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的、多样化的微生物群落，它们被包裹在同样复杂的多糖基质中。胞外多糖（EPS）是由微生物（变形链球菌）合成的，并促进基质的生化和结构变化，增强生物膜的致病性。龋齿的发生是由于生物膜内持续的低pH值环境，其中含有大量的细胞外多糖。除杀微生物剂外，开发影响EPS基质发育和致酸性的新型化疗方法是预防或减少与口腔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，通过将两种天然的抗龋/抗牙菌斑剂（芹菜素和tt-法尼醇）与氟化物结合，来减少牙齿生物膜和龋齿的发展和毒性。这些化合物减弱生物膜内变形链球菌致龋性的可能途径至少包括三种途径：(1)通过抑制葡萄糖基转移酶的活性和表达，这与生物膜中多糖基质的形成有关；(2)通过破坏变形链球菌膜完整性影响酸的产生；(3)通过减少IPS的合成和/或积累。这些生物活性在体外影响变形链球菌生物膜的多糖基质组成和致酸性，从而在不影响体内口腔菌群活力的情况下增强氟化物的抑牙性能。虽然我们之前的USPHS/NIH支持的研究产生了大量的数据，但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，并评估它们在体内的有效性。因此，我们提出了一个多学科的、逐步的研究项目，以研究它们对以下方面的影响：1)利用实时荧光定量PCR （real-time PCR）研究与细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分析生物膜中多糖基质的结构；3)通过PTS系统和糖酵解酶的特异性生化检测，确定变形链球菌的代谢途径。此外，我们将确定体内治疗方法的最有效剂量，这也可能减少氟化物暴露。通过将生物化学和分子技术与龋病体内模型相结合，我们希望加深我们对这些化合物如何调节变形链球菌生物膜发育的发病机制的理解，并扩大它们作为预防生物膜相关疾病的新型化疗方法的潜在用途，这可以在未来的临床试验中进行评估。

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.