Influence of Apigenin on gtf Gene Expression in Streptococcus mutans UA159

Abstract

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...

Symbiotic Relationship between Streptococcus mutans and Candida albicans Synergizes Virulence of Plaque Biofilms In Vivo

“…The resulting cDNAs were amplified with a Bio-Rad CFX96 system (Bio-Rad Laboratories, Inc., Hercules, CA) using previously published specific primers and TaqMan probes (15,47). A standard curve was plotted for each primer set, as described elsewhere (48). The standard curves were used to transform the critical threshold cycle (C T ) values to relative numbers of cDNA molecules.…”

Influences of trans‐trans farnesol, a membrane‐targeting sesquiterpenoid, on Streptococcus mutans physiology and survival within mixed‐species oral biofilms

“…Primers for fabM (acid tolerance) (5ʹ-ACTGATTAATGCCAATG GGAAAGTC-3ʹ and 5ʹ-TGCGAACAAGAGATTGTAC ATCATC-3ʹ), and glgP (intracellular sugar metabolism) (5ʹ-GACTTTAAAGACACTCTGCATGAAG-3ʹ and 5ʹ-A CGAACAACCTTAGCCAAAGAAG-3ʹ) were designed using Beacon Designer 2.0 software (Premier Biosoft International, Palo Alto, CA, USA). Standard curves were used to determine the relative number of cDNA molecules, which were normalized to the relative number of 16S rRNA cDNA molecules in each sample, as previously described [23]. These values were used to determine the fold of change between each treated sample and the vehicle control.…”

“…cDNAs were amplified with specific primers using a MyiQ real-time PCR detection system with iQ SYBR Green supermix (BioRad). The following genes were amplified in qPCR assays with specific primer sets that were used in previous studies: gtfB, gtfC, gtfD (associated with EPS synthesis), pdhA (associated with glycolysis), atpD (acid tolerance), 16S rRNA (reference gene) [21][22][23]. Primers for fabM (acid tolerance) (5ʹ-ACTGATTAATGCCAATG GGAAAGTC-3ʹ and 5ʹ-TGCGAACAAGAGATTGTAC ATCATC-3ʹ), and glgP (intracellular sugar metabolism) (5ʹ-GACTTTAAAGACACTCTGCATGAAG-3ʹ and 5ʹ-A CGAACAACCTTAGCCAAAGAAG-3ʹ) were designed using Beacon Designer 2.0 software (Premier Biosoft International, Palo Alto, CA, USA).…”

Isolation and purification of total RNA from Streptococcus mutans in suspension cultures and biofilms

“…6,14 In addition, cDNAs were synthesized from 1 µg of purified RNA samples using BioRad iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., CA, USA) which contains a MMLV RNase H+ reverse transcriptase and random hexamers. The resulting cDNA was used as template in the real-time PCR step; a reaction containing only the reagents (no template control) was also included.…”