Investigating SloR virulence gene metalloregulation in S. mutans.

描述(申请人提供)：变形链球菌引起的龋齿在发达国家和发展中国家仍然是一个主要问题，根据世界卫生组织的数据，这影响了全世界60%-90%的儿童(128人)。尽管美国普遍存在饮水加氟和旨在改善口腔健康的教育项目，但最近的报告显示，乳牙期龋齿的患病率或严重程度没有显著改善(，第120页)。此外，大约90%的美国成年人在他们的恒牙上有龋齿，这使得全国的牙科治疗费用在2002年超过700亿美元(44)。由于变形链球菌的生存和毒力直接关系到菌斑环境中必需金属离子的可获得性和运输，特别是铁和锰，因此金属离子摄取机制是抗龋齿药物设计的诱人靶点。在目前的拨款申请中，我们提出了一种减轻龋齿的新方法，该方法集中在研究变形链球菌金属调节蛋白对全球毒力基因的调控。在之前的资助期间，我们证实了当达到可持续的锰水平时(如在进餐期间)，变形链球菌金属离子转运基因和毒力因子受到Slo依赖的抑制，而当金属离子变得有限时(如在两餐之间)，这种抑制就会消失。这些观察结果使我们假设，在生理性锰浓度下增强Slo抑制将减弱变形链球菌毒力基因的表达，并阻碍龋病发生的过程。本申请旨在通过使用分子和生物化学方法阐明变形链球菌中SLOR激活和DNA结合的结构基础来解决这一假说。对野生型和突变型Slo蛋白的结晶学分析，结合DNA足迹和DNA弯曲实验，将阐明Slo：DNA相互作用的细节，体内实验将验证Slo介导的毒力基因调控及其与龋病结局的关系。综上所述，这些研究可以帮助设计针对SLOR活性的新治疗剂，以便控制或消除龋齿。 公共卫生相关性：该应用程序建议研究一种重要的人类病原体--变形链球菌--的SLOR介导的毒力基因调控的细节。为此，我们将表征Slor金属调节蛋白与变形链球菌基因组中特定保守DNA序列的结合，这可以促进空洞的形成过程。在无菌大鼠模型中，我们将探索SLOR促进基因激活和抑制的机制，并研究SLOR突变体的致龋性，从而最终将SLOR作为药物设计的靶点，以减轻变形链球菌引起的疾病。这项拟议的研究符合NIH/NIDCR的使命，因为它可以促进改进的龋病治疗和预防策略的发展，从而减少一种最普遍的传染病，这种疾病继续危及数百万人的生命。

DESCRIPTION (provided by applicant): Streptococcus mutans-induced caries formation continues to be a major problem in developed and developing nations which, according to the World Health Organization, impacts 60-90% of children world-wide (128). Despite widespread water fluoridation and the implementation of educational programs aimed at improving oral health in the United States, recent reports reveal no significant improvement in the prevalence or severity of dental caries in the primary dentition (64, 120). Moreover, approximately 90% of adult Americans have dental caries in their permanent teeth, contributing to the nation's estimated costs for dental treatment that exceeded $70 billion in 2002 (44). Since the survival and virulence of S. mutans is directly related to the availability and transport of essential metal ions in the plaque environment, particularly iron and manganese, metal ion uptake mechanisms represent attractive targets for drug design aimed at combating cavities. In the present grant application, we profess a novel approach to alleviating tooth decay that is centered on investigating global virulence gene regulation by the S. mutans SloR metalloregulatory protein. During the previous grant term, we confirmed SloR-dependent repression of S. mutans metal ion transport genes and virulence factors when sustainable levels of manganese are achieved (such as during a mealtime), and loss of this repression when metal ions become limiting (such as between meals). These observations led us to hypothesize that enhanced SloR repression at physiological manganese concentrations will attenuate S. mutans virulence gene expression and impede the process of cariogenesis. This application sets out to address this hypothesis by elucidating the structural basis for SloR activation and DNA binding in S. mutans using molecular and biochemical approaches. Crystallographic analysis of the wild-type and mutant SloR protein coupled with DNA footprinting and DNA bending experiments will elucidate the details of the SloR:DNA interaction, and in vivo experiments will validate SloR-mediated virulence gene regulation and its relationship with cariogenic outcome. Taken together, these studies can facilitate the design of new therapeutic agents that target SloR activity so that caries may be controlled or eliminated. PUBLIC HEALTH RELEVANCE: This application proposes to investigate the details of SloR-mediated virulence gene regulation in an important human pathogen, Streptococcus mutans. To this end, we will characterize the binding of a SloR metalloregulatory protein to specific conserved DNA sequences in the S. mutans genome that can facilitate the process of cavities formation. The mechanisms which promote gene activation and repression by SloR will be explored and the cariogenic potential of SloR mutant variants investigated in a germfree rat model so that ultimately SloR can be targeted for drug design to alleviate S. mutans-induced disease. The proposed research fits the mission of the NIH/NIDCR because it can promote the development of improved caries treatment and prevention strategies, and so curtail a most ubiquitous infectious disease that continues to compromise millions of human lives.