Supporting InformationJoyce et al. 10.1073/pnas.1323599111SI Materials and MethodsBile Salt Hydrolase Cloning. Bile salt hydrolases (BSHs) fromLactobacillus salivarius strains (1) were cloned independentlyinto the plasmid pBKminiTn7GM2 (2) under the control of theP44 promoter (3) using splicing by overlap extension PCR.Transposon integration was carried out as described previously(2). PCR downstream from the glmS region confirmed con-structions, as did sequence analysis (GATC Biotech).

Bile Salt Activity Assay. BSH-negative E. coli MG1655 (EC),E. coli clones expressing bsh genes from Lactobacillus salivariusJCM1046 (ECBSH1), and E. coli clones expressing bsh genesfrom Lactobacillus salivarius UCC118 (ECBSH2) were examinedfor their ability to deconjugate bile in vitro using the ninhydrinassay for free taurine (4) and by coincubation for 90 min inmurine gall bladder bile acid followed by ultra-performance(UP)LC-MS analysis. Protein concentrations were measuredwith the Bio-Rad Protein Assay (Bio-Rad), and BSA (Sigma)was used as the standard.

Mice.Germ-free Swiss Webster mice were maintained in the germ-free unit in theAlimentary Pharmabiotic Centre.Monocolonizationexperiments were initiated by oral dosing of appropriate strainsat 1 × 109 cfu per mouse. Monocolonized mice were housed inrelevant groups in individual germ-free isolators for the dura-tion of the experiment. For analysis of conventional mice,C57BL/6J male mice were purchased from Harlan and werehoused under barrier-maintained conditions at UniversityCollege Cork. Male 6-wk-old C57BL/6J mice were fasted for24 h and were supplied immediately with streptomycin-treateddrinking water (5 mg/mL final concentration) for the duration ofthe experiment. After 24 h mice were fed either a normal low-fatdiet (10% calories from fat; Research Diets InternationalD12450B) or a high-fat diet (45% calories from fat; ResearchDiets International D12451) for 10 wk (n = 20 in each group).These two groups were divided further into parallel groups (n =5 for each group) and were inoculated on two consecutive dayswith relevant E. coli strains in PBS at 1 × 106 cfu per mouse byoral gavage. Body weight and food intake were assessed weekly.Fecal samples were taken from each mouse on a weekly basisand were used for bacterial enumeration. At the end of thestudy, mice were killed, and internal organs (liver, spleen, in-testine) and fat pads (reproductive, renal, mesenteric, and in-guinal) were removed, weighed, and stored at −80 °C. Theexperiments outlined were approved by the University CollegeCork Animal Experimentation Ethics Committee.

Metabolic Markers.Mice were fasted for 5–6 h, and blood glucosewas measured using a Contour glucose meter (Bayer) usingblood collected from the tip of the tail vein. Blood was collectedby cardiac puncture, and plasma was extracted. Plasma insulinconcentrations were determined using an ELISA kit (Mercodia).Plasma and liver triglyceride levels were determined using in-finity triglyceride liquid stable reagent (Thermo Scientific), andcholesterol levels were determined from plasma using a choles-terol quantification kit (BioVision). Inflammasome activationwas assessed directly from plasma and from liver extracts usingthe 7-Plex Meso Scale Discovery Kit.

Chemicals. Standard conjugated bile acids and bile acids werepurchased from Sigma Aldrich or Steraloids and are listed inTable S1. Deuterated cholic acid (D-2452) and deuterated

chenodeoxycholic acid (D-2772) were purchased from CDN Iso-topes Inc. HPLC-grade methanol, acetonitrile, water, ammoniumacetate, ammonium formate, ammonium hydroxide, formic acid,and acetic acid and water were obtained from Fisher Scientific.Standards were constituted as 1 mg/mL. Stock solutions of in-dividual sulfated bile acids were prepared in water:MeOH (1:1)and combined to a final volume of 1.0 mL in water to give aconcentration of 40 μg/mL for each. Subsequent dilutions weremade as necessary.

Bile Acid Extractions. Bile acids were extracted from 100 μL ofplasma added to 50% ice-cold methanol. The extract was mixedand then was centrifuged at 16,000 × g for 10 min at 4 °C.The supernatant was retained and further extracted by theaddition of acetonitrile (5% NH4OH). The resultant superna-tant was dried under vacuum and reconstituted in 50% MeOH.The extracted bile acids were resuspended in 150 mL of ice cold50% MeOH.

UPLC-MS. UPLC-MS was performed using the method of Swannet al. (5), with modifications. Five microliters of extracted bileacids were injected onto a 50-mm T3 Acquity column (WatersCorp.) and were eluted using a 20-min gradient of 100% A to100% B (A, water, 0.1% formic acid; B, methanol, 0.1% formicacid) at a flow rate of 400 μL/min and column temperature of50 °C. Samples were analyzed using an Acquity UPLC system(Waters Ltd.) coupled online to an LCT Premier mass spec-trometer (Waters MS Technologies, Ltd.) in negative electro-spray mode with a scan range of 50–1,000 m/z. Bile acids ionizestrongly in negative mode, producing a prominent [M-H]negativeion. Capillary voltage was 2.4 Kv, sample cone was 35 V, des-olvation temperature was 350 °C, source temperature was 120 °C,and desolvation gas flow was 900 L/h.Principal components analysis (PCA) was performed in

Markerlynx (Waters) by limiting the number of elements (N,H, S, C) to be detected in individual analytes. Furthermorea template of defined known masses was applied to allow thedetection of bile acid only. The groups (types) are shown indifferent colors, and the separation of the groups is easilyvisible (Fig. S2D). Each analyte was identified according to itsmass and retention time. Standard curves were performedusing known bile acids, and each analyte was quantified ac-cording to the standard curve and normalized according to thedeuterated internal standards.

Microarrays. Tissues were stored in RNAlater (Qiagen) beforeRNA extraction using the RNAeasy Plus Universal kit (Qiagen).Microarrays were carried out using mouse Exon ST1.0 arrays(Affymetrix) by the Almac Group, Craigavon, Northern Ireland.Analysis and pathway mapping were carried out using SubioPlatform software (Subio, Inc.) and Genesis Software. The mi-croarray data reported in this paper have been deposited in theGene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no GSE46952).

Quantitative RT- PCR. Quantitative RT-PCR (qRT-PCR) usedRNA to generate cDNA. Primers and pairs designed by Uni-versal ProbeLibrary (Roche) were used for qPCR with theLightCycler 480 System (Roche). The 2-ΔΔC method (6) was usedto calculate relative changes in gene expression.

Statistical Analysis.Data for all variables were normally distributedand therefore allowed a parametric test of significance. Data are

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presented as mean values, and their SD is indicated. Statisticalanalyses were performed by ANOVA and Student t-test and,

where indicated, were corrected for false discovery using theBenjamini–Hochberg procedure.

1. Fang F, et al. (2009) Allelic variation of bile salt hydrolase genes in Lactobacillussalivarius does not determine bile resistance levels. J Bacteriol 191(18):5743–5757.

2. Koch B, Jensen LE, Nybroe O (2001) A panel of Tn7-based vectors for insertion of thegfp marker gene or for delivery of cloned DNA into Gram-negative bacteria ata neutral chromosomal site. J Microbiol Methods 45(3):187–195.

3. McGrath S, Fitzgerald GF, van Sinderen D (2001) Improvement and optimization of twoengineered phage resistance mechanisms in Lactococcus lactis. Appl Environ Microbiol67(2):608–616.

4. Lipscomb IP, Pinchin HE, Collin R, Harris K, Keevil CW (2006) The sensitivity of approvedNinhydrin and Biuret tests in the assessment of protein contamination on surgical steelas an aid to prevent iatrogenic prion transmission. J Hosp Infect 64(3):288–292.

5. Swann JR, et al. (2011) Systemic gut microbial modulation of bile acid metabolism inhost tissue compartments. Proc Natl Acad Sci USA 108(Suppl 1):4523–4530.

6. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4):402–408.

Fig. S1. Alterations of host bile acid signatures through gastrointestinal expression of cloned BSH in gnotobiotic mice. Taurocholic acid (TCA) and cholic acid(CA) plasma bile acids (assessed by UPLC-MS/MS) in GF mice, mice monocolonized with EC, ECBSH1, or ECBSH2, and conventionalized mice (CONV-D). Statisticalcomparisons were made relative to EC-colonized controls using the Student t test and were corrected for false discovery using the Benjamini–Hochbergprocedure. *P < 0.05 relative to EC controls (n = 5 per group).

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cBAucBA

cBAucBA

cBAucBA

GF

ECBSH1ECBSH2Conv-D

EC

A

B

C

D

Fig. S2. BSH activity alters the relative levels of conjugated (cBA) and unconjugated (ucBA) bile acid in gnotobiotic mice. (A–D) Bile acid ratios (assessed byUPLC-MS) in (A) plasma, (B) liver, and (C) feces of GF mice, mice monocolonized with EC, ECBSH1, or ECBSH2,and CONV-D mice. (D) PCA plot of the plasma bileacid signatures for each group normalized to the internal standard (n = 5 per group).

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Fig. S3. (Continued)

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Fig. S3. (Continued)

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Fig. S3. qPCR confirmation of selected microarray mRNA expression targets. Selected genes of interest identified from the microarray analysis were subjectedto qRT-PCR for independent confirmation. qPCR data are shown as expression relative to the β-actin housekeeper control in ileum or liver tissue samples frommice in the respective treatment groups. Treatment groups were GF mice, conventionalized (CONV-D) mice, and mice monocolonized with EC, ECBSH1, orECBSH2. Statistical significance was determined using ANOVA. Data are presented as means ± SEM, n = 5 per group. #P < 0.05 = vs. BSH1; ##P < 0.01 vs. BSH1;###P < 0.001 vs. BSH1; $P < 0.05 = vs. EC; $$P < 0.01 vs. EC; $$$P < 0.001 vs. EC; *P < 0.05 vs. GF; **P < 0.01 vs. GF; ***P < 0.001 vs. GF.

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Fig. S4. E. coli colonization in the gastrointestinal tract of conventional C57BL/6J mice administered streptomycin ad libitum. E. coli was enumerated bystandard plate counts from feces on the days indicated.

***

***

A

B

C

D

cBA ucBA

cBA ucBA

cBA ucBA

Fig. S5. Gastrointestinal expression of cloned BSH in conventional mice alters bile acid profiles. Conventional mice were either not treated (NT) or wereprovided with streptomycin (5 mg/mL) ad libitum in drinking water to promote stable high-level colonization of the host E. coli MG1655 StrepR strain andvariants as indicated (mice given antibiotic only (Ab), mice colonized with EC, ECBSH1, or ECBSH2; n = 5 per group). Bile acids were assessed by UPLC-MS. (A)Plasma levels of TCA and CA. Statistical comparisons were made relative to EC controls using the Student t test and were corrected for false discovery using theBenjamini–Hochberg procedure. ***P < 0.0005 relative to EC controls (n = 5 per group). (B–D) relative levels of conjugated (cBA) and unconjugated (ucBA) bileacid in (B) plasma, (C) liver, and (D) feces.

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Fig. S6. (A) BSH1 activity lowers weight gain in mice fed a high-fat diet (HFD; 45% calories from fat) or normal chow (LFD; 10% calories from fat). Experi-mental design is as in Fig. 5A. *P < 0.05, **P < 0.005, ***P < 0.0005, mice colonized with EC or with ECBSH1 (for HFD groups only); Student t test. (B) Weight oftotal excised fat from mice undergoing antibiotic treatment alone (Ab) or mice colonized with EC, ECBSH1, or ECBSH2. *P < 0.05 relative to the EC dataset ineach case. (C and D) ECBSH1 colonization lowers plasma cholesterol (C) and liver triglycerides (D) in mice fed either an LFD or a HFD. *P < 0.05, **P < 0.005relative to mice colonized with EC.

Fig. S7. Absolute levels of (A) plasma and (B) liver cytokines in conventional C57BL/6J mice colonized by EC in our model system as measured by the Meso ScaleDiscovery assay.

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ppar abcg8

cdkn1a nfat2

Fig. S8. Gastrointestinal expression of BSH influences gene-expression patterns in ileal tissue in conventional mice. Mice given streptomycin were colonized byE. coli MG1655 StrepR as outlined in our model system. Gene-expression patterns of selected genes were examined using qRT-PCR in ileal tissue from micecolonized by EC, ECBSH1, or ECBSH2 and from uncolonized GF mice (n = 5 per group). Statistical significance was determined using ANOVA. Data are presentedas means ± SEM; #P < 0.05 vs. ECBSH1; $P < 0.05 vs. EC; $$P < 0.01 vs. EC; *P < 0.05 vs. GF; **P < 0.01 vs. GF; ***P < 0.001 vs. GF.

TC-BAs

UC-BAs BSH

Lipid Metabolism & Transport

Signalling, Hormones, Adipokines

Peripheral Circadian Rhythm

Gut Homeostasis / barrier func�on?

Altera�ons to wider microbiota*

microbiota

defensins

Host physiology, weight gain, adiposity, cholesterol levels

Fig. S9. Schematic showing the central role of microbial BSH activity in host physiological processes. BSH activity generates unconjugated bile acids (UC-BAs)from tauroconjugated bile acids (TC-BAs) in the gastrointestinal tract and provides the gateway reaction for the further metabolism of BAs by the microbiota.BSH activity influences host processes known to play a role in adiposity and cholesterol levels in the host such as lipid metabolic pathways, signaling moleculesand adipokines, peripheral circadian rhythm, and homeostasis (and potentially barrier function).*Bile acid metabolism also has been shown previously toinfluence further the community structure of the gut microbiota (1).

1. Islam KB, et al. (2011) Bile acid is a host factor that regulates the composition of the cecal microbiota in rats. Gastroenterology 141(5):1773–1781.

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Table S1. Bile acid moieties analyzed by UPLC-MS in this study

Analyte Negative ion mass RT, min Formula R2 value

Taurine 124.0068 0.75 C24H7NO3S 0.997593Cholic acid 407.2797 11.76 C24H40O5 0.992983Chenodeoxycholic acid 391.2848 17.63 C24H40O4 0.988765Lithocholic acid 375.2855 21.72 C24H40O3 0.641440Deoxycholic acid 391.2848 18.86 C24H40O4 0.991164Dehydrocholic acid 401.239 1.71 C24H34O5 0.998547Ursodeoxycholic acid 391.2848 9.04 C24H40O4 0.992197Hyodeoxycholic acid 391.2848 10.04 C24H40O4 0.978549α-Muricholic acid 407.2898 5.5 C24H40O5 0.980232β-Muricholic acid 407.2898 6.44 C24H40O5 0.981049ω-Muricholic acid 407.2898 5.86 C24H40O5 0.915250Taurocholic acid 514.2838 4.95 C26H45NO7S 0.994024Taurochenodeoxycholic acid 498.2889 8.64 C26H44NO6S 0.993961Taurolithocholic acid 482.294 14.74 C26H45NO5S 0.988314Taurodeoxycholic acid 498.2889 3.54 C26H44NNaO 0.992574Tauroursodeoxycholic acid 498.2889 8.64 C26H44NO6S 0.992203Taurohyodeoxycholic acid 498.2889 3.49 C26H43NO5 0.988796Tauroα-Muricholic acid 514.2838 1.79 C26H45NO7S 0.965300Tauroβ-Muricholic acid 514.2838 1.69 C26H45NO7S 0.997434Tauroω-Muricholic acid 514.2838 1.89 C26H45NO7S 0.987685D4 cholic acid 411.3053 11.97 C24D4H36O5 NAD4 chenodeoxycholic acid 427.6565 17.82 C24D4H36O4 NA

RT, column (25 m) retention time.

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