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.
The multitude of different bacteria that live in our oral cavities form complicated interaction networks within the tissue attached microbial communities commonly known as plaque and to whom they bind (who their neighbors are) can greatly influence their behavior in health and disease. Despite being such an important feature only few of the interactions among the many possible ones are known because existing assays mainly employ tedious one-on-one approaches to identify which bacteria can bind to each other. The approach proposed in this application will allow simultaneous identification of the majority of binding partners for any bacterium of interest, which will ultimately provide a complete picture of the bacterial interaction network and provide novel information on the potential of bacterial interactions as therapeutic targets.
