A Metaproteomic Approach to Discovery and Translation of Protein Expression Biomarkers for Sucrose-Induced Dysbiosis
Although the etiology of dental caries is multifactorial, frequent consumption of fermentable carbohydrates, notably sucrose, appears to be a major factor driving the supragingival microbiota in the direction of dysbiosis. Recent 16S rDNA-based studies indicate that caries-associated communities were less diverse than healthy supragingival plaque overall, but still displayed considerable taxonomic diversity between individuals. A key finding of the Human Microbiome Project was that healthy sites from different people were broadly similar with respect to gene function, even though there was extensive individual variation in their taxonomic profiles. That pattern may also extend to dysbiotic communities. In that case, shifts in community-wide protein expression might provide more sensitive and specific biomarkers of dysbiosis than can be achieved through taxonomy alone. The preliminary 2-D MSMS metaproteomic data for this project suggest that sucrose–induced changes in protein expression patterns are conserved in taxonomically diverse oral microcosm biofilm communities. They also demonstrate that it is feasible to develop relatively low-cost quantitative targeted proteomics SRM-MS assays for proteins that were upregulated by sucrose. Those data provide the foundation for the long-range goals of this research, which are to use shotgun metaproteomic analysis of oral microcosm biofilm communities to define a suite of conserved protein function biomarkers of sucrose-induced dysbiosis (Discovery Phase), and then use targeted proteomics to develop a set of relatively low-cost assays which could allow early detection of those biomarkers in dental plaque, before obvious signs of demineralization occur on intact teeth (Translational Phase).
For the Discovery Phase, the researchers will: conduct metagenomic and metaproteomic analyses of biofilm microcosms grown from plaque collected from 45 caries-active children, to validate their preliminary observations that changes in protein expression are more consistent indicators of sucrose-induced dysbiosis than are shifts in the taxonomic diversity of potentially cariogenic species; conduct metagenomic/metaproteomic studies of biofilm microcosms grown from plaque collected from 45 caries-free children, to test the hypothesis that they may be more resistant to dysbiosis than microcosms from caries-active children; and use bioinformatic and biostatistical analysis of the Discovery Phase dataset to define a set of conserved protein function biomarkers that can be used as risk indicators for sucrose-induced dysbiosis.
For the Translational Phase the researchers will: optimize the preliminary targeted proteomics SRM-MS protocol to achieve absolute quantification of sucrose-induced differences in protein expression from plaque samples; use the optimized protocol to finalize a set of multiplexed SRM-MS assays for the target biomarkers defined in the Discovery Phase; and conduct a cross-sectional exploratory study to determine whether the SRM-MS assay suite detects sucrose-induced dysbiosis in plaque samples, and differentiates between independent samples of caries-active and caries-free children.
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