Systematic approach to explore the microbial interactome

摘要 微生物间的相互作用是多菌生物膜内社会行为的关键驱动力，最终 影响宿主的健康或疾病结局。这些交互的性质在很大程度上受到 “谁”、“在哪里”、“什么”、“何时”、“如何”和“与谁在一起”的因素。此应用程序旨在开发一种方法 对于与谁进行全面评估，最终解开复杂的互动网络 微生物种类(微生物相互作用体)。了解哪些细菌相互黏附尤其重要 对开放流动系统的重要性，如大多数定植于口腔的生物膜：物理性物种间 结合确保了有效的细胞间信号传递所需的接近，这通常发生在微米级。 因此，相互粘连的微生物更有可能影响彼此的行为，并有可能参与 在最近的研究中提出的针对单个物种的群落操纵。 在我们之前发表的研究的基础上，我们建议开发一个系统的方法，并开始建立 利用核梭杆菌构建口腔微生物群落的种级物理相互作用网络 (FN)是一种成熟的生物膜结构的桥梁生物，作为模式生物。通过建立这一点 对于FN的方法，我们将开始测试我们的工作假设，即物种间在 微生物相互作用组是建立与健康和疾病相关的口腔的主要因素 生物膜，通过规定不同物种亚集的共同定位，从而决定它们的组合 (有益的或致病的)功能潜力。 这将通过本申请中提出的两个目标来实现。在目标1中，我们将开发一个多功能的 使用超顺磁珠作为感兴趣的“诱饵”物种(FN)的锚定基质的下拉试验 允许从与健康和疾病相关的代表性口腔中捕获相互作用的伴侣物种 微生物“猎物”群落。相互作用的生物体将使用Illumina(MiSeq)下一代进行鉴定 测序。在目标2中，我们将应用荧光原位杂交(FISH)或组合标记和光谱 成像荧光原位杂交(Clasi-FISH)及其与 应用DAIME软件研究健康人和疾病人体内FN微生物相互作用的分布 龈下生物膜。对确认的体内相互作用物种的进一步序列生物信息学分析将 评估它们的功能基因组潜力。 物理微生物相互作用组是生物膜社会结构和行为的重要决定因素 可能决定相关宿主的健康和疾病结果的社区。这个 拟议的研究将建立一种全面调查微生物的通用方法 互动组不仅限于口腔，而且可以很容易地扩展到任何生物膜群落。

Abstract Intermicrobial interactions are key driving forces for social behavior within polymicrobial biofilms that ultimately influences health or disease outcome for the host. The nature of these interactions are greatly influenced by the “Who”, “Where”, “What”, “When”, “How” and “With whom” factors. This application aims to develop an approach for comprehensive assessment of the “With Whom” to ultimately unravel the complex network of interacting microbial species (microbial interactome). Understanding which bacteria adhere to each other is particularly important for open flow systems such as most of the biofilms colonizing the oral cavity: physical interspecies binding ensures the proximity necessary for efficient intercellular signaling, which often occurs on a micron scale. Thus, microorganisms that coadhere are more likely to influence each other’s behavior and potentially take part in the community manipulation that recent research proposed for individual species. Building on our previous published research, we propose to develop a systematic approach and start building the species-level physical interaction network for oral microbial communities using Fusobacterium nucleatum (Fn), a well-established bridging organism for biofilm architecture as model organism. By establishing this approach for Fn we will start testing our working hypothesis that interspecies adherence within the microbial interactome is a major factor for the establishment of health- and disease-associated oral biofilms by dictating the co-localization of distinct subsets of species and thus their combined (beneficial or pathogenic) functional potential. This will be accomplished via the two aims proposed in this application. In Aim 1, we will develop a versatile pulldown assay that employs superparamagnetic beads as anchoring matrix for the “bait” species of interest (Fn) to allow the capture of interacting partner species from representative health- and disease-associated oral microbial “prey” communities. Interacting organisms will be identified using Illumina (MiSeq) next generation sequencing. In Aim 2, we will apply fluorescent in situ hybridization (FISH) or combinatorial labeling and spectral imaging fluorescent in situ hybridization (CLASI-FISH) followed by quantitative analysis of co-localization with the software daime to investigate the in vivo distribution of the Fn microbial interactomes in healthy and diseased subgingival biofilms. Further sequence bioinformatics analysis of the confirmed in vivo interacting species will evaluate their functional genomic potential. The physical microbial interactome is an important determinant in the social structure and behavior of biofilm communities which can potentially determine the health and disease outcome for the associated host. The proposed research will establish a versatile approach for comprehensive investigation of the microbial interactome that is not limited to the oral cavity but can easily be expanded to any biofilm community.