ena-STUDY-Naseer-10-05-2021-23:40:46:926-97
Dietary patterns, particularly the consumption of a red meat-rich or western-type diet, are associated with an increased risk of cardiovascular disease (CVD).1–5 L-Carnitine is a nutrient abundant in red meat that both associates with CVD event risks in humans with high TMAO, and enhances atherosclerosis in animal models.6 Dietary L-carnitine is converted by gut microbes into trimethylamine (TMA), and then absorbed and converted in the host to TMAO by liver flavin-containing monooxygenases (FMOs), especially FMO3.7 Interestingly, chronic dietary patterns greatly influence the capacity of the gut microbial community to convert L-carnitine ? TMA, with vegetarians and vegans (in contrast to omnivores) showing a minimal capacity to generate TMA (and thus TMAO) from dietary carnitine
L-Carnitine, a nutrient abundant in red meat, is metabolized by gut microbiota into trimethylamine (TMA), a precursor of the atherothrombotic metabolite trimethylamine N-oxide (TMAO).  Gut microbiota conversion of carnitine into TMA occurs in omnivores, but far less so in vegetarians or vegans. Previous studies revealed the L-carnitine?TMA microbial transformation occurs via ?-butyrobetaine (?BB), and while multiple microbes convert dietary L-carnitine into ?BB, relatively few have capacity to transform ?BB into TMA. The heightened risk for cardiovascular disease (CVD) observed with omnivores compared to vegetarians/vegans is thought to be linked, in part, to gut microbiota-dependent TMAO generation from carnitine. Despite the central role of ?BB in this transformation, neither the relationship of ?BB to CVD risks, nor the gut microbial genes responsible for ?BB?TMA transformation in omnivores are known. Herein we show circulating ?BB is strongly associated with incident risks of heart attack, stroke or death amongst patients (n=2,918) undergoing elective cardiac evaluations, but only amongst those with elevated TMAO, suggesting the heightened risks associated with ?BB may correspond to its ability to be metabolized into TMAO. In animal studies, ingestion of ?BB, like carnitine, raised TMAO levels and enhanced thrombus formation in vivo. In vitro human fecal culturing studies, and parallel studies in germ-free mice with defined synthetic communities, show the introduction of Emergencia timonensis, which can metabolize ?BB into TMA, is sufficient to impart polymicrobial communities with the capacity to complete the carnitine??BB?TMA transformation, elevate TMAO levels, and enhance thrombosis potential in recipients following arterial injury.  RNAseq analyses of E. timonensis revealed a 6 gene cluster, herein named gamma-butyrobetaine utilization gene cluster (gbu), which is uniquely upregulated in response to ?BB. Subsequent combinatorial cloning and functional expression studies identified a minimum contingent of 4 genes (gbuA, gbuB, gbuC, and gbuE) that are necessary and sufficient to recapitulate the conversion of ?BB ?TMA when co-expressed in E. coli. Finally, metagenomics analysis of human fecal communities from subjects (n=50) enrolled in a randomized cross-over design diet intervention study comparing red meat versus alternative dietary protein sources revealed that the abundance of gbuA is both significantly correlated with plasma TMAO and a red-meat rich diet. The present studies reveal a novel bacterial gene cluster whose abundance is regulated by consumption of red-meat, critical to the transformation of the dietary carnitine intermediate ?BB into TMAO in hosts, and contributes to both heightened thrombosis potential in vivo and CVD risk.
L-Carnitine, a nutrient abundant in red meat, is metabolized by gut microbiota into trimethylamine (TMA), a precursor of the atherothrombotic metabolite trimethylamine N-oxide (TMAO).  Gut microbiota conversion of carnitine into TMA occurs in omnivores, but far less so in vegetarians or vegans. Previous studies revealed the L-carnitine?TMA microbial transformation occurs via ?-butyrobetaine (?BB), and while multiple microbes convert dietary L-carnitine into ?BB, relatively few have capacity to transform ?BB into TMA. The heightened risk for cardiovascular disease (CVD) observed with omnivores compared to vegetarians/vegans is thought to be linked, in part, to gut microbiota-dependent TMAO generation from carnitine. Despite the central role of ?BB in this transformation, neither the relationship of ?BB to CVD risks, nor the gut microbial genes responsible for ?BB?TMA transformation in omnivores are known. Herein we show circulating ?BB is strongly associated with incident risks of heart attack, stroke or death amongst patients (n=2,918) undergoing elective cardiac evaluations, but only amongst those with elevated TMAO, suggesting the heightened risks associated with ?BB may correspond to its ability to be metabolized into TMAO. In animal studies, ingestion of ?BB, like carnitine, raised TMAO levels and enhanced thrombus formation in vivo. In vitro human fecal culturing studies, and parallel studies in germ-free mice with defined synthetic communities, show the introduction of Emergencia timonensis, which can metabolize ?BB into TMA, is sufficient to impart polymicrobial communities with the capacity to complete the carnitine??BB?TMA transformation, elevate TMAO levels, and enhance thrombosis potential in recipients following arterial injury.  RNAseq analyses of E. timonensis revealed a 6 gene cluster, herein named gamma-butyrobetaine utilization gene cluster (gbu), which is uniquely upregulated in response to ?BB. Subsequent combinatorial cloning and functional expression studies identified a minimum contingent of 4 genes (gbuA, gbuB, gbuC, and gbuE) that are necessary and sufficient to recapitulate the conversion of ?BB ?TMA when co-expressed in E. coli. Finally, metagenomics analysis of human fecal communities from subjects (n=50) enrolled in a randomized cross-over design diet intervention study comparing red meat versus alternative dietary protein sources revealed that the abundance of gbuA is both significantly correlated with plasma TMAO and a red-meat rich diet. The present studies reveal a novel bacterial gene cluster whose abundance is regulated by consumption of red-meat, critical to the transformation of the dietary carnitine intermediate ?BB into TMAO in hosts, and contributes to both heightened thrombosis potential in vivo and CVD risk.