Cardiometabolic diseases affect millions of people worldwide and have numerous underlying risk factors. Commensal microbes that comprise the intestinal microbiome are implicated in the progression and onset of many of these diseases, including type 2 diabetes, obesity, and atherosclerosis. Gut microbes have extensive metabolic capabilities, allowing them to produce or modify molecules that influence disease risk. Members of the gut microbiota have been known to convert cholesterol into the poorly absorbable metabolite coprostanol for almost 100 years, however the microbial genes responsible for this metabolism were not known. In recent work, we analyzed paired gut metagenomic and metabolomic data to identify a novel group of cholesterol dehydrogenases that metabolize cholesterol when expressed in vitro. Using clinical and metagenomic data from the Framingham Heart Study (FHS), we observed lower serum cholesterol in subjects whose microbiomes encode these cholesterol dehydrogenases. The cholesterol dehydrogenases we originally described convert cholesterol to cholestenone and coprostanone to coprostanol, and we have since identified the intermediate enzyme that converts cholestenone to coprostanone. In this proposal, we will functionally characterize metabolism of sterols in the gut and determine the impact of this process on cardiometabolic disease. In Aim 1, we will identify determinants of host-microbe interactions by collecting and analyzing clinical variables with stool and serum samples from the Gen3/Omni2 FHS cohorts. We will generate coupled stool and serum metabolomics and metagenomics datasets, perform culturomics to assemble a microbial strain library, and identify host exposomes from clinical data. We will then utilize these data to identify and prioritize microbially- derived or modified circulating metabolites associated with CVD for further mechanistic investigations. In Aim 2, We will couple bioinformatics with microbiology and biochemistry for targeted identification of enzymes/proteins that alter sterol structures. This effort will allow us to probe the diversity of gut microbial sterol metabolizing enzymes and their substrates in order to determine specific microbes and genes with the capacity to modify cholesterol. Taking a systems-level approach, we will colonize mice with cholesterol-metabolizing microbial communities to determine how microbial metabolism modulates serum cholesterol and pathways central to cardiovascular disease. In Aim 3, we will functionally link microbiome enzyme activity to sterol metabolism and metabolic disease. We will employ cell-based transcriptional and proteomic assays to interrogate the effects of microbially-modified sterols on local sterol sensing pathways in epithelial cells and measure the effects of circulating sterol metabolites on human immune cells.