Establishing a novel 13C-labeled metabolite library for the study of dental caries - PROJECT SUMMARY Bacteria contain a vast diversity of metabolites due to rich accessory genomes 100 times greater than that found in humans. Most of these metabolites are unknown metabolites and are often excluded from current metabolite studies that use standard metabolomics references limited to common human, drug or industrial chemicals, providing an incomplete perspective in these investigations. There is currently no oral bacterial metabolite reference library available for the study of dental disease by metabolomics. The overarching goal of this research plan is to establish a novel 13C-labeled oral bacterial metabolite reference library with known oral bacterial strains and to propose an unprecedented integrated workflow approach combining the 13C-labeled oral bacterial metabolite library with clinical samples to select targeted unknown metabolites to be analyzed for differential LC-MS, transcriptomics and genetic modification leading to full characterization of unknown oral metabolites related to dental caries or health. In Aim 1, a 13C-labeled oral bacterial metabolite reference library for Streptococcus mutans and Streptococuss gordonii will be created using SWATH LC-MS, with dual peaks identified and curated to improve rigor and reproducibility in oral metabolomics studies. Under Aim 2, the newly generated oral bacterial metabolite reference library will be evaluated for feasibility and optimized for its application in the study of clinical saliva and plaque samples for the study of caries versus health. The goal of Aim 3 will be to establish the unprecedented workflow using the 13C-labeled oral bacterial metabolite library combined differential metabolomics, transcriptomics analysis, and genetic modification leading to isolation, purification and full characterization of previously unknown bacterial metabolites relevant to caries versus health. Creation of a 13C-labeled oral metabolite reference library is crucial for the generation of quality oral metabolomics analysis by allowing for differentiation and removal of noise or artifacts, quantification of labeled metabolites, preliminary identification of unknown compounds, normalization across multiple laboratories/equipment, corrections for ion suppression and, by our novel approach, will allow unknown metabolites to be linked back to specific oral bacteria for full structural elucidation. Most importantly, this approach will allow the inclusion of oral bacterial metabolites previously missed, overlooked, or excluded in other studies. This research will resolve many of the issues of quality metabolomics in oral studies, while limiting the unknowns to target for isolation, purification and characterization to manageable numbers. Solving these challenges in oral bacterial metabolomics will greatly advance the field of oral metabolomics investigations, leading to novel natural products discovery that can be used as treatment or prevention of oral (e.g., childhood dental caries) or other human disease.
