Gene expression and protein synthesis of esterase from Streptococcus mutans are affected by biodegradation by-product from methacrylate resin composites and adhesives.

An esterase from S. mutans UA159, SMU_118c, was shown to hydrolyze methacrylate resin-based dental monomers. OBJECTIVE: To investigate the association of SMU_118c to the whole cellular hydrolytic activity of S. mutans toward polymerized resin composites, and to examine how the bacterium adapts its hydrolytic activity in response to environmental stresses triggered by the presence of a resin composites and adhesives biodegradation by-product (BBP). MATERIALS AND METHODS: Biofilms of S. mutans UA159 parent wild strain, SMU_118c knockout strain (ΔSMU_118c), and SMU_118c complemented strain (ΔSMU_118cC) were incubated with photo-polymerized resin composite. High performance liquid chromatography was used to quantify the amount of a universal 2,2-Bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane (bisGMA)-derived BBP, bishydroxy-propoxy-phenyl-propane (bisHPPP) in the media. Fluorescence in situ hybridization (FISH) and quantitative proteomic analysis were used to measure SMU_118c gene expression and production of SMU_118c protein, respectively, from biofilms of S. mutans UA159 wild strain that were cultured with bisHPPP. RESULTS: The levels of bisHPPP released from composite were similar for ΔSMU_118c and media control, and these were significantly lower compared to the parent wild-strain UA159 and complemented strain (ΔSMU_118cC) (p < 0.05). Gene expression of SMU_118c and productions of SMU_118c protein were higher for bisHPPP incubated biofilms (p < 0.05). SIGNIFICANCE: This study suggests that SMU_118c is a dominant esterase in S. mutans and capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to BBP was to increase the expression of the esterase gene and enhance esterase production, potentially accelerating the biodegradation of the restoration, adhesive and restoration-tooth interface, ultimately contributing to premature restoration failure. STATEMENT OF SIGNIFICANCE: We recently reported (Huang et al., 2018) on the isolation and initial characterization of a specific esterase (SMU_118c) from S. mutans that show degradative activity toward the hydrolysis of dental monomers. The current study further characterize this enzyme and shows that SMU_118c is a dominant degradative esterase activity in the cariogenic bacterium S. mutans and is capable of catalyzing the hydrolysis of the resinous matrix of polymerized composites and adhesives. In turn, the bacterial response to biodegradation by-products from composites and adhesives was to increase the expression of the esterase gene and enhance esterase production, accelerating the biodegradation of the restoration, adhesive and the restoration-tooth interface, potentially contributing to the pathogenesis of recurrent caries around resin composite restorations.

Mechanistic, genomic and proteomic study on the effects of BisGMA-derived biodegradation product on cariogenic bacteria.

OBJECTIVES: Investigate the effects of a Bis-phenyl-glycidyl-dimethacrylate (BisGMA) biodegradation product, bishydroxypropoxyphenyl-propane (BisHPPP), on gene expression and protein synthesis of cariogenic bacteria. METHODS: Quantitative real-time polymerase chain reaction was used to investigate the effects of BisHPPP on the expression of specific virulence-associated genes, i.e. gtfB, gtfC, gbpB, comC, comD, comE and atpH in Streptococcus mutans UA159. Possible mechanisms for bacterial response to BisHPPP were explored using gene knock-out and associated complemented strains of the signal peptide encoding gene, comC. The effects of BisHPPP on global gene and protein expression was analyzed using microarray and quantitative proteomics. The role of BisHPPP in glucosyltransferase (GTF) enzyme activity of S. mutans biofilms was also measured. RESULTS: BisHPPP (0.01, 0.1mM) up-regulated gtfB/C, gbpB, comCDE, and atpH most pronounced in biofilms at cariogenic pH (5.5). The effects of BisHPPP on the constructed knock-out and complemented strains of comC from quorum-sensing system, implicated this signaling pathway in up-regulation of the virulence-associated genes. Microarray and proteomics identified BisHPPP-regulated genes and proteins involved in biofilm formation, carbohydrate transport, acid tolerance and stress-response. GTF activity was higher in BisHPPP-exposed biofilms when compared to no-BisHPPP conditions. SIGNIFICANCE: These findings provide insight into the genetic and physiological pathways and mechanisms that help explain S. mutans adaptation to restorative conditions that are conducive to increased secondary caries around resin composite restorations and may provide guidance to clinicians' decision on the selection of dental materials when considering the long term oral health of patients and the interactions of composite resins with oral bacteria.

Triethylene Glycol Up-Regulates Virulence-Associated Genes and Proteins in Streptococcus mutans.

Triethylene glycol dimethacrylate (TEGDMA) is a diluent monomer used pervasively in dental composite resins. Through hydrolytic degradation of the composites in the oral cavity it yields a hydrophilic biodegradation product, triethylene glycol (TEG), which has been shown to promote the growth of Streptococcus mutans, a dominant cariogenic bacterium. Previously it was shown that TEG up-regulated gtfB, an important gene contributing to polysaccharide synthesis function in biofilms. However, molecular mechanisms related to TEG's effect on bacterial function remained poorly understood. In the present study, S. mutans UA159 was incubated with clinically relevant concentrations of TEG at pH 5.5 and 7.0. Quantitative real-time PCR, proteomics analysis, and glucosyltransferase enzyme (GTF) activity measurements were employed to identify the bacterial phenotypic response to TEG. A S. mutans vicK isogenic mutant (SMΔvicK1) and its associated complemented strain (SMΔvicK1C), an important regulatory gene for biofilm-associated genes, were used to determine if this signaling pathway was involved in modulation of the S. mutans virulence-associated genes. Extracted proteins from S. mutans biofilms grown in the presence and absence of TEG were subjected to mass spectrometry for protein identification, characterization and quantification. TEG up-regulated gtfB/C, gbpB, comC, comD and comE more significantly in biofilms at cariogenic pH (5.5) and defined concentrations. Differential response of the vicK knock-out (SMΔvicK1) and complemented strains (SMΔvicK1C) implicated this signalling pathway in TEG-modulated cellular responses. TEG resulted in increased GTF enzyme activity, responsible for synthesizing insoluble glucans involved in the formation of cariogenic biofilms. As well, TEG increased protein abundance related to biofilm formation, carbohydrate transport, acid tolerance, and stress-response. Proteomics data was consistent with gene expression findings for the selected genes. These findings demonstrate a mechanistic pathway by which TEG derived from commercial resin materials in the oral cavity promote S. mutans pathogenicity, which is typically associated with secondary caries.