Title: Biogeography of microbial bile acid transformations along the murine gut
Abstract: Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition. Bile acids, which are synthesized from cholesterol by the liver, are chemically transformed along the intestinal tract by the gut microbiota, and the products of these transformations signal through host receptors, affecting overall host health. These transformations include bile acid deconjugation, oxidation, and 7α-dehydroxylation. An understanding of the biogeography of bile acid transformations in the gut is critical because deconjugation is a prerequisite for 7α-dehydroxylation and because most gut microorganisms harbor bile acid transformation capacity. Here, we used a coupled metabolomic and metaproteomic approach to probe in vivo activity of the gut microbial community in a gnotobiotic mouse model. Results revealed the involvement of Clostridium scindens in 7α-dehydroxylation, of the genera Muribaculum and Bacteroides in deconjugation, and of six additional organisms in oxidation (the genera Clostridium, Muribaculum, Bacteroides, Bifidobacterium, Acutalibacter, and Akkermansia). Furthermore, the bile acid profile in mice with a more complex microbiota, a dysbiosed microbiota, or no microbiota was considered. For instance, conventional mice harbor a large diversity of bile acids, but treatment with an antibiotic such as clindamycin results in the complete inhibition of 7α-dehydroxylation, underscoring the strong inhibition of organisms that are capable of carrying out this process by this compound. Finally, a comparison of the hepatic bile acid pool size as a function of microbiota revealed that a reduced microbiota affects host signaling but not necessarily bile acid synthesis. In this study, bile acid transformations were mapped to the associated active microorganisms, offering a systematic characterization of the relationship between microbiota and bile acid composition. The gut is a complex ecosystem hosting a wide diversity of microorganisms. The gut microbiome is sometimes referred to as the forgotten endocrine organ because of its profound influence on host physiology. Gut microbes convert dietary and other exogenous molecules into signaling metabolites to communicate with the host (1Schroeder B.O. Bäckhed F. Signals from the gut microbiota to distant organs in physiology and disease.Nat. Med. 2016; 22: 1079-1089Crossref PubMed Scopus (632) Google Scholar). Bile acids (BAs) are one of these microbiota-derived signaling metabolites. Primary BAs are synthesized in the liver from cholesterol (dietary or endogenous). In the hepatocytes, they are conjugated to glycine or taurine and stored continuously in the gall bladder as the main component of bile. The human liver synthesizes only two primary BAs [cholic acid (CA) and chenodeoxycholic acid (CDCA)], whereas the rodent liver synthesizes five [CA, CDCA, two muricholic acids (αMCA and βMCA), and ursodeoxycholic acid, the 7β epimer of CDCA (2Wahlström A. Sayin S.I. Marschall H.U. Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.Cell Metab. 2016; 24: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar)]. After food intake, the presence of fat molecules in the duodenum stimulates the release of a hormone, cholecystokinin, which triggers the contraction of the gall bladder and the relaxation of the Oddi sphincter, leading to the release of bile into the small intestine (3Hofmann A.F. The continuing importance of bile acids in liver and intestinal disease.Arch. Intern. Med. 1999; 159: 2647-2658Crossref PubMed Scopus (632) Google Scholar). Along the intestinal tract, primary BAs undergo several chemical transformations that are catalyzed by gut microorganisms, leading to the formation of secondary BAs. Thus, the activity of the gut microbiota increases the diversity of the BA pool. The microbial transformation of the BA pool is essential, as it modifies their detergent properties (i.e., their cytotoxicity) and their affinity for host BA receptors (2Wahlström A. Sayin S.I. Marschall H.U. Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.Cell Metab. 2016; 24: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar). For instance, the secondary BAs deoxycholic acid (DCA) and lithocholic acid (LCA), resulting from microbial 7α-dehydroxylation of CA and CDCA, have a higher affinity for the membrane receptor Takeda G-protein receptor 5 (TGR5) compared with the host liver-derived primary BAs (2Wahlström A. Sayin S.I. Marschall H.U. Bäckhed F. Intestinal crosstalk between bile acids and microbiota and its impact on host metabolism.Cell Metab. 2016; 24: 41-50Abstract Full Text Full Text PDF PubMed Scopus (1056) Google Scholar, 4Ridlon J.M. Kang D.J.D. Hylemon P.B.P. Bajaj J.J.S. Bile acids and the gut microbiome.Curr. Opin. Gastroenterol. 2014; 30: 332-338Crossref PubMed Scopus (652) Google Scholar). Gut microorganisms act on the side chain, the hydroxyl groups, and the cyclohexane rings in the BA structure. Thus, microbial BA transformations include deconjugation or hydrolysis (i.e., the removal of the taurine/glycine moiety on the side chain); oxidation and epimerization of hydroxyl groups (at the C3, C7, and C12 positions) and of the C5 hydrogen; reduction of ketone groups; dehydroxylation at C7 and C12 (5Edenharder R. Dehydroxylation of cholic acid at C12 and epimerization at C5 and C7 by Bacteroides species.J. Steroid Biochem. 1984; 21: 413-420Crossref PubMed Scopus (16) Google Scholar); desulfation (6Huijghebaert S. Parmentier G. Eyssen H. Specificity of bile salt sulfatase activity in man, mouse and rat intestinal microflora.J. Steroid Biochem. 1984; 20: 907-912Crossref PubMed Scopus (16) Google Scholar); esterification of hydroxyl groups (7Kelsey M.I. Molina J.E. Huang S.K. Hwang K.K. The identification of microbial metabolites of sulfolithocholic acid.J. Lipid Res. 1980; 21: 751-759Abstract Full Text PDF PubMed Google Scholar); and the oxidation of a steroid ring to form unsaturated BAs (8Macdonald I.A. Bokkenheuser V.D. Winter J. McLernon A.M. Mosbach E.H. Degradation of steroids in the human gut.J. Lipid Res. 1983; 24: 675-700Abstract Full Text PDF PubMed Google Scholar, 9Prabha V. Ohri M. Review: bacterial transformations of bile acids.World J. Microbiol. Biotechnol. 2006; 22: 191-196Crossref Scopus (20) Google Scholar) (Fig. 1). A new microbial BA transformation was recently identified in both humans and mice: reconjugation. After being deconjugated by bacteria carrying bile salt hydrolase (BSH), the unconjugated BA can be reconjugated by bacteria with amino acids such as tyrosine, phenylalanine, leucine forming tyrosocholic acid, phenylalanocholic acid, and leucocholic acid (10Quinn R.A. Vrbanac A. Melnik A.V. Patras K.A. Christy M. Nelson A.T. Aksenov A. Tripathi A. Humphrey G. da Silva R. et al.Global chemical effects of the microbiome include new bile-acid conjugations.Nature. 2020; 579: 123-129Crossref PubMed Scopus (141) Google Scholar). The abundance and diversity of bacteria carrying out the various BA transformations vary enormously. Deconjugating bacteria, synthesizing BSH, are reported to be abundant in the gut and widespread across bacterial phyla (11Urdaneta V. Casadesús J. Interactions between bacteria and bile salts in the gastrointestinal and hepatobiliary tracts.Front. Med. (Lausanne). 2017; 4: 163Crossref PubMed Scopus (151) Google Scholar, 12Foley M.H. O'Flaherty S. Barrangou R. Theriot C.M. Bile salt hydrolases: gatekeepers of bile acid metabolism and host-microbiome crosstalk in the gastrointestinal tract.PLoS Pathog. 2019; 15: e1007581Crossref PubMed Scopus (83) Google Scholar). Similarly, oxidation of the hydroxyl groups in BAs is also a common activity in gut microorganisms and is catalyzed by hydroxysteroid dehydrogenases (HSDHs) (13Doden H. Sallam L.A. Devendran S. Ly L. Doden G. Daniel S.L. Alves J.M.P. Ridlon J.M. Metabolism of oxo-bile acids and characterization of recombinant 12α-hydroxysteroid dehydrogenases from bile acid 7α-dehydroxylating human gut bacteria.Appl. Environ. Microbiol. 2018; 84: e00235-18Crossref PubMed Scopus (28) Google Scholar). In contrast, 7α-dehydroxylating bacteria are considered as rare organisms in the gut and so far only belong to the order Clostridiales (14Ridlon J.M. Kang D.J. Hylemon P.B. Bile salt biotransformations by human intestinal bacteria.J. Lipid Res. 2006; 47: 241-259Abstract Full Text Full Text PDF PubMed Scopus (1556) Google Scholar). The 7α-dehydroxylation reaction is catalyzed by a series of proteins (Bai proteins) encoded by genes belonging to the bai operon (15Ridlon J.M. Harris S.C. Bhowmik S. Kang D-J.J. Hylemon P.B. Consequences of bile salt biotransformations by intestinal bacteria.Gut Microbes. 2016; 7: 22-39Crossref PubMed Scopus (427) Google Scholar). However, because deconjugation is a prerequisite for BA 7α-dehydroxylation, it is important to consider the relative localization of these two BA transformations (16Devendran S. Shrestha R. Alves J.M.P. Wolf P.G. Ly L. Hernandez A.G. Méndez-García C. Inboden A. Wiley J. Paul O. et al.Clostridium scindens ATCC 35704: integration of nutritional requirements, the complete genome sequence, and global transcriptional responses to bile acids.Appl. Environ. Microbiol. 2019; 85: e00052-19Crossref PubMed Scopus (19) Google Scholar, 17Batta A.K. Salen G. Arora R. Shefer S. Batta M. Person A. Side chain conjugation prevents bacterial 7-dehydroxylation of bile acids.J. Biol. Chem. 1990; 265: 10925-10928Abstract Full Text PDF PubMed Google Scholar). Connecting the spatial organization of the gut microorganisms to biological function remains challenging because of the high diversity of the gut microbial community (18Tropini C. Earle K.A. Huang K.C. Sonnenburg J.L. The gut microbiome: connecting spatial organization to function.Cell Host Microbe. 2017; 21: 433-442Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). For BA transformations, this difficulty is compounded by the large number of BA chemical species that can be generated by the gut microbiota. The original study of microbial BA transformation biogeography dates back to 1968, when Midtvedt and Norman sampled luminal content along the intestinal tract of three rats and probed for in vitro BA transformations in each sample (19Midtvedt T. Norman A. Anaerobic, bile acid transforming microorganisms in rat intestinal content.Acta Pathol. Microbiol. Scand. 1968; 72: 337-344Crossref PubMed Scopus (20) Google Scholar). First, they noticed variability in the distribution of microbial BA transformations among the three rats. For instance, one rat had very little deconjugating activity in the small intestine (ileum) compared with the two other rats. Two rats showed 7α-HSDH activity (oxidation of the 7α-hydroxyl) in the ileum, while the third rat did not. Since then, other studies have investigated BA profiles longitudinally (20Sayin S.I. Wahlström A. Felin J. Jäntti S. Marschall H-U. Bamberg K. Angelin B. Hyötyläinen T. Orešič M. Bäckhed F. Gut microbiota regulates bile acid metabolism by reducing the levels of tauro-beta-muricholic acid, a naturally occurring FXR antagonist.Cell Metab. 2013; 17: 225-235Abstract Full Text Full Text PDF PubMed Scopus (1239) Google Scholar, 21Selwyn F.P. Csanaky I.L. Zhang Y. Klaassen C.D. Importance of large intestine in regulating bile acids and glucagon-like peptide-1 in germ-free mice.Drug Metab. Dispos. 2015; 43: 1544-1556Crossref PubMed Scopus (57) Google Scholar). However, probing for the presence/absence of specific BAs may not accurately reflect the in vivo microbial BA transformation activity or the presence/absence of the organisms responsible for the synthesis of that particular BA. For instance, it was recently observed that BA 7α-dehydroxylation activity was absent in the ileum of gnotobiotic mice despite colonization of the 7α-dehydroxylating organism Clostridium scindens (22Marion S. Studer N. Desharnais L. Menin L. Escrig S. Meibom A. Hapfelmeier S. Bernier-Latmani R. In vitro and in vivo characterization of Clostridium scindens bile acid transformations.Gut Microbes. 2019; 10: 481-503Crossref PubMed Scopus (37) Google Scholar). It was hypothesized that the high level of tauro-conjugated BAs in the ileum likely precluded BA 7α-dehydroxylation (22Marion S. Studer N. Desharnais L. Menin L. Escrig S. Meibom A. Hapfelmeier S. Bernier-Latmani R. In vitro and in vivo characterization of Clostridium scindens bile acid transformations.Gut Microbes. 2019; 10: 481-503Crossref PubMed Scopus (37) Google Scholar). Furthermore, the ability to transform BAs in vitro does not necessarily reflect the in vivo activity. For instance, Narushima et al. reported that the 7α-dehydroxylating bacterium C. hiranonis (formerly Clostridium strain TO-931), known to deconjugate TCA to CA and to 7α-dehydroxylate cholic acid in vitro, did not show any activity when amended to germ-free (GF) mice (23Narushima S. Itoh K. Takamine F. Uchida K. Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.Microbiol. Immunol. 1999; 43: 893-897Crossref PubMed Scopus (18) Google Scholar). Thus, in vivo studies are needed to identify organisms active in BA metabolism within the microbial community of the gut. In this study, we explored the longitudinal distribution of BA transformations in gnotobiotic mouse models and identified the microorganisms responsible for these transformations using a combined metabolomic and metaproteomic approach. BA 7α-dehydroxylation was a transformation of particular interest because of its connection to TGR5 signaling. We contrasted gnotobiotic mice lacking BA 7α-dehydroxylating activity with gnotobiotic mice containing the same base microbiota but complemented with the 7α-dehydroxylating organism C. scindens ATCC 35704. Finally, for comparison, we considered the BA profile in mice with either a complex microbiota [conventional, specific pathogen-free (SPF) mice], a reduced microbiota (antibiotic-treated SPF mice), or no microbiota (GF mice). Groups of age-matched C57BL/6 mice (6–12 weeks old) were used. GF and gnotobiotic sDMDMm2 mice were established and maintained at the University of Bern Clean Mouse Facility. sDMDMm2 mice were colonized with a mouse-intestine-derived 12-species mouse microbiota [Oligo-MM12] (supplemental Table S1) (24Eberl C. Ring D. Münch P.C. Beutler M. Basic M. Slack E.C. Schwarzer M. Srutkova D. Lange A. Frick J.S. et al.Reproducible colonization of germ-free mice with the oligo-mouse-microbiota in different animal facilities.Front. Microbiol. 2020; 10: 299Crossref Scopus (24) Google Scholar). All Oligo-MM12 strains are available at http://www.dsmz.de/miBC. In-depth descriptions of the Oligo-MM12 consortium species and novel taxa are provided elsewhere (25Lagkouvardos I. Pukall R. Abt B. Foesel B.U. Meier-Kolthoff J.P. Kumar N. Bresciani A. Martínez I. Just S. Ziegler C. et al.The Mouse Intestinal Bacterial Collection (miBC) provides host-specific insight into cultured diversity and functional potential of the gut microbiota.Nat. Microbiol. 2016; 1: 16131Crossref PubMed Scopus (219) Google Scholar, 26Brugiroux S. Beutler M. Pfann C. Garzetti D. Ruscheweyh H-J. Ring D. Diehl M. Herp S. Lötscher Y. Hussain S. et al.Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium.Nat. Microbiol. 2016; 2: 16215Crossref PubMed Scopus (168) Google Scholar, 27Garzetti D. Brugiroux S. Bunk B. Pukall R. McCoy K.D. Macpherson A.J. Stecher B. High-quality whole-genome sequences of the oligo-mouse-microbiota bacterial community.Genome Announc. 2017; 5: e00758-17Crossref PubMed Scopus (22) Google Scholar). SPF mice were purchased from Envigo and acclimatized to the local animal facility for 7 days before the start of experiments. SPF mice were pretreated with the antibiotic clindamycin (100 µl by i.p. injection, 2 mg/ml in PBS) or streptomycin (100 µl by gavage, 200 mg/ml in PBS) 24 h before being euthanized. sDMDMm2 mice were inoculated with C. scindens ATCC 35704 by gavage of 107 to 109 colony-forming units of C. scindens and colonized for 7 days before being euthanized. C. scindens precolonization was performed in flexible film isolators. These mice are denoted sDMDMm2+Cs. All animals were euthanized inside a sterile laminar flow hood, and an aseptic technique was used to collect the specific tissues and contents (liver, terminal ileum tissue, distal duodenum content, ileum content, cecum content, and colon content). The collected samples were flash-frozen in liquid nitrogen and kept at −80°C until subsequent analyses. All animal experiments were performed in accordance with the Swiss Federal and the Bernese Cantonal regulations and were approved by the Bernese Cantonal ethical committee for animal experiments under license numbers BE 82/13 and BE 111/16. The liver (30 mg of the caudate lobe) and terminal ileum tissue were lysed and homogenized in tubes filled with 2.8 mm zirconium oxide beads using Precellys 24 Tissue Homogenizer (Bertin Instruments) at 6,500 rpm for 3 × 10 s. Total RNA was isolated with the RNeasy Plus Mini Kit (QIAGEN). The QuantiTect Reverse Transcription Kit (QIAGEN) was used to synthesize 20 µl cDNA templates from 1 µg purified RNA. cDNA templates were diluted 10× before use in subsequent reactions. SensiFAST SYBR No-ROX Kit (Bioline) was used for quantitative real-time PCR with a final reaction volume of 10 µl (7.5 µl mix and 2.5 µl diluted cDNA template). Gene-specific primers (400 nM) were used in each reaction, and all results were normalized to the ribosomal protein L32 mRNA (primer sequences can be found in supplemental Table S2). PCR product specificity was verified by performance of a melting curve for each primer set. Assays were performed using Mic qPCR Cycler (Bio Molecular Systems) with a three-step program (2 min at 95°C followed by 40 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 10 s). Four replicates of each sample for each primer set were performed, as well as negative controls (no reverse transcription and no template controls). Relative quantification analysis using the REST method was performed with the Mic qPCR analysis software (Bio Molecular Systems). Stock solutions (10 mM) of each BA standard were prepared in methanol (supplemental Table S3). Individual standard solutions (100 µM) were mixed together and diluted with a 50:50 (v/v) mix of ammonium acetate (5 mM) and methanol to construct external standard curves between 1 and 10,000 nM. Deuterated CDCA and DCA were used as recovery standards. Approximately 10 mg of a freeze-dried intestinal sample was homogenized with 150 µl H2O and spiked with 40 µl recovery standard (100 μM) in 2 ml tubes filled with 2.8 mm zirconium oxide beads. Homogenization was performed using an automated Precellys 24 Tissue Homogenizer (Bertin Instruments) at 5,000 rpm for 20 s. Mixed samples were equilibrated on ice for 1 h. A volume of 500 µl ice-cold alkaline acetonitrile (5% ammonia in acetonitrile) was added to the homogenates, which were then vigorously vortexed and continuously shaken for 1 h at room temperature. The mixtures were centrifuged at 16,000 g for 10 min, and the supernatants were collected. The pellets were extracted with another 500 µl of ice-cold alkaline acetonitrile. Supernatants from the two extraction steps were pooled and lyophilized in a rotational vacuum concentrator (Christ) before reconstitution in 100 µl of a 50:50 (v/v) mix of 5 mM ammonium acetate and methanol (pH 6) and stored at −20°C. Supernatants were diluted according to the intestinal compartment (4,000-fold for the small intestine and liver samples and 100-fold for the cecum and colon samples) before LC/MS injection. The extraction recovery ratios ranged between 75% and 85%. Both qualitative and quantitative analyses were conducted on an Agilent 6530 Accurate-Mass Q-TOF LC/MS mass spectrometer coupled to an Agilent 1290 series UHPLC system. The separation was achieved using a Zorbax Eclipse-Plus C18 column (2.1 × 100 mm, 1.8 μm; Agilent) heated at 50°C. A binary gradient system consisted of 5 mM ammonium acetate (pH 6) in water as eluent A and acetonitrile as eluent B. The sample separation was carried out at 0.4 ml/min over a 22 min total run time using the following program: 0–5.5 min, isocratic 21.5% B; 5.5–6 min, 21.5–24.5% B; 6–10 min, 24.5–25% B; 10–10.5 min, 25–29% B; 10.5–14.5 min, isocratic 29% B; 14.5–15 min, 29–40% B; 15–18 min, 40–45% B; 18–20.5 min, 45–95% B; 20.5–22 min, isocratic 95%. The system was reequilibrated to initial conditions for 3 min. The sample manager system temperature was maintained at 4°C, and the injection volume was 5 μl. Mass spectrometer detection was operated in the negative ionization mode using the Dual AJS Jet stream ESI Assembly. The QTOF instrument was operated in the 4 GHz high-resolution mode [typical resolution 17,000 (full width at half maximum) at m/z 1,000] in profile mode and calibrated in negative full-scan mode using ESI-L solution (Agilent). Internal calibration was performed during acquisition via continuous infusion of a reference mass solution [5 mM purine, 1 mM HP-921 (Agilent reference mass kit) in 95% methanol acidified with 0.1% formic acid] and allowed to permanently achieve a mass accuracy better than 5 ppm. High-resolution mass spectra were acquired over the range of m/z 300–700 at an acquisition rate of three spectra. AJS settings were as follows: drying gas flow, 8 l/min; drying gas temperature, 300°C; nebulizer pressure, 35 psi; capillary voltage, 3,500 V; nozzle voltage, 1,000 V; fragmentor voltage, 175 V; skimmer voltage, 65 V; octopole 1 RF voltage, 750 V. Data were processed using the MassHunter Workstation (Agilent). According to this method, 36 BAs (supplemental Table S3) were quantified by external calibration curves. Extracted ion chromatograms were based on a retention-time window of ±0.5 min with mass-extraction windows of ±30 ppm centered on the m/ztheor of each BA. Whole genomes for the 12 Oligo-MM12 species (27Garzetti D. Brugiroux S. Bunk B. Pukall R. McCoy K.D. Macpherson A.J. Stecher B. High-quality whole-genome sequences of the oligo-mouse-microbiota bacterial community.Genome Announc. 2017; 5: e00758-17Crossref PubMed Scopus (22) Google Scholar) and C. scindens ATCC 35704 (16Devendran S. Shrestha R. Alves J.M.P. Wolf P.G. Ly L. Hernandez A.G. Méndez-García C. Inboden A. Wiley J. Paul O. et al.Clostridium scindens ATCC 35704: integration of nutritional requirements, the complete genome sequence, and global transcriptional responses to bile acids.Appl. Environ. Microbiol. 2019; 85: e00052-19Crossref PubMed Scopus (19) Google Scholar) were obtained from the NCBI and EBI (accession numbers available in supplemental Table S4). Sequences were processed by Prodigal (28Hyatt D. Brugiroux S. Bunk B. Pukall R. McCoy K.D. Macpherson A.J. Stecher B. Prodigal: prokaryotic gene recognition and translation initiation site identification.BMC Bioinformatics. 2010; 11: 119Crossref PubMed Scopus (4548) Google Scholar) to produce the gene sequences forming the BLASTP search database. Reference protein sequences for BSH, 3α-HSDH, 3β-HSDH, 7α-HSDH, 7β-HSDH, and 12α-HSDH proteins were obtained from NCBI via the accession numbers presented in supplemental Table S5. These sequences were subsequently blasted against the produced protein database. The top five matches, as identified by bit score, were selected for manual review. The final sequences with the highest scores were selected and are presented in supplemental Table S6. Proteins were extracted from the small intestine, cecum, and colon content samples of sDMDMm2 (n = 3) and sDMDMm2+Cs mice (n = 3) at the Oak Ridge National Laboratory. Protein extraction and digestion followed by LC/MS/MS analysis were performed as described previously (29Patnode M.L. Beller Z.W. Han N.D. Cheng J. Peters S.L. Terrapon N. Henrissat B. Le Gall S. Saulnier L. Hayashi D.K. et al.Interspecies competition impacts targeted manipulation of human gut bacteria by fiber-derived glycans.Cell. 2019; 179: 59-73.e13Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) with slight modifications. Briefly, intestinal content samples were suspended in SDS lysis buffer (4% SDS, 100 mM Tris-HCl, 10 mM dithiothreitol, pH 8.0) and subjected to bead beating. The proteins were isolated by chloroform-methanol extraction and resuspended in 4% sodium deoxycholate (SDC) in 100 mM ammonium bicarbonate buffer. The crude protein concentration was estimated using a Nanodrop (Thermo Fisher Scientific). Protein samples (250 μg) were then transferred to a 10 kDa MWCO spin filter (Vivaspin 500; Sartorius), washed with ammonium bicarbonate buffer, and digested with sequencing-grade trypsin. After proteolytic digestion, the tryptic peptide solution was adjusted to 1% formic acid to precipitate the remaining SDC. The precipitated SDC was removed using water-saturated ethyl acetate. For each sample, 9 μg of peptides were analyzed by automated 2D LC/MS/MS using a Vanquish UHPLC system with the autosampler plumbed directly in-line with a Q Exactive Plus mass spectrometer (Thermo Fisher Scientific) across three successive salt cuts of ammonium acetate (35, 100, and 500 mM), each followed by a 155-min organic gradient. Eluting peptides were measured and sequenced by data-dependent acquisition on the Q Exactive Plus as described previously (30Clarkson S.M. Giannone R.J. Kridelbaugh D.M. Elkins J.G. Guss A.M. Michener J.K. Construction and optimization of a heterologous pathway for protocatechuate catabolism in Escherichia coli enables bioconversion of model aromatic compounds.Appl. Environ. Microbiol. 2017; 83: e01313-17Crossref PubMed Scopus (32) Google Scholar). Peptide identification and protein inference were performed as follows: MS raw data files were searched against the protein database of 12 bacterial species genomes published by Garzetti et al. (27Garzetti D. Brugiroux S. Bunk B. Pukall R. McCoy K.D. Macpherson A.J. Stecher B. High-quality whole-genome sequences of the oligo-mouse-microbiota bacterial community.Genome Announc. 2017; 5: e00758-17Crossref PubMed Scopus (22) Google Scholar) and the C. scindens ATCC 35704 genome published by Devendran et al. (16Devendran S. Shrestha R. Alves J.M.P. Wolf P.G. Ly L. Hernandez A.G. Méndez-García C. Inboden A. Wiley J. Paul O. et al.Clostridium scindens ATCC 35704: integration of nutritional requirements, the complete genome sequence, and global transcriptional responses to bile acids.Appl. Environ. Microbiol. 2019; 85: e00052-19Crossref PubMed Scopus (19) Google Scholar) (supplemental Table S4) along with common contaminates (e.g., trypsin, human keratin) appended with a decoy database consisting of reverse protein sequences to control the false-discovery rate (FDR) to 1% at the spectral level (31Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2727) Google Scholar). MS/MS spectra data analysis (searching and filtering) was done by the Crux MS analysis toolkit (version 3.2-f7929ba) (32Park C.Y. Klammer A.A. Käli L. MacCoss M.J. Noble W.S. Rapid and accurate peptide identification from tandem mass spectra.J. Proteome Res. 2008; 7: 3022-3027Crossref PubMed Scopus (133) Google Scholar). Tide-search (33Diament B.J. Noble W.S. Faster SEQUEST searching for peptide identification from tandem mass spectra.J. Proteome Res. 2011; 10: 3871-3879Crossref PubMed Scopus (115) Google Scholar) was used for searching. For Tide-search, default settings were used except for the following parameters: allowed clip N-terminal methionine, allowed four missed cleavages, a precursor mass tolerance of 10 ppm, a static modification on cysteines (iodoacetamide; +57.0214 Da), dynamic modifications on methionine (oxidation; +15.9949), and a fragment ion mass tolerance of 0.02 Da. Trypsin was used as the digestion enzyme that cleaves the C terminus to argini