Molecular analysis of F. nucleatum interspecies interactions in biofilms

口腔内有一个复杂的微生物生物膜群落，能够介导急性和慢性多微生物感染，包括牙周病，这仍然是世界范围内导致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，而核梭杆菌（Fn）已被确定为中心的“桥接”生物，能够将众多口腔细菌物种（“早期”和“晚期”定植者）排列成组织良好的群落，以形成生物膜和发病机制。通过对浮游细胞的体外共聚集研究，Fn粘附多种口腔物种的能力已经得到了清楚的证明，但只有有限的研究探讨了这种能力如何在多物种微生物生物膜中表现出来。更重要的是，缺乏有效的基因操作工具和明确的分子靶点阻碍了对这些由fn介导的迷人物种间相互作用背后的分子机制的研究。我们最近首次获得了梭杆菌黏附素（RadD）的遗传特征，该黏附素介导了早期口腔定植菌的种间黏附。在我们的初步研究中，我们证明了RadD依赖于其伴侣种血链球菌（Ss）的结合刺激Fn的明显表型变化和差异基因表达。基于这些发现，我们提出了我们的工作假设，即“Fn通过RadD和相关的rad蛋白与早期定植的伴侣物种相互作用，这触发了Fn对生物膜形成重要的形态学和生理变化”。为了验证这一假设，我们提出了一项详细的研究RadD在fn介导的种间相互作用中的作用，使用血链球菌（Ss）作为模型早期定植的伴侣物种。方法将包括表征生物膜行为、口腔群落整合、基因表达模式和鉴定Ss中参与omp介导的与Fn相互作用的细胞成分。这种全面的方法将在分子水平上为生物膜形成过程提供详细的知识，包括物理细胞-细胞接触和信号传导。该研究将为改善广泛存在的人类传染病的治疗干预提供新的靶点，并为Fn在晚期定殖伴侣物种或动物模型中的未来研究奠定基础。

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. PUBLIC HEALTH RELEVANCE: Fusobacterium nucleatum (Fn) is a bacterium with key functions in building oral biofilms in the gingival pocket through its ability to stick to a large variety of other bacteria. These bacteria include the ones that grow on oral tissue and tooth surfaces as well as important periodontal pathogens. The detailed understanding of how Fn binds to the surface attached bacteria will provide an important basis for improved therapeutics against periodontitis since it enables the recruitment of pathogenic bacteria into the oral bacterial plaque community.