Evaluation of a Novel Anti-Caries Approach to Modulate Virulence of S. mutans

摘要 牙科生物膜是一个动态和多样化的微生物群落，它被一种同样复杂的多糖包裹着。 矩阵。胞外多糖(EPS)是由微生物(变形链球菌，a)合成的 主要贡献者)，并促进基质中的生化和结构变化，增强 生物膜。龋齿的发生是由于生物膜内持续的低pH环境，包括 胞外多糖量增加。新的化疗方法的发展， 除了微生物杀菌剂之外，影响EPS基质形成和产酸能力的方法也是很有希望的 预防或减少与牙科生物膜有关的口腔疾病。最近，我们确定了一种新的战略来 通过将两种天然产生的抗菌素结合起来，减少牙科生物膜和龋齿的形成和毒力 龋齿/抗菌斑剂(芹菜素和TT-法尼醇)与氟化物。它们可能通过的途径 化合物在生物膜内减弱变形链球菌的致龋性至少涉及三条途径：(1)通过 抑制葡萄糖转移酶的活性和表达，而葡萄糖转移酶与 生物膜中的多糖基质，(2)通过破坏变形链球菌膜完整性而影响产酸， 和(3)通过减少IPS的合成和/或积累。这些生物活动影响了 变形链球菌生物膜的多糖基质组成和体外产酸能力 增强氟化物的防龋性，而不影响活体口腔菌群的生存能力。 尽管我们之前的USPHS/NIH支持的研究产生了大量数据，但进一步 需要分析来阐明这些制剂的分子和生理作用机制，以及 以评估它们在体内的有效性。因此，我们提出了一个跨学科、循序渐进的研究项目，以 研究它们在以下方面的影响：1)与卵巢癌形成相关的特定基因的表达 实时荧光定量聚合酶链式反应检测胞外多糖基质，2)胞外多糖基质的结构 用GC-MS、MALDI-TOF-MS和核磁共振技术研究生物膜；3)变形链球菌的特定生化代谢途径 PTS系统和糖酵解酶的检测。此外，我们将确定我们的最有效剂量 体内治疗方法，这也可能减少氟暴露。通过将生物化学和 通过活体龋齿模型的分子技术，我们希望加强我们对如何 这些化合物调节变形链球菌生物被膜的形成，并拓展其潜力。 作为预防生物膜相关疾病的一种新的化疗方法的有用性，这可能是 在未来的临床试验中进行评估。项目叙事 该项目提出了一种新的预防龋齿的治疗方法，这种最普遍和最常见的 在美国代价高昂的口腔传染病。我们的方法使用天然化合物，是非常有效的 而不会抑制口腔内的菌群(非微生物杀灭)，并可能减少接触氟化物。

Abstract Dental biofilm is a dynamic and diverse microbial community enmeshed in an equally complex polysaccharide matrix. The extracellular polysaccharides (EPS) are synthesized by microorganisms (Streptococcus mutans, a key contributor) and promote biochemical and structural changes in the matrix enhancing the cariogenicity of the biofilm. Dental caries occurs as a result of persistent low pH environment within biofilms containing elevated amounts of extracellular polysaccharides. The development of novel chemotherapeutic approaches, other than microbiocides, that affect the development of EPS matrix and acidogenicity are promising routes to prevent or reduce oral diseases related to dental biofilm. Recently, we have identified a novel strategy to reduce the development and virulence of dental biofilms and caries by combining two naturally occurring anti- caries/anti-plaque agents (apigenin and tt-farnesol) with fluoride. The putative pathways by which these compounds attenuate the cariogenicity of S. mutans within biofilms involve, at least, three routes: (1) by inhibiting the activity and expression of glucosyltransferases, which are associated with the formation of the polysaccharide matrix in biofilms, (2) by affecting acid production by disrupting S. mutans membrane integrity, and (3) by reducing the synthesis and/or accumulation of IPS. These biological activities influenced the composition of the polysaccharide matrix and acidogenicity of S. mutans biofilms in vitro, which resulted in enhanced cariostatic properties of fluoride without affecting the viability of oral flora population in vivo. Although significant amount of data were generated from our previous USPHS/NIH supported studies, further analyses are required to elucidate the molecular and physiological mechanisms of action of these agents, and to evaluate their effectiveness in vivo. Therefore, we propose a multi-disciplinary, step-wise research project to investigate their influence on: 1) the expression of specific genes associated with the formation of the extracellular polysaccharide matrix using real-time PCR, 2) structure of the polysaccharides matrix in the biofilm using GC-MS, MALDI-TOF-MS and NMR; 3) metabolic pathway of S. mutans by specific biochemical assays on PTS system and glycolytic enzymes. Furthermore, we will identify the most effective dosage of our therapeutic approach in vivo, which may also reduce fluoride exposure. By integrating biochemical and molecular techniques with an in vivo model of dental caries, we expect to enhance our understanding of how these compounds modulate the pathogenesis of S. mutans biofilm development, and expand their potential usefulness as a novel chemotherapeutic approach to prevention of biofilm-related diseases, which could be evaluated in future clinical trials. Project Narrative This project proposes a novel therapeutic approach to prevent dental caries, the single most prevalent and costly oral infectious diseases in the United States. Our approach uses natural compounds, is highly effective without suppressing the resident oral flora (non-microbiocidal) and may decrease exposure to fluoride.