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.二甲基丙烯酸三乙二醇酯（Triethylene glycol dimethacrylate, TEGDMA）是一种广泛应用于牙科复合树脂的稀释单体。该物质经口腔内复合树脂的水解降解后，会生成亲水性降解产物三乙二醇（triethylene glycol, TEG），已有研究表明，该产物可促进主要致龋菌变形链球菌（Streptococcus mutans）的生长。既往研究已证实，TEG可上调gtfB基因的表达——该基因是生物膜中多糖合成功能的关键调控基因。但目前学界对TEG调控细菌功能的分子机制仍知之甚少。 本研究以变形链球菌UA159（S. mutans UA159）为研究对象，在pH 5.5和pH 7.0的条件下，将其与临床相关浓度的TEG共孵育。本研究采用实时荧光定量PCR（quantitative real-time PCR）、蛋白质组学分析（proteomics analysis）以及葡萄糖基转移酶（glucosyltransferase, GTF）活性检测等手段，探究细菌对TEG的表型应答反应。我们构建了变形链球菌vicK基因敲除同源突变株（SMΔvicK1）及其互补菌株（SMΔvicK1C）——vicK是调控生物膜相关基因的关键调控基因——以此明确该信号通路是否参与变形链球菌毒力相关基因的调控。我们对添加与未添加TEG条件下培养的变形链球菌生物膜提取的总蛋白进行质谱分析（mass spectrometry），以实现蛋白的鉴定、表征与定量。 在致龋pH（5.5）及特定浓度条件下，TEG可显著上调生物膜中gtfB/C、gbpB、comC、comD及comE基因的表达。vicK敲除株（SMΔvicK1）与互补菌株（SMΔvicK1C）的应答差异表明，该信号通路参与了TEG介导的细胞应答调控。TEG可提升GTF酶活性，而该酶负责合成参与致龋生物膜形成的不溶性葡聚糖。此外，TEG可上调与生物膜形成、碳水化合物转运、耐酸性及应激反应相关的蛋白丰度。蛋白质组学数据与所选基因的基因表达检测结果相一致。本研究结果阐明了口腔内商用树脂材料释放的TEG通过何种分子通路促进变形链球菌致病力——该致病过程通常与继发龋相关。