PROJECT SUMMARY/ABSTRACT For over a decade, driven in part by the Human Microbiome Project and related international programs, there have been extensive research efforts focused on characterizing the healthy composition and diseased microbiome states. These studies have shown that changes in the gut microbiome can provoke phenotypic changes in the host and promote or suppress the development of various chronic diseases. In humans and animals, imbalances in the gut microbiome have been associated with type 1 and 2 diabetes, obesity, high plasma cholesterol levels, inflammatory bowel disease, autism, Parkinson's disease, atherosclerosis, and other ailments. The plasticity that allows the microbiome to progress into maladaptive states, also implies that it might be possible to remodel a diseased microbiome within a living human or animal to treat or prevent disease progression. Yet, despite considerable promise for advancing a new generation of personalized therapeutics, methods that can achieve selective remodeling of a diseased gut microbiome into a healthy state have not yet been developed. The proposed research program is designed to directly tackle this central issue. We provide extensive proof-of-principle Preliminary Results data to show, for the first time, that a dysfunctional gut microbiome induced by a high fat diet (HFD) can be remodeled in vitro and in vivo by selected compounds to prevent the development of atherosclerosis in a mouse model of the disease. These compounds elicited marked reductions in plasma cholesterol levels and atherosclerotic lesions in high-fat-fed, low-density lipoprotein receptor (LDLr)-null mice in a 10-week daily oral-dosing study. Mechanistic studies, including microbiome 16S rRNA sequencing, RNA-Seq, flow cytometry, and metabolomics, provide strong support that the lead compound functionally alters gene expression levels within the microbial community as well as the host (mouse), and rebalances levels of various metabolites and anti-inflammatory immune Treg cells. Building on these advances, the proposed studies seek to exploit our in vitro method for screening of complex gut microbial populations as a whole to identify molecules that can modulate its overall composition without significantly reducing species diversity. We use high throughput 16S rRNA sequencing as readout for this process to simultaneously determine the activity and selectivity of each tested compound for remodeling the gut microbiota. Complementary to the screening efforts are studies carried out in vivo in animal models of atherosclerosis, obesity, and diabetes. Comparative analyses of the microbial metagenomics and host transcriptomics for treated vs. control animals would provide data for developing scoring and categorization tools based on novel connectivity mapping and functional gene network reconstructions. In short, these studies should enable discovery of new disease-associated targets and biological pathways operating in the microbiota or the host. It is our hope that the proposed studies will serve as a catalyst for advancing novel therapeutics.