Molecular analysis of F. nucleatum interspecies interactions in biofilms

描述（由申请人提供）：口腔中含有复杂的微生物生物膜群落，能够介导急性和慢性多种微生物感染，包括牙周病，牙周病仍然是全球牙齿脱落的最常见原因。特定的种间相互作用是构建这些群落的关键因素，具核梭杆菌（Fn）已被确立为中心的“桥接”生物体，使许多口腔细菌物种（“早期”和“晚期”定植者）排列成组织良好的群落，用于生物膜形成和发病。Fn粘附于多种口腔微生物的能力已经通过体外共聚集研究得到了清楚的证明，但只有有限的研究探讨了这种能力如何在多种微生物生物膜中表现出来。更重要的是，缺乏有效的遗传操作工具和明确的分子靶点，阻碍了这些Fn介导的迷人的种间相互作用背后的分子机制的调查。我们最近实现了第一个遗传特性的梭菌粘附素（RadD），介导种间粘附与早期口腔殖民者。在我们的初步研究中，我们证明了RadD依赖性结合其伴侣物种血链球菌（Ss）刺激Fn中不同的表型变化和差异基因表达。基于这些发现，我们开发了我们的工作假设，即"Fn通过RadD和相关的Rad-protein与早期定殖伴侣物种相互作用，这引发了Fn中对生物膜形成重要的显著形态和生理变化"。为了验证这一假设，我们提出了一个详细的检查RadD在Fn介导的种间相互作用的作用，血链球菌（Ss）作为模型早期殖民合作伙伴物种。方法将包括生物膜行为的表征，口腔社区整合，基因表达模式和识别参与OMP介导的与Fn相互作用的细胞成分。这种全面的方法将提供详细的知识，在分子水平上的生物膜的形成，包括物理细胞接触和信号。这项研究将提供新的目标，为改善治疗干预的广泛的人类传染病，并作为未来的研究Fn与晚期殖民合作伙伴物种或动物模型的基础。

DESCRIPTION (provided by applicant): The oral cavity harbors a complex microbial biofilm community capable of mediating acute and chronic polymicrobial infections including periodontal diseases which are still the most common reason for tooth loss worldwide. Specific interspecies interactions are key factors in constructing these communities and Fusobacterium nucleatum (Fn) has been established as the central "bridging" organism that enables arrangement of numerous oral bacterial species (both "early" and "late" colonizers) into well organized communities for biofilm formation and pathogenesis. The ability of Fn to adhere to a large variety of oral species has been clearly demonstrated through elaborate in vitro co-aggregation studies with planktonic cells, yet only limited studies have explored how this ability manifests in multispecies microbial biofilms. More importantly, lack of effective genetic manipulation tools and clear molecular targets has hampered investigation of the molecular mechanisms behind these Fn-mediated fascinating interspecies interactions. We recently achieved the first genetic characterization of a fusobacterial adhesin (RadD) that mediates interspecies adhesion with early oral colonizers. In our preliminary studies, we demonstarted that RadD- dependent binding to its partner species Streptococcus sanguinis (Ss) stimulates distinct phenotypic changes and differential gene expression in Fn. Based on these findings, we developed our working hypothesis that "Fn interacts via RadD and associated Rad-proteins with early colonizing partner species, which triggers significant morphological and physiological changes in Fn important for biofilm formation". To test this hypothesis, we propose a detailed examination of the role of RadD in Fn-mediated interspecies interactions using Streptococcus sanguinis (Ss) as model early colonizing partner species. Approaches will include characterization of biofilm behavior, oral community integration, gene expression patterns and identification of cellular components in Ss involved in the OMP-mediated interaction with Fn. This comprehensive approach will provide detailed knowledge at the molecular level for the events involved in biofilm formation including physical cell-cell contact and signaling. This research will provide novel targets for improved therapeutic intervention of a widespread human infectious disease and serve as a basis for future studies of Fn with late colonizing partner species or animal models.