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

描述（由申请人提供）：摘要牙齿生物膜是一个动态的和多样化的微生物群落，陷入同样复杂的多糖基质。胞外多糖（EPS）由微生物（变形链球菌，一种关键贡献者）合成，并促进基质中的生化和结构变化，从而增强生物膜的致龋性。龋齿的发生是由于含有升高量的胞外多糖的生物膜内的持续低pH环境。除了杀微生物剂之外，影响EPS基质和产酸性的发展的新型化学治疗方法的开发是预防或减少与牙齿生物膜相关的口腔疾病的有希望的途径。最近，我们已经确定了一种新的策略，以减少牙齿生物膜和龋齿的发展和毒力，通过组合两种天然存在的抗龋齿/抗菌斑剂（芹菜素和TT-法尼醇）与氟化物。这些化合物减弱S.生物膜内的变形链球菌至少涉及三种途径：（1）通过抑制葡糖基转移酶的活性和表达，葡糖基转移酶与生物膜中多糖基质的形成有关，（2）通过破坏S.变形菌膜完整性，和（3）减少IPS的合成和/或积累。这些生物活性影响了S.在体外的变形杆菌生物膜，这导致增强的防龋性能的氟化物，而不影响生活力的口腔植物群人口在体内。虽然我们以前的USPHS/NIH支持的研究产生了大量的数据，但还需要进一步的分析来阐明这些药物的分子和生理作用机制，并评估其体内有效性。因此，我们提出了一个多学科、分阶段的研究项目，以探讨它们对以下方面的影响：1）利用实时荧光PCR技术研究与胞外多糖基质形成相关的特定基因的表达; 2）利用GC-MS、MALDI-TOF-MS和NMR技术研究生物膜中多糖基质的结构; 3）S.通过对PTS系统和糖酵解酶的特异性生化测定，研究了变形杆菌的PTS系统和糖酵解酶的活性。此外，我们将确定我们的治疗方法在体内的最有效剂量，这也可能减少氟化物暴露。通过将生物化学和分子技术与体内龋齿模型相结合，我们希望加深对这些化合物如何调节链球菌发病机制的了解。变形菌生物膜的发展，并扩大其潜在的有用性作为一种新的化学治疗方法，以预防生物膜相关的疾病，这可以在未来的临床试验进行评估。 项目叙述：该项目提出了一种新的治疗方法来预防龋齿，这是美国最流行和最昂贵的口腔传染病。我们的方法使用天然化合物，非常有效，不会抑制口腔内的常驻植物群（非杀微生物剂），并可能减少氟化物的暴露。

DESCRIPTION (provided by applicant): 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.