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

描述（由申请人提供）：牙菌斑是细菌多物种生物膜群落的一个实例。在清洁牙齿表面上的发育由细菌间粘附事件的紧密协调和高度可再现（从人-人）时空连续介导。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，病原体如牙龈卟啉单胞菌和伴放线放线杆菌可以迅速分裂并克服牙龈生物膜群体。牙周病的症状有哪些？病原体在共生生物膜中成功整合和扩张的机制尚不清楚。在这个探索性的提议中，假设种间细菌交流有助于从健康的牙菌斑群落转变为导致疾病的菌斑群落。最近的证据表明，autoinducer-2（AI-2），种间细菌的信号分子，可能介导这样的社区变化。AI-2是一种细菌酶LuxS的产物，由嗜热菌以皮摩尔浓度产生，由病原体以更高浓度产生。我们提出了一个探索性计划，通过在体外流动池中培养野生型和luxS突变体（不产生AI-2），开始阐明基于AI-2的通讯的作用。将评估AI-2对（I）由健康细菌物种组成的生物膜的时空发育和（II）口腔病原体整合到这些生物膜中的贡献。为了实现这一目标，我们已经从世界各地的研究小组获得了口腔细菌和致病性口腔细菌的野生型和luxS突变体。我们建议在单，双，和三分物种的体外生物膜中生长野生型和luxS突变体，这些生物膜代表了在人体腔中发现的条件。将使用人唾液作为营养源，并在流动池中形成生物膜，以通过共聚焦激光扫描显微镜（CLSM）进行研究。对每个物种都有特异性的双标记抗体将与IMARIS（R）成像软件串联使用，以辨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，这项R21探索性计划将大大有助于理解AI-2在牙菌斑生物膜中的作用，如果发现生物膜定植中的作用，将有助于开发新型治疗化合物来治疗牙周病。 公共卫生相关性：牙菌斑是由数百种细菌组成的，它们生活在密切的关系中，有些细菌会导致牙周病。我们提出，健康和致病物种通过产生各种小信号分子相互交流，这些小信号分子统称为自诱导物-2（AI-2）。致病菌比健康菌产生更多的AI-2，并且牙菌斑中AI-2浓度的增加以健康细菌为代价促进病原体的生长。

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.