Apigenin, a potent inhibitor of glucosyltransferase activity, affects the accumulation of Streptococcus mutans biofilms in vitro by reducing the formation of insoluble glucans and enhancing the soluble glucan content of the polysaccharide matrix. In the present study, we investigated the influence of apigenin on gtfB, gtfC, and gtfD expression in S. mutans UA159. Apigenin (0.1 mM) significantly decreased the expression of gtfB and gtfC mRNA (P < 0.05); in contrast, it increased the expression of gtfD in S. mutans growing in the planktonic state. The protein levels of GTF B, GTF C, and GTF D in culture supernatants were also affected; less GTF B and C were detected, whereas the level of GTF D was significantly elevated (P < 0.05). A similar profile of gtf expression was obtained with biofilms, although an elevated concentration (1 mM) of apigenin was required to elicit the effects. The influence of apigenin on gtf gene expression was independent of any effect on GTF activity, did not involve inhibition of growth or effects on pH, and was not affected by addition of sucrose. The data show that apigenin modulates the genetic expression of virulence factors in S. mutans.The ability of Streptococcus mutans to synthesize extracellular glucans is a critical virulence factor involved in the pathogenesis of dental caries in animals and humans (19,22,26). This bacterium harbors three distinct gtf genes expressing glucosyltransferase (GTF) activity (17). The gtfB and gtfC genes are in an operon-like arrangement and encode enzymes that produce mostly water-insoluble ␣-(133)-linked glucans, whereas the gtfD gene, which is not linked to the gtfBC locus, encodes an enzyme that synthesizes water-soluble ␣-(136)-linked glucans.Glucans provide binding sites for, and promote accumulation of, cariogenic streptococci (and other oral microorganisms) on the tooth surface, and they contribute to the establishment of the extracellular polysaccharide matrix, which provides bulk and structural integrity to dental biofilms (known as dental plaque) (4). By preventing glucan production, therapeutic approaches to influencing the formation or virulence of dental biofilms related to caries could be precise and selective and would not necessarily suppress the resident oral flora. Mutant strains of S. mutans defective in the gtf genes, especially gtfB and gtfC, are far less cariogenic than the parent strains in vivo (26). Therefore, agents that affect the expression of gtf genes offer a very attractive route for prevention of dental biofilm-related diseases, such as dental caries.Apigenin, a nontoxic bioflavonoid ubiquitously found in plant-derived foods and propolis (a honeybee product), is a novel anticaries and antiplaque agent in vitro and in vivo (13,14,15). Apigenin effectively disrupted the formation and accumulation of S. mutans UA159 biofilms without killing the organism (15). Analysis of the polysaccharide content of the matrix from a biofilm revealed that apigenin remarkably diminished the amounts of insoluble glucans but increased the...

The presence of extracellular polysaccharides matrix makes extraction and purification of RNA from Streptococcus mutans within biofilms challenging. In this study, several approaches to purify RNA extracted from S. mutans in suspension cultures and biofilms were examined. The combination of sonication (3 pulses of 30 s at 7 W), suspension in NAES buffer (50 mM sodium acetate buffer, 10 mM EDTA and 1% SDS; pH 5.0) and homogenization-mechanical cells disruption in NAESacid phenol:chloroform, yielded 9.04 mg (or 0.52 mg) of crude preparation of RNA per 100 mg of total cell (or biofilm) dry-weight. The crude RNA preparations were subjected to various DNAse I treatments. The combination of DNAse I in silica-gel based column followed by recombinant DNase I in solution provided the best genomic DNA removal, resulting in 4.35 mg (or 0.06 mg) of purified RNA per 100 mg of total cell (or biofilm) dry-weight. The cDNAs generated from the purified RNA sample were efficiently amplified using gtfB S. mutans-specific primers. The results showed a method that yields high-quality RNA from both planktonic cells and biofilms of S. mutans in sufficient quantity and quality for real-time RT-PCR analyses.