Characterization of interactions of dietary cholesterol with the murine and human gut microbiome

<jats:title>Abstract</jats:title><jats:p>Consumption of dietary lipids, such as cholesterol, modulates the gut microbiome with consequences for host health through the production of microbiome-derived metabolites. Despite the implications for host metabolism, a limited number of specific interactions of the gut microbiome with diet-derived lipids have been characterized. This is partially because obtaining species-level resolution of the responsible taxa can be challenging and additional approaches are needed to identify health-relevant metabolites produced from cholesterol–microbiome interactions. Here we performed bio-orthogonal labelling sort sequence spectrometry, a click chemistry based workflow, to profile cholesterol-specific host–microbe interactions. Mice were exposed to an alkyne-functionalized variant of cholesterol and 16S ribosomal RNA gene amplicon sequencing of faecal samples identified diet-derived cholesterol-interacting microbes from the genera <jats:italic>Bacteroides</jats:italic>, <jats:italic>Bifidobacterium</jats:italic>, <jats:italic>Enterococcus</jats:italic> and <jats:italic>Parabacteroides</jats:italic>. Shotgun metagenomic analysis provided species-level resolution of diet-derived cholesterol-interacting microbes with enrichment of bile acid-like and sulfotransferase-like activities. Using untargeted metabolomics, we identify that cholesterol is converted to cholesterol sulfate in a <jats:italic>Bacteroides</jats:italic>-specific manner via the enzyme BT_0416. Mice monocolonized with <jats:italic>Bacteroides thetaiotaomicron</jats:italic> lacking Bt_0416 showed altered host cholesterol and cholesterol sulfate compared with wild-type mice, identifying a previously uncharacterized microbiome-transformation of cholesterol and a mechanism for microbiome-dependent contributions to host phenotype. Moreover, identification of a cholesterol-responsive sulfotransferase in <jats:italic>Bacteroides</jats:italic> suggests diet-dependent mechanisms for altering microbiome-specific cholesterol metabolism. Overall, our work identifies numerous cholesterol-interacting microbes with implications for more precise microbiome-conscious regulation of host cholesterol homeostasis.</jats:p>