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Comparative Study
. 2008 Sep 9;105(36):13580-5.
doi: 10.1073/pnas.0804437105. Epub 2008 Aug 29.

Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome

Brian V Jones  1 , Máire Begley, Colin Hill, Cormac G M Gahan, Julian R Marchesi
Affiliations
Comparative Study

Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome (VSports在线直播)

Brian V Jones et al. Proc Natl Acad Sci U S A. .

Abstract

Bile salt hydrolases (BSHs) catalyze the "gateway" reaction in a wider pathway of bile acid modification by the gut microbiota. Because bile acids function as signaling molecules regulating their own biosynthesis, lipid absorption, cholesterol homeostasis, and local mucosal defenses in the intestine, microbial BSH activity has the potential to greatly influence host physiology. However, the function, distribution, and abundance of BSH enzymes in the gut community are unknown. Here, we show that BSH activity is a conserved microbial adaptation to the human gut environment with a high level of redundancy in this ecosystem. Through metagenomic analyses we identified functional BSH in all major bacterial divisions and archaeal species in the gut and demonstrate that BSH is enriched in the human gut microbiome. Phylogenetic analysis illustrates that selective pressure in the form of conjugated bile acid has driven the evolution of members of the Ntn_CGH-like family of proteins toward BSH activity in gut-associated species VSports手机版. Furthermore, we demonstrate that BSH mediates bile tolerance in vitro and enhances survival in the murine gut in vivo. Overall, we demonstrate the use of function-driven metagenomics to identify functional anchors in complex microbial communities, and dissect the gut microbiome according to activities relevant to survival in the mammalian gastrointestinal tract. .

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Abundance of putative BSH and related proteins in the human gut metagenome. Amino acid sequences of putative BSH and related amidases described in Fig. S3, from 15 human gut metagenomes (6, 7), Sargasso sea (24), soil (25), and the combined gut metagenomes of lean and obese mice (8) were used to calculate and compare the relative abundance of these proteins in each metagenome. Abundance was calculated as hits per megabase (Mb) for individual and combined metagenomes and revealed variation between individuals and a general enrichment of BSH and related proteins in the human gut metagenome. Japanese individuals: InA, male 45years; InB, male 6 months; InD, male 35 years; InE, male 3 months; InM, female 4 months; F1-S, T, U, Family I, male 30 years, female 28 years, female 7 months, respectively; F2-V, W, X, Y, Family II, male 37 years, female 36 years, male 3 years, female 1.5 years, respectively. American metagenomes: H 7 & 8, female 28 years combined with male 37 years; HUM COMB, all human metagenomes combined; MUS COMB, all murine metagenomes combined; SARG SEA, Sargasso sea metagenome; SOIL, soil metagenome. Colors indicate putative origin of BSH homologues retrieved from each metagenome or combined metagenomes. Values above bars represent overall hits per Mb for each category.
Fig. 2.
Fig. 2.
Phylogenetic analysis of putative BSH and PVA amino acid sequences. A set of 367-aa sequences belonging to the Ntn_CGH-like family (Ntn_PVA and Ntn_CGH families), and displaying all putative conserved critical catalytic amino acids common to both BSH and PVA (based on alignments of our functional BSH and previous analysis of BSH and PVA crystal structures) (23) were used to construct phylogenetic trees and examine the evolution of BSH activity. Sequences originated from a diverse range of bacterial divisions and full-length sequences retrieved from human (7) and Sargasso sea (24) metagenomes were also included. Supporting bootstrap values for deep branching nodes are indicated with black filled diamonds identifying nodes supported by bootstrap values of 50 or over, dark-gray filled diamonds identifying nodes with bootstraps values of 40 or over, and light-gray filled diamonds nodes with bootstrap values <40. Green branches represent clades containing sequences with proven activity against glyco-CBA and tauro-CBA, whereas yellow branches indicate clades containing sequences proven to exhibit activity against tauro-CBA only. Purple branches represent clades with sequences that have been proven to exhibit no BSH activity. Red circles indicate positions of sequences derived from human gut metagenomes. Blue circles indicate sequences retrieved from the Sargasso sea metagenome. Arrows show positions of Archaeal sequences included in this analysis: 1→, Methanosphera stadmanae (human gut); 2→, Methanobrevibacter smithii (human gut); 3→, Methanosarcina acetovorans (marine sediment). Brackets indicate regions dominated by major bacterial divisions comprising the human gut microbiota, and are generally composed of sequences from gut-associated genera. A1, Firmicutes: Eubacterium, Coprococcus, Clostridium, Ruminococcus, Dorea, Lactobacills, Enterococcus, Listeria, Lactococcus; A2, Firmicutes: Lactobacillus, Clostridium, Listeria, Staphylococcus, Oenococcus, Bacillus; B, Actinobacteria: Bifidobcterium, Collinsella; C1, C2, and C3, Bacteroidetes: Parabacteroides, Bacteroides. (Scale bar, 0.1 aa substitution per site.)
Fig. 3.
Fig. 3.
Elucidation of BSH function in the human gut microbiota. bsh genes from functional metagenomic clones FM1, FM5, FM7, and FM62 (Table S2), corresponding to BSH types A, D, E, F, respectively (Table S3) were cloned in L. innocua FH2333 by using plasmid pNZ44. The resulting clones were designated BSH1, BSH5, BSH7, and BSH62, respectively. Empty plasmid and plasmid-containing bsh from Lactobacillus plantarum were used as negative (−ve) and positive (+ve) controls, respectively. (A) Confirmation of expression of cloned BSH. An agar plate assay (22, 33) was used to examine the bile hydrolase activity of strains. Cultures that were grown in brain heart infusion (BHI) broth were spotted (10 μl) onto LB bile agar supplemented with 0.5% bile acids. Strains with bile salt hydrolase activity were surrounded with white precipitate halos of deconjugated bile acids. (B) Bile survival assays. Overnight cultures were inoculated (3%) into broth supplemented with 25% (wt/vol) bovine bile (Oxgall, B3883 Sigma). Viable cell counts were performed after 3 h by diluting cultures in one-quarter-strength Ringer's solution and enumeration on BHI. Statistical differences from the negative control were determined by Student's t test. *, P < 0.01. (C) Murine experiments. Spontaneous rifampicin-resistant mutants of strains were isolated as described (15). For each strain, five BALB/c mice were administered with ≈109 cells by oral gavage on two consecutive days. On the third day the small and large intestines were removed and homogenized in PBS and bacterial numbers were determined by spread plating homogenized organs BHI on agar supplemented with 50 μg/ml rifampicin. Horizontal bars indicate the mean. Statistical differences were determined by Student's t test. *, P < 0.05
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