The intestinal microbiota play a pivotal role in human health. Microbiota functionalities include energy production, immune priming, and regulation of energy expenditure. In metabolic diseases, such as type 2 diabetes (T2D) and obesity, the composition and functionality of the gut microbiota is altered, with profound implications for communication with the human host. Recently, it has been suggested that bacterial membrane vesicles (bMVs), produced by intraluminal bacteria, partake in this communication. These bMVs and their cargo; e.g. metabolites, nucleic acids and proteins, are believed to cross the intestinal barrier in health and disease. As such, they can reach organs such as adipose tissue (AT), skeletal muscle, and the liver where they can affect glucose and fatty acid metabolism. However, the context in which gut bMVs readily cross the intestinal barrier to have a marked influence on host metabolism is unknown. In this project we aim to elucidate bMV-mediated effects of the microbiome on the host in health and (metabolic) disease. Purification of bMVs from feces/plasma from healthy and obese/overweight/T2D subjects will be performed through (ultra)centrifugation and size exclusion chromatography steps. Initial characterization using analytical methods (e.g. Brownian motion detection and qPCR) will be used as quality control whereas 16S rRNA sequencing of purified bMVs will be used to determine vesicle producer strains. Proteomics and assessment of bMV immunogenicity will be performed to characterize vesicle cargo. Relative abundancies and bMV characteristics will be compared between groups and interventions to determine correlations in bMV/bacterial abundancy and  metabolic health (outcomes). Causal relationships between gut-derived bMV types and host tissue dysfunction will subsequently be validated using (co)culture tissue studies in which e.g. insulin signaling and inflammation will be characterized. These findings will advance our understanding of the communication between the microbiota and host metabolism, with implications for modulation of the microbiota through e.g. nutrition.