REFINED MULTI-OMIC APPROACHES TOWARDS GUT MICROBIAL METABOLISM

Within the human gastrointestinal (GI) tract lies a complex and diverse community of ~1013 bacterial cells that collectively encode roughly 200-fold more genes than the human genome and play an integral role in physiological processes ranging from immunity to promoting the synthesis of neurotransmitters.1,2,3,4 While these microbes have evolved with the host, many proteins they synthesize are capable of chemistry distinct from host enzymes. We combine metagenomics, targeted metaproteomics, structural biology, and animal studies to investigate the role of gut microbial enzymes in host endobiotic metabolism. We show that structurally and functionally distinct -Glucuronidase (GUS) enzymes produced by gut bacteria can catabolize chemically distinct endobiotic-glucuronides, and we further demonstrate that this activity can be inhibited by FDA-approved drugs, as well as potent novel chemotypes, at physiological concentrations. Together, these data establish the role of the gut microbiota in mediating endobiotic levels and demonstrate the extent to which drugs may interact with the microbiota to alter host homeostasis. We then compared the fecal microbiomes of transplant recipients receiving the immunosuppressant Mycophenolate Mofetil (MMF), an immunosuppressant prodrug prescribed to prevent organ transplant rejection and to treat autoimmune diseases. MMF usage is limited by severe gastrointestinal (GI) toxicity that is observed in approximately nearly of MMF recipients. We hypothesized that the GI toxicity may be related to the actions of GUS enzymes, as the active form of the drug, mycophenolic acid (MPA), undergoes extensive enterohepaticrecirculation by GUS enzymes. Neither microbial composition nor the presence of specific structural classes of GUS genes were sufficient to explain the differences in MPA reactivation measured between fecal samples from the two cohorts. We employed targeted metaproteomics to identify and quantify the GUS proteins present in the human fecal samples, which was improved by using the metagenomics data collected from the fecal samples. These results indicated that FMN-binding GUS enzymes may be responsible for reactivation of MPA and could be a driving force behind MPA-induced GI toxicity.We next explore the metagenomic landscape of AzoRs across the human gut microbiome and characterize four of the 160 novel proteins. We found that the most prevalent AzoRs are produced by Firmicutes, and AzoRs produced by this phylum appear to be remarkably efficient compared to AzoRs produced by relatively well-characterized phyla. This study provides initial data to fuel for future endeavors in characterizing the functional diversity of gut microbial AzoR enzymes, particularly those using targeted metaproteomics to assess whether our observations extend to ex vivo protein abundance of AzoRs from individual phyla. Finally, we present a catalog of tools that can be widely used for structural metagenomics and demonstrated their use with two additional gut microbial enzyme families, bile salt hydrolases enzymes and tryptophanase enzymes. The structure-guided bioinformatics methods described here provide a road map for exploring bacterial metabolism with empirically driven certainty, in the context of the gut and beyond.