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

摘要 牙齿生物膜是一个动态的、多样性的微生物群落， 矩阵胞外多糖（EPS）由微生物（变形链球菌， 关键贡献者），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， 细胞外多糖含量升高。新的化疗方法的发展， 除了杀微生物剂，影响EPS基质和产酸性的发展是有前途的途径， 预防或减少与牙齿生物膜有关的口腔疾病。最近，我们发现了一种新的策略， 通过结合两种天然存在的抗- 龋齿/抗牙斑剂（芹菜素和TT-法尼醇）与氟化物。这些疾病的可能途径 化合物减弱S.生物膜内的变异体涉及至少三种途径：（1）通过 抑制葡糖基转移酶的活性和表达，所述葡糖基转移酶与葡萄糖基转移酶的形成相关， 生物膜中的多糖基质，（2）通过破坏S.变形菌膜完整性， 和（3）通过减少IPS的合成和/或积累。这些生物活动影响了 多糖基质的组成和S.体外变形杆菌生物膜，这导致 增强氟化物的防龋性能，而不影响体内口腔植物群种群的活力。 虽然大量的数据是从我们以前的USPHS/NIH支持的研究中产生的，但进一步 需要进行分析以阐明这些药剂的分子和生理作用机制， 以评估其在体内的有效性。因此，我们提出了一个多学科，逐步研究项目， 研究它们对以下方面的影响：1）与细胞形成相关的特定基因的表达， 使用实时PCR的胞外多糖基质的结构，2）胞外多糖基质中的多糖基质的结构， 利用GC-MS、MALDI-TOF-MS和NMR研究了S.特异性生化变异株 PTS系统和糖酵解酶的测定。此外，我们将确定最有效的剂量， 体内治疗方法，这也可能减少氟化物暴露。通过整合生物化学和 分子技术与体内模型的龋齿，我们希望提高我们的理解如何 这些化合物调节S.变形菌生物膜的发展，并扩大其潜力 作为预防生物膜相关疾病的新的化学治疗方法的有用性， 在未来的临床试验中进行评估。项目叙述 该项目提出了一种新的治疗方法来预防龋齿，这是最普遍和最有效的方法。 在美国，昂贵的口腔传染病。我们的方法使用天然化合物，非常有效 而不抑制口腔常驻植物群（非杀微生物剂），并可减少对氟化物的暴露。

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.