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例子。清洁牙齿表面的发育是由细菌间黏附事件的紧密协调和高度可复制（从人-人）的时空序列介导的。在这个过程中，病原体会成为与牙齿相关的生物膜的一部分。如果能够整合，病原体如牙龈卟啉单胞菌和放线菌（放线菌）放线菌可以迅速分裂并克服共生生物膜群体。这会导致牙周病。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，假设物种间的细菌交流有助于从健康的牙菌斑群落到引起疾病的菌斑群落的转变。最近的证据表明，自诱导剂-2 (AI-2)，一种种间细菌信号分子，可能介导了这种群落变化。AI-2是细菌酶LuxS的产物，共生细菌以皮摩尔浓度产生AI-2，病原体则以更高浓度产生AI-2。我们提出了一个探索性计划，通过在体外流动细胞中培养野生型和luxS突变体（不产生AI-2）来开始阐明基于AI-2的通信的作用。AI-2对(I)由健康细菌物种组成的生物膜的时空发展和（II）口腔病原体融入这些生物膜的贡献将进行评估。为了实现这一目标，我们从世界各地的研究小组获得了野生型和luxS突变体的共生和致病口腔细菌。我们建议在单种、双种和三种体外生物膜中培养野生型和luxS突变体，这些生物膜代表了在人类腔中发现的条件。利用人唾液作为营养源，利用共聚焦激光扫描显微镜（CLSM）在流动细胞中形成生物膜。荧光标记抗体，针对每个物种，将与IMARIS(R)成像软件串联使用，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，这个R21探索性项目将显著有助于理解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型治疗化合物。

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.