Investigation of the role of AI-2 in the development of dental plaque biofilms

描述(申请人提供)：牙菌斑是细菌多物种生物膜群落的一个例子。清洁牙齿表面的发育是由紧密协调和高度可重复性(人与人)的细菌间黏附事件的时空序列所调节的。在这个过程中，病原体可以成为牙齿相关生物膜的一部分。如果能够整合，牙龈卟啉单胞菌和伴生放线菌等病原体可以迅速分裂并克服共生生物膜种群。这会导致牙周病。病原体在共生生物膜中成功整合和扩展的机制尚不清楚。在这项探索性的提案中，它被假设为物种间的细菌交流有助于从健康的牙菌斑群落过渡到导致疾病的群落。最近的证据表明，自身诱导物-2(AI-2)是一种跨物种的细菌信号分子，可能介导了这种群落变化。AI-2是细菌酶LuxS的产物，由共生细菌在皮摩尔浓度下产生，病原体在较高浓度下产生。我们提出了一个探索性的计划，通过在体外培养的流动细胞中生长野生型和LuxS突变体(不产生AI-2)来开始阐明基于AI-2的沟通的作用。将评估AI-2对(I)由健康细菌物种组成的生物膜的时空发展和(Ii)口腔病原体与这些生物膜的整合的贡献。为了实现这一点，我们从世界各地的研究小组获得了共生和致病口腔细菌的野生型和LuxS突变体。我们建议在单物种、双重物种和三部分物种的体外生物膜中种植野生型和LuxS突变体，这些生物膜代表了在人体空洞中发现的条件。人类唾液将被用作营养源，生物膜将在流动细胞中开发，用于共聚焦激光扫描显微镜(CLSM)的研究。每种生物特有的荧光标记抗体将与IMARIS(R)成像软件一起使用，以识别时空生物膜的发展。因此，可以确定AI-2在生物膜发育和病原体整合中的作用。最终，这一R21探索性计划将大大有助于了解AI-2在牙菌斑生物膜中的作用，如果发现AI-2在生物膜定植中的作用，将有助于开发治疗牙周病的新的治疗化合物。 公共卫生相关性：牙菌斑是由数百种密切相关的细菌组成的，有些细菌会导致牙周病。我们认为，健康和致病物种之间的交流是通过产生各种小信号分子来实现的，这些小信号分子统称为自动诱导剂-2(AI-2)。致病物种比健康物种产生更多的AI-2，牙菌斑中AI-2浓度的增加促进了病原体的生长，但损害了健康细菌。

DESCRIPTION (provided by applicant): Dental plaque is an example of a bacterial multi-species biofilm community. Development on a clean tooth surface is mediated by a tightly orchestrated and highly reproducible (from person- person) spatiotemporal succession of inter-bacterial adhesion events. During this process, pathogens can become part of the tooth-associated biofilm. If able to integrate, pathogens such as Porphyromonas gingivalis and Aggregatibacter (Actinobacillus) actinomycetemcomitans can rapidly divide and overcome the commensal biofilm population. This results in periodontal disease. The mechanism by which the pathogens can successfully integrate and expand in commensal biofilms is unknown. It is hypothesized in this exploratory proposal that inter-species bacterial communication contributes to the transition from a healthy dental plaque community to one that causes disease. Recent evidence suggests that autoinducer-2 (AI-2), an inter-species bacterial signal molecule, may mediate such a community change. AI-2, a product of the bacterial enzyme LuxS, is produced at picomolar concentrations by commensal bacteria and at higher concentration by pathogens. We propose an exploratory program to begin to elucidate the role of AI-2 based communication by growing wild-type and luxS mutants (that do not produce AI-2) in in-vitro flowcells. The contribution of AI-2 to (I) the spatiotemporal development of biofilms consisting of healthy bacterial species and (II) the integration of oral pathogens into these biofilms will be assessed. To achieve this, we have obtained wild-type and luxS mutants of commensal and pathogenic oral bacteria from research groups from around the world. We propose to grow wild-type and luxS mutants in single-, dual-, and tripartite-species in-vitro biofilms that are representative of conditions that are found in the human cavity. Human saliva will be used as the nutrient source and biofilms will be developed in flowcells for study by Confocal Laser Scanning Microscopy (CLSM). Fluorescently labeled antibodies, specific for each species, will be used in tandem with IMARIS(R) imaging software to discern spatiotemporal biofilm development. Thus a role for AI-2 in biofilm development and pathogen integration can be determined. Ultimately, this R21 exploratory program will significantly contribute to the understanding of the role of AI-2 in dental plaque biofilms and, if a role in biofilm colonization is discovered, facilitate in the development of novel therapeutic compounds to treat periodontal disease. PUBLIC HEALTH RELEVANCE: Dental plaque is composed of hundreds of species of bacteria living in close association, and some can cause periodontal disease. We propose that healthy and disease- causing species communicate with one-another by producing various small signal molecules that are collectively called autoinducer-2 (AI-2). Pathogenic species produce more AI-2 than the healthy species and an increased AI-2 concentration in dental plaque promotes the growth of pathogens at the expense of the healthy bacteria.