PROJECT SUMMARYThe central goal of our proposed research is to obtain a functional understanding of the native and toxin-exposed gutmicrobiome in xenometabolism from phylum to taxa to specific enzymes. The genomes of the microorganisms within thegut microbiome encode an extensive capacity for the metabolism of dietary compounds, pharmaceutical drugs,environmental chemicals, persistent organic pollutants, and other xenobiotics. Gut microbiota-xenobiotic interactions arealso reciprocal, as exposure to xenobiotics can perturb the metabolic function of the microbiome. Our currentunderstanding of microbiome functions are largely inferred from corollary measurements that can ascribe the potential fora function, but can not actually confirm that the function is present and active. We will make a significant advance in theunderstanding of microbial metabolic functions and how they influence human health by applying an activity-basedprotein profiling (ABPP) platform for in situ analysis of taxa and enzyme functional activities. By being able to identifythe functionally active cells and enzymes within the microbiome, meaningful conclusions can be drawn about theconnection between the gut microbiome and xenobiotic metabolism. Our ABPP platform will deploy chemical probesthat target only catalytically active enzymes involved in xenometabolism in the gut microcbiome. The probes target thefunctional enzymes, but will enable isolation, characterization, and quantification of both the cells expressing thosefunctions, and the specific enzymes catalyzing xenobiotic metabolism. With our ABPP platform we will address thecentral hypothesis that xenometabolism in the gut microbiome can be performed with phylogenetically disparatepopulations and different enzymes. We will initially develop a suite of new activity-based probes for five centralxenobiotic metabolizing enzyme families in the gut microbiome. We will apply these to mouse gut and fecalmicrobiomes, and use flow cytometry, imaging, and proteomics to characterize functional cells and enzymes. We willalso evaluate how exposure to a toxin, which is also a strong agonist of the aryl hydrocarbon receptor, impacts thecompositional and functional environment of the microbiome. Completion of this project will provide a rich newfunctional understanding of microbial xenobiotic metabolism at the level of specific cells and enzymes. Our research willalso yield a platform for future microbiome studies to evaluate individual variability and susceptibility to diseases, tounderstand consequences associated with xenobiotic exposures in adults and developing children, as well as for the futureof precision medicine.