Bile Acid 7α-Dehydroxylating Gut Bacteria Secrete Antibiotics that Inhibit Clostridium difficile: Role of Secondary Bile Acids.

Clostridium scindens biotransforms primary bile acids into secondary bile acids, and is correlated with inhibition of Clostridium difficile growth in vivo. The aim of the current study was to determine how C. scindens regulates C. difficile growth in vitro and if these interactions might relate to the regulation of gut microbiome structure in vivo. The bile acid 7α-dehydroxylating gut bacteria, C. scindens and C. sordellii, were found to secrete the tryptophan-derived antibiotics, 1-acetyl-ß-carboline and turbomycin A, respectively. Both antibiotics inhibited growth of C. difficile and other gut bacteria. The secondary bile acids, deoxycholic acid and lithocholic acid, but not cholic acid, enhanced the inhibitory activity of these antibiotics. These antibiotics appear to inhibit cell division of C. difficile. The results help explain how endogenously synthesized antibiotics and secondary bile acids may regulate C. difficile growth and the structure of the gut microbiome in health and disease.

Bile acid oxidation by Eggerthella lenta strains C592 and DSM 2243T.

Strains of Eggerthella lenta are capable of oxidation-reduction reactions capable of oxidizing and epimerizing bile acid hydroxyl groups. Several genes encoding these enzymes, known as hydroxysteroid dehydrogenases (HSDH) have yet to be identified. It is also uncertain whether the products of E. lenta bile acid metabolism are further metabolized by other members of the gut microbiota. We characterized a novel human fecal isolate identified as E. lenta strain C592. The complete genome of E. lenta strain C592 was sequenced and comparative genomics with the type strain (DSM 2243) revealed high conservation, but some notable differences. E. lenta strain C592 falls into group III, possessing 3α, 3ß, 7α, and 12α-hydroxysteroid dehydrogenase (HSDH) activity, as determined by mass spectrometry of thin layer chromatography (TLC) separated metabolites of primary and secondary bile acids. Incubation of E. lenta oxo-bile acid and iso-bile acid metabolites with whole-cells of the high-activity bile acid 7α-dehydroxylating bacterium, Clostridium scindens VPI 12708, resulted in minimal conversion of oxo-derivatives to lithocholic acid (LCA). Further, Iso-chenodeoxycholic acid (iso-CDCA; 3ß,7α-dihydroxy-5ß-cholan-24-oic acid) was not metabolized by C. scindens. We then located a gene encoding a novel 12α-HSDH in E. lenta DSM 2243, also encoded by strain C592, and the recombinant purified enzyme was characterized and substrate-specificity determined. Genomic analysis revealed genes encoding an Rnf complex (rnfABCDEG), an energy conserving hydrogenase (echABCDEF) complex, as well as what appears to be a complete Wood-Ljungdahl pathway. Our prediction that by changing the gas atmosphere from nitrogen to hydrogen, bile acid oxidation would be inhibited, was confirmed. These results suggest that E. lenta is an important bile acid metabolizing gut microbe and that the gas atmosphere may be an important and overlooked regulator of bile acid metabolism in the gut.

Metabolism of hydrogen gases and bile acids in the gut microbiome.

The human gut microbiome refers to a highly diverse microbial ecosystem, which has a symbiotic relationship with the host. Molecular hydrogen (H2 ) and carbon dioxide (CO2 ) are generated by fermentative metabolism in anaerobic ecosystems. H2 generation and oxidation coupled to CO2 reduction to methane or acetate help maintain the structure of the gut microbiome. Bile acids are synthesized by hepatocytes from cholesterol in the liver and are important regulators of host metabolism. In this Review, we discuss how gut bacteria metabolize hydrogen gases and bile acids in the intestinal tract and the consequences on host physiology. Finally, we focus on bile acid metabolism by the Actinobacterium Eggerthella lenta. Eggerthella lenta appears to couple hydroxyl group oxidations to reductive acetogenesis under a CO2 or N2 atmosphere, but not under H2 . Hence, at low H2 levels, E. lenta is proposed to use NADH from bile acid hydroxyl group oxidations to reduce CO2 to acetate.

Identification of a gene encoding a flavoprotein involved in bile acid metabolism by the human gut bacterium Clostridium scindens ATCC 35704.

BACKGROUND: The multi-step bile acid 7α-dehydroxylating pathway by which a few species of Clostridium convert host primary bile acids to toxic secondary bile acids is of great importance to gut microbiome structure and host physiology and disease. While genes in the oxidative arm of the 7α-dehydroxylating pathway have been identified, genes in the reductive arm of the pathway are still obscure. METHODS: We identified a candidate flavoprotein-encoding gene predicted to metabolize steroids. This gene was cloned and overexpressed in E. coli and affinity purified. Reaction substrate and product were separated by thin layer chromatography and identified by liquid chromatograph mass spectrometry-ion trap-time of flight (LCMS-IT-TOF). Phylogenetic analysis of the amino acid sequence was performed. RESULTS: We report the identification of a gene encoding a flavoprotein (EDS08212.1) involved in secondary bile acid metabolism by Clostridium scindens ATCC 35704 and related species. Purified rEDS08212.1 catalyzed formation of a product from 3-dehydro-deoxycholic acid that UPLC-IT-TOF-MS analysis suggests loses 4amu. Our phylogeny identified this gene in other bile acid 7α-dehydroxylating bacteria. CONCLUSIONS: These data suggest formation of a product, 3-dehydro-4,6-deoxycholic acid, a recognized intermediate in the reductive arm of bile acid 7α-dehydroxylation pathway and the first report of a gene in the reductive arm of the bile acid 7α-dehydroxylating pathway.

Consequences of bile salt biotransformations by intestinal bacteria.

Emerging evidence strongly suggest that the human "microbiome" plays an important role in both health and disease. Bile acids function both as detergents molecules promoting nutrient absorption in the intestines and as hormones regulating nutrient metabolism. Bile acids regulate metabolism via activation of specific nuclear receptors (NR) and G-protein coupled receptors (GPCRs). The circulating bile acid pool composition consists of primary bile acids produced from cholesterol in the liver, and secondary bile acids formed by specific gut bacteria. The various biotransformation of bile acids carried out by gut bacteria appear to regulate the structure of the gut microbiome and host physiology. Increased levels of secondary bile acids are associated with specific diseases of the GI system. Elucidating methods to control the gut microbiome and bile acid pool composition in humans may lead to a reduction in some of the major diseases of the liver, gall bladder and colon.