-P1 interactions. Recently, small molecules have been found to successfully inhibit biofilms by targeting amyloid fibrils. Thus, our study aimed to screen small molecules targeting C3 segment with the capacity to influence amyloid fibrils and S. mutans biofilms. In silico screening was utilized to discover promising small molecules, which were evaluated for their effects on bacterial cells and amyloid fibrils. We selected 99 small molecules and enrolled 55 small molecules named D1–D55 for crystal violet staining. Notably, D25 selectively inhibit S. mutans biofilms but had no significant influence on biofilms formed by Streptococcus gordonii and Streptococcus sanguinis, and D25 showed no bactericidal effects and low cytotoxicity. In addition, amyloid fibrils in free-floating bacteria, biofilms and purified C123 were quantified with ThT assays, and the differences were not statistically significant in the presence or absence of D25. Morphological changes of amyloid fibrils were visualized with TEM images, where amorphous aggregates were obvious coupled with long and atypical amyloid fibrils. Moreover, amyloid-related genes were upregulated in response to D25. In conclusion, D25 is a promising antimicrobial agent with the capacity to influence amyloid fibrils and inhibit S. mutans biofilms.

Amyloid Aggregates Are Localized to the Nonadherent Detached Fraction of Aging Streptococcus mutans Biofilms

“…Second, once nonamyloid-related background reactivity stemming from the presence of cell wall material was eliminated by filtration of biofilm samples, ThT fluorescence assays also identified amyloid material only in the nonadherent fraction of the older biofilm. Although other studies have reported, based on ThT uptake alone, that amyloid formation is an early event associated with adherent S. mutans biofilms ( 53 – 55 ), our more rigidly controlled experiments indicate otherwise. The third line of evidence comes from confocal microscopy and immunostaining with two different C123-reactive monoclonal antibodies that can discriminate between amyloid and nonamyloid forms of the polypeptide.…”

“…While some studies have utilized in situ uptake and fluorescence intensity of the amyloidophilic dye ThT as a direct indicator of the presence of amyloid within growing S. mutans biofilms ( 53 – 55 ), ThT fluorescence can be misleading due to an interaction of ThT with nonamyloid components of the biofilms. Although amyloid formation is a biofilm-associated process for S. mutans ( 27 , 28 ), we have shown previously that planktonically grown wild-type S. mutans , as well as mutants lacking P1, WapA, and Smu_63c, or Sortase A, all demonstrate an identical uptake of ThT ( 30 ).…”

The Application of Small Molecules to the Control of Typical Species Associated With Oral Infectious Diseases

“…Besingi et al screened and identified a benzoquinone derivative AA-861, which exhibited antibiofilm effects against S. mutans by targeting amyloid fibrils, an important scaffold in S. mutans biofilms (Besingi et al, 2017). Chen et al also screened and identified a small molecule, namely D25, which targeted amyloid fibrils and selectively inhibited S. mutans biofilms (Chen et al, 2021).…”

“…Chen et al. also screened and identified a small molecule, namely D25, which targeted amyloid fibrils and selectively inhibited S. mutans biofilms ( Chen et al., 2021 ).…”

Novel antimicrobial agents targeting the Streptococcus mutans biofilms discovery through computer technology

“…The interactions between C123 (C-terminal segment) and P1 contribute to biofilm-related events such as amyloid fibrils formation, suggesting that C3 would serve as a promising anti-amyloid target ( Rivière et al., 2020 ). Chen ( Chen et al., 2021 ) selected small molecules targeting C3 through structure-based virtual screening, and found that D25 selectively inhibited amyloid fibrils and S. mutans biofilms but had little influence on biofilms formed by S. gordonii and S. sanguinis .…”