Date of Award
Doctor of Philosophy (PhD)
The gastrointestinal tract contains the largest number and greatest diversity of microbes which are referred to collectively as the gut microbiota. Alterations in microbiota dysbiosis are associated with diverse disease states, including colorectal cancer (CRC), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS). However, the causative mechanisms mediated by the gut microbiota are still to be illustrated. Particularly, the gut commensals encode ~150 times more genes than the human genome and thousands of metabolites that mostly lack functional annotations or structural identification. Therefore, the functional profiling of human commensal metabolites is promising to reveal the molecular mechanisms of the gut microbiota regulating homeostasis or disease outcomes. Through systematic forward-screening methods on a large collection of human gut commensals from divergent phylogenies, we investigated how the gut microbiota influences physiological outcomes through small molecule metabolites in CRC colorectal cancer (CRC) and pain sensation. 1) We established an electrophoresis-based pipeline to evaluate the genotoxicity of microbial small molecule metabolites causing DNA damage. Using comparative metabolomics and bioactivity-guided natural product-discovery techniques, we discovered a previously undescribed family of genotoxic metabolites—termed the indolimines—produced by the CRC-associated species Morganella morganii whose ability to exacerbate CRC independent of inflammation was confirmed in gnotobiotic mice. Through transposon-based random mutagenesis, we identified the gene aat (encoding AAT_I protein, aspartate aminotransferase fold type I) responsible for the synthesis of primary amines, precursors of indolimines. The att- M. morganii exhibited defects of genotoxicity. This project reveals the existence of a previously unexplored universe of genotoxic small molecules from the human microbiome and implies a broader role for microbiota-derived genotoxins in CRC. In addition, the cancer-promoting mechanism of M. morganii is dissected at genetic, molecular and physiological levels. The understanding about genotoxins could instruct the diagnoses or therapies of CRC in the future. 2) We established a calcium influx-based pipeline to investigate the endogenous agonists of TRPV1 ion channel derived from the gut microbiota. We found that Klebsiella species and Accidaminococcus intestini enabled to produce heat-stable, small-molecule metabolites to activate TRPV1 channel. Particularly, A. intestini was confirmed to sensitize TRPV1+ nociceptive dorsal root ganglion (DRG) neurons, suggesting the effects of commensal metabolites in pain sensation and relevant diseases like IBS. Untargeted global metabolomics and screening of human small molecule metabolite library implied that microbial-specific phospholipids might be a novel class of endogenous ligands of TRPV1. We are still working on bacteria engineering to identify the genes or gene clusters responsible for the synthesis of TRPV1 agonists. This study indicates a potential mechanism by which the gut commensals regulate visceral pain sensation and provides a new direction for IBS diagnoses or therapies. In summary, both projects underline the significance of functional profiling of the gut microbiota in dissecting causative mechanisms in human diseases, especially those with complex etiology such as cancer, pain syndromes and neurological disorders.
Cao, Yiyun, "Functional Profiling of Human Commensal Metabolites in Colorectal Cancer and Pain Sensation" (2022). Yale Graduate School of Arts and Sciences Dissertations. 569.