Berberine alters gut microbial function through modulation of bile acids.

BACKGROUND: Berberine (BBR) is a plant-based nutraceutical that has been used for millennia to treat diarrheal infections and in contemporary medicine to improve patient lipid profiles. Reduction in lipids, particularly cholesterol, is achieved partly through up-regulation of bile acid synthesis and excretion into the gastrointestinal tract (GI). The efficacy of BBR is also thought to be dependent on structural and functional alterations of the gut microbiome. However, knowledge of the effects of BBR on gut microbiome communities is currently lacking. Distinguishing indirect effects of BBR on bacteria through altered bile acid profiles is particularly important in understanding how dietary nutraceuticals alter the microbiome. RESULTS: Germfree mice were colonized with a defined minimal gut bacterial consortium capable of functional bile acid metabolism (Bacteroides vulgatus, Bacteroides uniformis, Parabacteroides distasonis, Bilophila wadsworthia, Clostridium hylemonae, Clostridium hiranonis, Blautia producta; B4PC2). Multi-omics (bile acid metabolomics, 16S rDNA sequencing, cecal metatranscriptomics) were performed in order to provide a simple in vivo model from which to identify network-based correlations between bile acids and bacterial transcripts in the presence and absence of dietary BBR. Significant alterations in network topology and connectivity in function were observed, despite similarity in gut microbial alpha diversity (P = 0.30) and beta-diversity (P = 0.123) between control and BBR treatment. BBR increased cecal bile acid concentrations, (P < 0.05), most notably deoxycholic acid (DCA) (P < 0.001). Overall, analysis of transcriptomes and correlation networks indicates both bacterial species-specific responses to BBR, as well as functional commonalities among species, such as up-regulation of Na+/H+ antiporter, cell wall synthesis/repair, carbohydrate metabolism and amino acid metabolism. Bile acid concentrations in the GI tract increased significantly during BBR treatment and developed extensive correlation networks with expressed genes in the B4PC2 community. CONCLUSIONS: This work has important implications for interpreting the effects of BBR on structure and function of the complex gut microbiome, which may lead to targeted pharmaceutical interventions aimed to achieve the positive physiological effects previously observed with BBR supplementation.

Development of a novel model of cholecystectomy in subsequently ovariectomized mice and characterization of metabolic and gastrointestinal phenotypes: a pilot study.

BACKGROUND: Cholecystectomy (XGB) is the most common abdominal surgery performed in the United States and is associated with an increased post-surgery incidence of metabolic and gastrointestinal (GI) diseases. Two main risk factors for XGB are sex (female) and age (40-50 yr), corresponding with onset of menopause. Post-menopausal estrogen loss alone facilitates metabolic dysfunction, but the effects of XGB on metabolic and GI health have yet to be investigated in this population. Study objectives were to (1) identify possible short-term effects of XGB and (2) develop a novel murine model of XGB in human menopause via subsequent ovariectomy (OVX) and assess longitudinal effects of OVX on metabolism, GI physiology, and GI microbiota in XGB mice. METHODS: Female C57BL/6 mice were utilized in two parallel studies (S1&S2). In S1, XGB mice were compared to a non-XGB baseline group after six wk. In S2, mice were XGB at wk0, either sham (SHM) or OVX at wk6, and sacrificed at wk12, wk18, and wk24. Body composition assessment and fresh fecal collections were conducted periodically. Serum and tissues were collected at sacrifice for metabolic and GI health endpoints. RESULTS: Compared to baseline, XGB increased hepatic CYP7A1 and decreased HMGCR relative expression, but did not influence BW, fat mass, or hepatic triglycerides after six wk. In S2, XGB/OVX mice had greater BW and fat mass than XGB/SHM. Cecal microbiota alpha diversity metrics were lower in XGB/OVX mice at wk24 compared the XGB/SHM. No consistent longitudinal patterns in fasting serum lipids, fecal microbial diversity, and GI gene expression were observed between S2 groups. CONCLUSIONS: In addition to developing a novel, clinically-representative model of XGB and subsequent OVX, our results suggest that OVX resulted in the expected phenotype to some extent, but that XGB may modify or mask some responses and requires further investigation.

Clostridium scindens ATCC 35704: Integration of Nutritional Requirements, the Complete Genome Sequence, and Global Transcriptional Responses to Bile Acids.

In the human gut, Clostridium scindens ATCC 35704 is a predominant bacterium and one of the major bile acid 7α-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 H2. 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.IMPORTANCEC. scindens is one of a few identified gut bacterial species capable of converting host cholic acid into disease-associated secondary bile acids such as deoxycholic acid. The current work represents an important advance in understanding the nutritional requirements and response to bile acids of the medically important human gut bacterium, C. scindens ATCC 35704. A defined medium has been developed which will further the understanding of bile acid metabolism in the context of growth substrates, cofactors, and other metabolites in the vertebrate gut. Analysis of the complete genome supports the nutritional requirements reported here. Genome-wide transcriptomic analysis of gene expression in the presence of cholic acid and deoxycholic acid provides a unique insight into the complex response of C. scindens ATCC 35704 to primary and secondary bile acids. Also revealed are genes with the potential to function in bile acid transport and metabolism.

Metabolism of Oxo-Bile Acids and Characterization of Recombinant 12α-Hydroxysteroid Dehydrogenases from Bile Acid 7α-Dehydroxylating Human Gut Bacteria.

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 Clostridium leptum; however, this activity has not been reported for high-activity bile acid 7α-dehydroxylating bacteria, such as Clostridium scindens, Clostridium hylemonae, and Clostridium hiranonis 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 Firmicutes, Actinobacteria in the Coriobacteriaceae family, and human gut ArchaeaIMPORTANCE 12α-HSDH activity has been established in the medically important bile acid 7α-dehydroxylating bacteria C. scindens, C. hiranonis, and C. hylemonae 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.

Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes.

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-cis orientation in which the sterol rings are "kinked", as well as small quantities of A/B-trans oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7α-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5α-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-Δ4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals.

Gut feelings about bacterial steroid-17,20-desmolase.

Advances in technology are only beginning to reveal the complex interactions between hosts and their resident microbiota that have co-evolved over centuries. In this review, we present compelling evidence that implicates the host-associated microbiome in the generation of 11ß-hydroxyandrostenedione, leading to the formation of potent 11-oxy-androgens. Microbial steroid-17,20-desmolase cleaves the side-chain of glucocorticoids (GC), including cortisol (and its derivatives of cortisone, 5α-dihydrocortisol, and also (allo)- 3α, 5α-tetrahydrocortisol, but not 3α-5ß-tetrahydrocortisol) and drugs (prednisone and dexamethasone). In addition to side-chain cleavage, we discuss the gut microbiome's robust potential to transform a myriad of steroids, mirroring much of the host's metabolism. We also explore the overlooked role of intestinal steroidogenesis and efflux pumps as a potential route for GC transport into the gut. Lastly, we propose several health implications from microbial steroid-17,20-desmolase function, including aberrant mineralocorticoid, GC, and androgen receptor signaling in colonocytes, immune cells, and prostate cells, which may exacerbate disease states.

Infection with Bacteroides Phage BV01 Alters the Host Transcriptome and Bile Acid Metabolism in a Common Human Gut Microbe.

Gut-associated phages are hypothesized to alter the abundance and activity of their bacterial hosts, contributing to human health and disease. Although temperate phages constitute a significant fraction of the gut virome, the effects of lysogenic infection are underexplored. We report that the temperate phage, Bacteroides phage BV01, broadly alters its host's transcriptome, the prominent human gut symbiont Bacteroides vulgatus. This alteration occurs through phage-induced repression of a tryptophan-rich sensory protein (TspO) and represses bile acid deconjugation. Because microbially modified bile acids are important signals for the mammalian host, this is a mechanism by which a phage may influence mammalian phenotypes. Furthermore, BV01 and its relatives in the proposed phage family Salyersviridae are ubiquitous in human gut metagenomes, infecting a broad range of Bacteroides hosts. These results demonstrate the complexity of phage-bacteria-mammal relationships and emphasize a need to better understand the role of temperate phages in the gut microbiome.

Bacterial steroid-17,20-desmolase is a taxonomically rare enzymatic pathway that converts prednisone to 1,4-androstanediene-3,11,17-trione, a metabolite that causes proliferation of prostate cancer cells.

The adrenal gland has traditionally been viewed as a source of "weak androgens"; however, emerging evidence indicates 11-oxy-androgens of adrenal origin are metabolized in peripheral tissues to potent androgens. Also emerging is the role of gut bacteria in the conversion of C21 glucocorticoids to 11-oxygenated C19 androgens. Clostridium scindens ATCC 35,704 is a gut microbe capable of converting cortisol into 11-oxy-androgens by cleaving the side-chain. The desA and desB genes encode steroid-17,20-desmolase. Our prior study indicated that the urinary tract bacterium, Propionimicrobium lymphophilum ACS-093-V-SCH5 encodes desAB and converts cortisol to 11ß-hydroxyandrostenedione. We wanted to determine how widespread this function occurs in the human microbiome. Phylogenetic and sequence similarity network analyses indicated that the steroid-17,20-desmolase pathway is taxonomically rare and located in gut and urogenital microbiomes. Two microbes from each of these niches, C. scindens and Propionimicrobium lymphophilum, respectively, were screened for activity against endogenous (cortisol, cortisone, and allotetrahydrocortisol) and exogenous (prednisone, prednisolone, dexamethasone, and 9-fluorocortisol) glucocorticoids. LC/MS analysis showed that both microbes were able to side-chain cleave all glucocorticoids, forming 11-oxy-androgens. Pure recombinant DesAB from C. scindens showed the highest activity against prednisone, a commonly prescribed glucocorticoid. In addition, 0.1 nM 1,4-androstadiene-3,11,17-trione, bacterial side-chain cleavage product of prednisone, showed significant proliferation relative to vehicle in androgen-dependent growth LNCaP prostate cancer cells after 24 h (2.3 fold; P <  0.01) and 72 h (1.6 fold; P < 0.01). Taken together, DesAB-expressing microbes may be an overlooked source of androgens in the body, potentially contributing to various disease states, such as prostate cancer.

The 'in vivo lifestyle' of bile acid 7α-dehydroxylating bacteria: comparative genomics, metatranscriptomic, and bile acid metabolomics analysis of a defined microbial community in gnotobiotic mice.

The formation of secondary bile acids by gut microbes is a current topic of considerable biomedical interest. However, a detailed understanding of the biology of anaerobic bacteria in the genus Clostridium that are capable of generating secondary bile acids is lacking. We therefore sought to determine the transcriptional responses of two prominent secondary bile acid producing bacteria, Clostridium hylemonae and Clostridium hiranonis to bile salts (in vitro) and the cecal environment of gnotobiotic mice. The genomes of C. hylemonae DSM 15053 and C. hiranonis DSM 13275 were closed, and found to encode 3,647 genes (3,584 protein-coding) and 2,363 predicted genes (of which 2,239 are protein-coding), respectively, and 1,035 orthologs were shared between C. hylemonae and C. hiranonis. RNA-Seq analysis was performed in growth medium alone, and in the presence of cholic acid (CA) and deoxycholic acid (DCA). Growth with CA resulted in differential expression (>0.58 log2FC; FDR < 0.05) of 197 genes in C. hiranonis and 118 genes in C. hylemonae. The bile acid-inducible operons (bai) from each organism were highly upregulated in the presence of CA but not DCA. We then colonized germ-free mice with human gut bacterial isolates capable of metabolizing taurine-conjugated bile acids. This consortium included bile salt hydrolase-expressing Bacteroides uniformis ATCC 8492, Bacteroides vulgatus ATCC 8482, Parabacteroides distasonis DSM 20701, as well as taurine-respiring Bilophila wadsworthia DSM 11045, and deoxycholic/lithocholic acid generating Clostridium hylemonae DSM 15053 and Clostridium hiranonis DSM 13275. Butyrate and iso-bile acid-forming Blautia producta ATCC 27340 was also included. The Bacteroidetes made up 84.71% of 16S rDNA cecal reads, B. wadsworthia, constituted 14.7%, and the clostridia made up <.75% of 16S rDNA cecal reads. Bile acid metabolomics of the cecum, serum, and liver indicate that the synthetic community were capable of functional bile salt deconjugation, oxidation/isomerization, and 7α-dehydroxylation of bile acids. Cecal metatranscriptome analysis revealed expression of genes involved in metabolism of taurine-conjugated bile acids. The in vivo transcriptomes of C. hylemonae and C. hiranonis suggest fermentation of simple sugars and utilization of amino acids glycine and proline as electron acceptors. Genes predicted to be involved in trimethylamine (TMA) formation were also expressed.

A collection of bacterial isolates from the pig intestine reveals functional and taxonomic diversity.

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.

Draft Genome Sequences of Pseudomonas fluorescens Strains SF39a and SF4c, Potential Plant Growth Promotion and Biocontrol Agents.

Pseudomonas fluorescens SF4c and SF39a, strains isolated from wheat rhizosphere, have potential applications in plant growth promotion and biocontrol of fungal diseases of crop plants. We report the draft genome sequences of SF4c and SF39a with estimated sizes of 6.5 Mb and 5.9 Mb, respectively.