Gaskins (2020) The 'invivo lifestyle' of bile acid 7αdehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice,

Our knowledge about the gut microbiota of pigs is still scarce, despite the importance of these animals for biomedical research and agriculture. Here, we present a collection of cultured bacteria from the pig gut, including 110 species across 40 families and nine phyla. We provide taxonomic descriptions for 22 novel species and 16 genera. Meta-analysis of 16S rRNA amplicon sequence data and metagenome-assembled genomes reveal prevalent and pig-specific species within Lactobacillus, Streptococcus, Clostridium, Desulfovibrio, Enterococcus, Fusobacterium, and several new genera described in this study. Potentially interesting functions discovered in these organisms include a fucosyltransferase encoded in the genome of the novel species Clostridium porci, and prevalent gene clusters for biosynthesis of sactipeptide-like peptides. Many strains deconjugate primary bile acids in in vitro assays, and a Clostridium scindens strain produces secondary bile acids via dehydroxylation. In addition, cells of the novel species Bullifex porci are coccoidal or spherical under the culture conditions tested, in contrast with the usual helical shape of other members of the family Spirochaetaceae. The strain collection, called ‘Pig intestinal bacterial collection’ (PiBAC), is publicly available at www.dsmz.de/pibac and opens new avenues for functional studies of the pig gut microbiota.

Bacteria in the gut can modulate the availability and efficacy of therapeutic drugs. Yet, the systematic mapping of the respective interactions has only started recently 1 and the main underlying mechanism proposed is chemical transformation of drugs by microbes (biotransformation). Here, we investigated the depletion of 15 structurally diverse drugs by 25 representative gut bacterial strains. This revealed 70 bacteria-drug interactions, 29 of which had not been reported before. Over half of the new interactions can be ascribed to bioaccumulation, that is bacteria storing the drug intracellularly without chemically modifying it, and in most cases without their growth being affected. As a case in point, we studied the molecular basis of bioaccumulation of the widely used antidepressant duloxetine by using clickchemistry, thermal proteome profiling and metabolomics. We find that duloxetine binds to several metabolic enzymes and changes metabolite secretion of the respective bacteria. When tested in a defined microbial community of accumulators and non-accumulators, duloxetine markedly altered the community composition through metabolic cross-feeding. We further validated our findings in an animal model, showing that bioaccumulating bacteria attenuate the behavioral response of Caenorhabditis elegans to duloxetine. Taken together, bioaccumulation by gut bacteria may be a common mechanism that alters drug availability and bacterial metabolism, with implications for microbiota composition, pharmacokinetics, side effects and drug responses, likely in an individual manner.Therapeutic drugs can have a strong impact on the gut microbiome and vice versa 2-5 . The underlying drug-bacteria interactions can reduce microbial fitness 6 or alter the drug availability through biotransformation 7-14 . The latter can have either a positive or a negative impact on drug activity and efficacy. While drugs like lovastatin and sulfasalazine are chemically transformed by gut bacteria into their active forms, bacterial metabolism can inactivate drugs such as digoxin 15,16 , or cause toxic effects as in the case of irinotecan 17 .Furthering the diversity of susceptible drugs, over one hundred molecules were recently reported to be chemically modified by gut bacteria 1 . Yet, the mechanistic view on these interactions is largely confined to drug biotransformation 12,13 . Drug accumulation without metabolizationTo expand the knowledge of bacterial effect on drug availability, we systematically profiled interactions between 15 human-targeted drugs and 25 representative human gut bacterial strains (21 species; with additional subspecies or conspecific strains of Bifidobacterium longum, Escherichia coli and Bacteroides uniformis) (Supplementary Table 1). The bacterial species were selected to cover a broad phylogenetic and metabolic diversity representative of the healthy microbiota 18 (Extended Data Fig. 1a, Supplementary Table 1). On the drug side, 12 orally administered small molecule drugs (MW<500 Da), amenable to UPLC-UV-based quantificat...

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, 3β, 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; 3β,7α-dihydroxy-5β-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.

Bile acids are important cholesterol-derived nutrient signaling hormones, synthesized in the liver, that act as detergents to solubilize dietary lipids. Bile acid 7α-dehydroxylating gut bacteria generate the toxic bile acids deoxycholic acid and lithocholic acid from host bile acids. The ability of these bacteria to remove the 7-hydroxyl group is partially dependent on 7α-hydroxysteroid dehydrogenase (HSDH) activity, which reduces 7-oxo-bile acids generated by other gut bacteria. 3α-HSDH has an important enzymatic activity in the bile acid 7α-dehydroxylation pathway. 12α-HSDH activity has been reported for the low-activity bile acid 7α-dehydroxylating bacterium ; however, this activity has not been reported for high-activity bile acid 7α-dehydroxylating bacteria, such as, , and Here, we demonstrate that these strains express bile acid 12α-HSDH. The recombinant enzymes were characterized from each species and shown to preferentially reduce 12-oxolithocholic acid to deoxycholic acid, with low activity against 12-oxochenodeoxycholic acid and reduced activity when bile acids were conjugated to taurine or glycine. Phylogenetic analysis suggests that 12α-HSDH is widespread among , in the family, and human gut 12α-HSDH activity has been established in the medically important bile acid 7α-dehydroxylating bacteria ,, and Experiments with recombinant 12α-HSDHs from these strains are consistent with culture-based experiments that show a robust preference for 12-oxolithocholic acid over 12-oxochenodeoxycholic acid. Phylogenetic analysis identified novel members of the gut microbiome encoding 12α-HSDH. Future reengineering of 12α-HSDH enzymes to preferentially oxidize cholic acid may provide a means to industrially produce the therapeutic bile acid ursodeoxycholic acid. In addition, a cholic acid-specific 12α-HSDH expressed in the gut may be useful for the reduction in deoxycholic acid concentration, a bile acid implicated in cancers of the gastrointestinal (GI) tract.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7␣-dehydroxylating anaerobes. While this organism is well-studied relative to bile acid metabolism, little is known about the basic nutrition and physiology of C. scindens ATCC 35704. To determine the amino acid and vitamin requirements of C. scindens, the leave-one-out (one amino acid group or vitamin) technique was used to eliminate the nonessential amino acids and vitamins. With this approach, the amino acid tryptophan and three vitamins (riboflavin, pantothenate, and pyridoxal) were found to be required for the growth of C. scindens. In the newly developed defined medium, C. scindens fermented glucose mainly to ethanol, acetate, formate, and H 2. The genome of C. scindens ATCC 35704 was completed through PacBio sequencing. Pathway analysis of the genome sequence coupled with transcriptome sequencing (RNA-Seq) under defined culture conditions revealed consistency with the growth requirements and end products of glucose metabolism. Induction with bile acids revealed complex and differential responses to cholic acid and deoxycholic acid, including the expression of potentially novel bile acid-inducible genes involved in cholic acid metabolism. Responses to toxic deoxycholic acid included expression of genes predicted to be involved in DNA repair, oxidative stress, cell wall maintenance/metabolism, chaperone synthesis, and downregulation of one-third of the genome. These analyses provide valuable insight into the overall biology of C. scindens which may be important in treatment of disease associated with increased colonic secondary bile acids.

No abstract

Members of the gastrointestinal microbiota are known to convert glucocorticoids to androstanes, which are subsequently converted to potent androgens by other members of the gut microbiota or host tissues. Butyricicoccus desmolans and Clostridium cadaveris have previously been reported for steroid-17,20-desmolase and 20β-hydroxysteroid dehydrogenase (HSDH) activities that are responsible for androstane formation from cortisol; however, the genes encoding these enzymes have yet to be reported. In this work, we identified and located a gene encoding 20β-HSDH in both B. desmolans and C. cadaveris. The 20β-HSDH of B. desmolans was heterologously overexpressed and purified from Escherichia coli. The enzyme was determined to be a homotetramer with subunit molecular mass of 33.8 ± 3.7 kDa. The r20β-HSDH displayed pH optimum in the reductive direction at pH 9.0 and in the oxidative direction at pH 7.0–7.5 with (20β-dihydro)cortisol and NAD(H) as substrates. Cortisol is the preferred substrate with Km, 0.80 ± 0.06 μM; Vmax, 30.36 ± 1.97 μmol·min−1; Kcat, 607 ± 39 μmol·μM−1·min−1; Kcat/Km, 760 ± 7.67. Phylogenetic analysis of the 20β-HSDH from B. desmolans suggested that the 20β-HSDH is found in several Bifidobacterium spp., one of which was shown to express 20β-HSDH activity. Notably, we also identified a novel steroid-17,20-desmolase-elaborating bacterium, Propionimicrobium lymphophilum, a normal inhabitant of the urinary tract.