Inhibitory effect of natural flavone luteolin on Streptococcus mutans biofilm formation

Streptococcus mutans is one of the key pathogens responsible for dental caries, which is known to be one of the most prevalent biofilm-associated diseases worldwide. S. mutans virulence strongly depends on its biofilm formation and enamel demineralization abilities due to the production of surface adhesins, exopolysaccharides, and acid in the presence of sugar. Luteolin is an abundant natural flavone with a prominent anti-bacterial function. However, it remains unclear how luteolin affects S. mutans pathogenicity including its acidogenicity and biofilm formation. In this study, the effect of luteolin on S. mutans growth, acid production, and its early and late biofilm formation and biofilm disruption was tested. Luteolin shows strong anti-biofilm activity, while it remains non-toxic for bacterial cell viability. In the biofilm, luteolin reduces the expression of S. mutans virulence genes such as gbpC, spaP, gtfBCD, and ftf encoding for surface adhesins and extracellular polysaccharides (EPS)-producing enzymes, which reflects in the strong reduction of bacteria and EPS. Further, it reduces water-insoluble glucan production in the biofilm, potentially, via direct interference with glucosyltransfereases (Gtfs). Moreover, at biofilm inhibitory concentrations, luteolin significantly reduces acid production by S. mutans. Finally, luteolin could target S. mutans amyloid proteins to disrupt the biofilm based on the observation that it inhibits the uptake of the amyloid dye, thioflavin T, by S. mutans extracellular proteins and failed to inhibit biofilm formation by the mutant strain lacking three main amyloid proteins. In conclusion, luteolin appears to be a potent natural compound with pleiotropic anti-biofilm properties against one of the main cariogenic human pathogens, S. mutans. IMPORTANCE Flavonoids are natural compounds with proven anti-bacterial and anti-biofilm properties. Here, we describe the anti-biofilm properties of natural flavone luteolin against the main cariogenic bacteria, S. mutans. Luteolin inhibited gene expression of cell surface adhesins, fructosyltransferases, and glucosyltransferases, which promotes a significant reduction of bacterial and EPS biomass in early and late biofilms. Moreover, luteolin could directly target S. mutans Gtfs and functional amyloids to modulate pathogenic biofilms. These observations provide important insights into the anti-biofilm properties of luteolin while laying out a framework for future therapeutic strategies targeting biofilm-associated virulence factors of oral pathogens.