Adaptation Mechanisms of Streptococcus mutans to Stresses Induced by Resin Composite Biodegradation By-products

Resin composites are currently the most popular dental restorative materials. While providing several beneficial properties such as superior esthetics and excellent adhesive strength to dentin and enamel, resin composites undergo biological breakdown in the oral cavity that results in secondary/recurrent caries and reduced restoration longevity compared to amalgam. The breakdown of the resin-dentin interface by salivary and bacterial esterases generates biodegradation by-products (BBPs) including triethylene glycol (TEG) and bis-hydroxy-propoxy-phenyl-propane (BisHPPP). This chemical degradation contributes to gap formation between the restoration and the tooth, providing a path for microleakage of cariogenic microorganisms such as Streptococcus mutans, potentially resulting in recurrent/secondary caries, which is a major cause for resin composite restorations replacement. To test the hypothesis that BBPs activate signaling pathways that are involved in Streptococcus mutans pathogenicity and understand the molecular pathways that regulate the BBPsâ effects on cariogenic bacteria, this thesis quantitatively and mechanistically studied the effect of TEG or BisHPPP at clinically-relevant concentrations on the virulence properties of S. mutans UA159 in a relevant in vitro model. Quantitative real-time polymerase chain reaction showed up-regulation of seven dominant virulence-associated genes i.e. gtfB, gtfC, gbpB, comCDE and atpH, more significantly in biofilm vs. planktonic cells, only at cariogenic pH (5.5). Exposure of the constructed knock-out and complemented strains of S. mutans for the key regulatory genes, vicK and comC, to BBPs suggested these signalling pathways as potential mechanisms for up-regulation of the virulence-associated genes. S. mutans biofilm proteome analysis after exposure to TEG/BisHPPP confirmed activation of specific bacterial adaptation mechanisms such as biofilm formation and adhesion, acid tolerance, optimization of phosphotransferase system and stress-response pathways in response to BBPs. Microarray data were consistent with the proteomics findings for BisHPPP. Furthermore, both BBPs increased glucosyltransferases enzyme activity, involved in synthesizing exopolysaccharide glucan associated with the formation of cariogenic biofilms. This thesis provides significant insight into the adaptive response of S. mutans to the dental biomaterial-derived degradation products and elucidates the potential molecular mechanisms that could contribute in pathogenesis of secondary caries in resin composite restorations. It also provides a rationale for development of dental restorative materials that are more biostable and biocompatible.Ph.D

Triethylene Glycol Up-Regulates Virulence-Associated Genes and Proteins in <i>Streptococcus mutans</i>

<div><p>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 <i>Streptococcus mutans</i>, a dominant cariogenic bacterium. Previously it was shown that TEG up-regulated <i>gtfB</i>, 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, <i>S</i>. <i>mutans</i> 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 <i>S</i>. <i>mutans vicK</i> 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 <i>S</i>. <i>mutans</i> virulence-associated genes. Extracted proteins from <i>S</i>. <i>mutans</i> biofilms grown in the presence and absence of TEG were subjected to mass spectrometry for protein identification, characterization and quantification. TEG up-regulated <i>gtfB/C</i>, <i>gbpB</i>, <i>comC</i>, <i>comD</i> and <i>comE</i> more significantly in biofilms at cariogenic pH (5.5) and defined concentrations. Differential response of the <i>vicK</i> 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 <i>S</i>. <i>mutans</i> pathogenicity, which is typically associated with secondary caries.</p></div

Effects of different concentrations of TEG (0.01, 0.1 and 1.0 mM) on <i>S</i>. <i>mutans</i> UA159 glucosyltransferase activity.

<p>The total amount of insoluble glucan synthesized by biofilm cells grown in the presence of 1 mM TEG was significantly increased compared to the no-TEG control (P<0.05). Data are plotted with standard error of the mean (±SE), n = 4.</p

Biofilm-related proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH 5.5.

<p>Biofilm-related proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH 5.5.</p

Relative expression of the <i>S</i>. <i>mutans</i> virulence genes: <i>gtfB</i>, <i>gtfC</i>, <i>gbpB</i>, <i>comC</i>, <i>comD</i>, <i>comE</i> and <i>atpH</i> for planktonic and biofilm growth conditions with different concentrations of TEG (0.001, 0.01 and 0.1 mM) at pH 5.5 relative to the no TEG control.

<p>* represents significant difference between individual TEG concentrations compared to the control (no TEG) in either growth mode (P<0.05). # Represents significant difference between biofilm and planktonic cultures at the same TEG concentration (P<0.001). Data are plotted with standard error of the mean (±SE), n = 4.</p

Distribution of <i>S</i>.<i>mutans</i> UA159 proteins into specific gene ontology (GO) of biological processes aftter exposure to TEG.

<p>A total number of 125 proteins were differentially expressed in <i>S</i>. <i>mutans</i> biofilm grown in the presence of different TEG concentrations (0.01, 0.1 and 1.0 mM), among which 116 proteins were more abundant and 9 proteins were less abundant compared to the no-TEG control.</p

Relative expression of <i>gtfB</i>, <i>comD and atpH</i> in wild-type, knock-out (SMΔvicK1) and complemented (SMΔvicK1C) <i>vicK</i> strains of <i>S</i>. <i>mutans</i> UA159 in the presence of different concentrations of TEG (0.01 and 0.1 mM) at pH 5.5.

<p>One-way analysis of variance (ANOVA) and Tukey <i>post hoc</i> analyses were performed to determine the differences in gene expression between individual TEG concentration and the no-TEG control (P<0.05). Expression of the related genes in complemented strain was similar to that of wild-type. Data are plotted with standard error of the mean (±SE), n = 4.</p

Carbohydrate transport proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH5.5.

<p>Carbohydrate transport proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH5.5.</p

Primers used for construction of <i>vicK</i> knock-out and complemented strains.

<p>Primers used for construction of <i>vicK</i> knock-out and complemented strains.</p

Acid tolerance-response proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH 5.5.

<p>Acid tolerance-response proteins from <i>S</i>. <i>mutans</i> UA159 grown in the presence of TEG at pH 5.5.</p