The microbial gbu gene cluster links cardiovascular disease risk associated with red meat consumption to microbiota L-carnitine catabolism

The heightened cardiovascular disease (CVD) risk observed with omnivores is thought to be linked, in part, to gut microbiota-dependent generation trimethylamine-N-oxide (TMAO) from L-carnitine, a nutrient abundant in red meat. Gut microbial transformation of L-carnitine into trimethylamine (TMA), the precursor of TMAO, occurs via the intermediate γ-butyrobetaine (γBB). However, the relationship between γBB, red meat ingestion and CVD risks, as well as the gut microbial genes responsible for the transformation of γBB to TMA, are unclear. Here we show plasma γBB levels in individuals from a clinical cohort (n=2,918) are strongly associated with incident CVD event risks. Culture of human fecal samples and microbial transplantation studies in gnotobiotic mice with defined synthetic communities showed that the introduction of Emergencia timonensis, a human gut microbe that can metabolize γBB into TMA, is sufficient to complete the carnitine→γBB→TMA transformation, elevate TMAO levels, and enhance thrombosis potential in recipients following arterial injury. RNAseq analyses of E. timonensis identified a 6 gene cluster, herein named gamma-butyrobetaine utilization gene cluster (gbu), which is upregulated in response to γBB. Combinatorial cloning and functional studies identified 4 genes (gbuA, gbuB, gbuC, and gbuE) that are necessary and sufficient to recapitulate the conversion of γBB to TMA when co-expressed in E. coli. Finally, reanalysis of samples (n=113) from a clinical randomized diet intervention study showed that the abundance of fecal gbuA correlates with plasma TMAO and a red meat-rich diet. Our findings reveal a microbial gene cluster that is critical to dietary carnitine→γBB→TMA→TMAO transformation in hosts and contributes to CVD risk.