Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces

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1 FEMS Microbiology Letters 235 (2004) Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces Pascale Lepercq a, Philippe Gerard a, Fabienne Beguet a, Pierre Raibaud a, Jean-Pierre Grill b, Purification Relano c, Chantal Cayuela c, Catherine Juste a, * a Unite d Ecologie et de Physiologie du Systeme Digestif, Institut National de la Recherche Agronomique, Domaine de Vilvert, Jouy-en-Josas Cedex, France b Laboratoire des BioSciences de l Aliment, Faculte des Sciences et Techniques Vandoeuvre-les-Nancy Cedex, France c Danone Vitapole, Nutrivaleur, Groupe Probiotiques et Fonctions Digestives, Route Departementale 128, Palaiseau Cedex, France Received 11 February 2004; received in revised form 6 April 2004; accepted 7 April 2004 First published online 20 April 2004 Abstract Ursodeoxycholic acid-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health. This work reports the first isolation of 7-epimerizing bacteria from feces of a healthy volunteer, on the basis of their capacity to epimerize the primary bile acid, chenodeoxycholic acid, to ursodeoxycholic acid. Five isolates were found to be active starting from unconjugated chenodeoxycholic acid and its tauro-conjugated homologue, but none of these strains could epimerize the glyco-conjugated form. Biochemical testing and 16S ribosomal DNA sequencing converged to show that all five isolates were closely related to Clostridium baratii (99% sequence similarity), suggesting that this bacterial species could be responsible at least partially, for this bioconversion in the human gut. Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Clostridium baratii; Bile acid epimerization; Chenodeoxycholic acid; Ursodeoxycholic acid; Human intestinal microbiota 1. Introduction Ursodeoxycholic acid (UDCA, 3a,7b-dihydroxy-5bcholan-24-oic acid) is a naturally low-occurring bile acid in man, representing normally less than 4% of total biliary and fecal bile acids [1,2]. Administered orally, UDCA has important therapeutic uses related to its ability to solubilize small radio-transparent gallstones [3] and to improve liver function in cholestatic diseases [4 6]. Supplemental dietary UDCA significantly reduces experimentally induced colon [7 9] and liver [10] carcinogenesis in rats. The tauro-conjugated form of UDCA was shown to be neuroprotective in a transgenic animal model of Huntington s disease [11]. UDCA has no proven adverse effects on health. * Corresponding author. Tel.: ; fax: address: juste@jouy.inra.fr (C. Juste). UDCA is the 7b-OH epimer of the primary (i.e., synthesized by the liver) bile acid chenodeoxycholic acid (CDCA, 3a,7a-dihydroxy-5b-cholan-24-oic acid). Epimerization proceeds in two subsequent steps: oxidation of the 7a-hydroxyl group by a 7a-hydroxysteroid dehydrogenase (7a-HSDH) and stereospecific reduction of the 7-keto functionality by a 7b-HSDH, thus generating the corresponding 7b-hydroxyl group (Fig. 1). Three genes encoding for three 7a-HSDH have been cloned and sequenced from E. coli HB 101 [12], Clostridium scindens (formerly Eubacterium sp VPI 12708) [13], Clostridium sordelii [14] and Bacteroides fragilis [15]. The enzyme 7b-HSDH was partially purified [16 18] but the encoding genes remain unknown. Two soil isolates, Clostridium absonum [19] and Clostridium limosum [20], as well as the aerobic opportunistic pathogen Stenotrophomonas (formerly Pseudomonas and Xanthomonas) maltophilia [21], are known to epimerize CDCA to UDCA. The intestinal microbiota is responsible for this /$22.00 Ó 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi: /j.femsle

2 66 P. Lepercq et al. / FEMS Microbiology Letters 235 (2004) Fig. 1. Biotransformations of CDCA (chenodeoxycholic acid), including its 7-epimerization into UDCA (ursodeoxycholic acid) via the intermediate 7keto-LCA (7-keto-lithocholic acid), and its 7-dehydroxylation into LCA (lithocholic acid). Enzymes are 7a-hydroxysteroid dehydrogenase (7a-HSDH), and 7b-hydroxysteroid dehydrogenase (7b-HSDH). epimerization in the large bowel, as inferred from UDCA formation in CDCA-containing cultures of human feces [22,23] and increased fecal excretion of UDCA in patients under CDCA therapy [24]. In vitro epimerization of CDCA to UDCA can proceed from the combination of two kinds of intestinal microorganisms, each carrying one of both 7-HSDH [25,26]. Moreover, six epimerizing lecithinase-lipase-negative clostridia were isolated from stools of patients with colon cancer [27], whose intestinal microbiota was shown to be altered compared to that of healthy controls [28,29], with a higher abundance of lecithinase-negative clostridia [30]. The present work was therefore designed to isolate CDCA to UDCA epimerizing bacteria from the intestinal microbiota of a healthy man. Since CDCA is mainly in a conjugated form when it enters the colon, bacterial epimerizing activities were assayed on CDCA and both its glyco(g)- and tauro(t)-conjugated homologues, GCDCA and TCDCA. 2. Materials and methods 2.1. Source and isolation of microorganisms A fresh stool was provided in an anaerobic box (Anaerocult, Merck, Darmstadt, Germany) by a healthy elderly male volunteer on an unrestricted Western diet, without any evidence of gastrointestinal or hepatic disorders, and without laxative or antibiotic use for the prior six months. Within 30 min after defecation, 1 g of feces (wet weight) was transferred to an anaerobic chamber (85% N 2, 10% H 2,5%CO 2 ), thoroughly mixed with 9 ml of anaerobic Liquid Casein Yeast (LCY) (casitone 2 g l 1 ; yeast extract 2 g l 1 ; NaCl 5 g l 1 ; KH 2 PO 4 1gl 1, ph 7.0) and serially 10-fold diluted to A 0.1 ml aliquot of each of the 10 5 to 10 8 dilutions was plated on 8H agar (peptone 15 g l 1 ; tryptone 10 g l 1 ; liver extract 5 g l 1 ; agar 14 g l 1, ph 8.0). After four days of incubation at 37 C within the chamber incubator, 108 well-isolated colonies were picked from the appropriate dilution plate (10 7 ) and subcultured in streaks on nine 8H agar plates labeled from A to I, each divided into 12 sectors. This set of plates was prepared in duplicate and incubated as described above. The first set was used for assaying the activity of each plate. The second set of plates was used for assaying the activity of each streak from the previously identified positive plates. Finally, positive streaks were resuspended and plated again and single colonies were subcultured twice to ensure isolation Bile acid transformation tests Sodium salts of CDCA and UDCA were purchased from Sigma (St. Quentin Fallavier, France) and sodium salts of 7 keto-lithocholic acid (7-keto-litho cholic acid; 3-hydroxy,7-keto-5b-cholan-24-oic acid), lithocholic acid (LCA; 3a-hydroxy-5b-cholan-24-oic acid), GCDCA (3a,7a-dihydroxy-5b-cholanoyl glycine) and TCDCA (3a,7a-dihydroxy-5b-cholanoyl taurine) were supplied by Steraloids (Newport, Rhode Island, USA). All bile acids were chromatographically pure (thin layer chromatography and gas liquid chromatography). The activity of each plate was assessed by re-suspending the 12 streaks together in 2 ml of brain heart infusion-yeast extract (BHI-YE: BHI, 37 g l 1, yeast extract, 5 g l 1, L-cysteine, 0.5 g l 1, and hemin, 0.01 g l 1, ph 7.4) containing 10 4 M CDCA. The activity of each streak from the positive plates, was then assessed by re-suspension in 2 ml of the same broth as above. Cultures of positive streaks were maintained in BHI-YE broth and further assayed by subculture in BHI-YE broth containing 10 4 M of the bile acid substrate, CDCA, GCDCA or TCDCA. For all transformation tests, cultures were grown anaerobically at 37 C for 48 h (corresponding to the late stationary phase). They were then acidified to ph 2.0 with 1M HCl, and bile acids were extracted three times with diethyl ether (3 5 ml). The combined solvent fractions were evaporated to dryness and the bile acid extract was reconstituted with 2 ml methanol and stored at )20 C until analysis. For pure cultures, aliquots were aspirated before acidification, for final ph measurement and enumeration on BHI-YE agar plates. C. absonum ATCC was used as a positive control in all transformation tests.

3 P. Lepercq et al. / FEMS Microbiology Letters 235 (2004) Bile acid analyses Detection of bile acid metabolites in the cultures was first carried out by thin layer chromatography (TLC) making it possible to visualize conjugated and unconjugated bile acids. Samples which contained CDCA, GCDCA or TCDCA transformation products on TLC were further analyzed through gas liquid chromatography (GLC). For TLC analysis, 100 ll of bile acid extract were methylated with 200 ll of diazomethane and the solution was allowed to stand at room temperature overnight. The methylated samples were evaporated to dryness under nitrogen, and the residue was reconstituted with 50 ll of dichloromethane. The plates (LK6DF silica gel 60 A, Whatman International, Maidstone, UK) were activated for 1 h at 110 C. The totality of the methylated samples was spotted on a plate and chromatographed in a solvent system (70% chloroform, 25% acetone, 5% methanol). After migration, the plate was dried, then sprayed with 10% sulphuric acid in ethanol, and heated at 160 C for 5 min. Bands were identified by comparison with bile acid standards spotted on the same plate [31]. For GLC analysis, 50 ll of bile acid extract were supplemented with 5 ll of the external standard 5 a- cholestane (0.5 g l 1 in hexane). Methylation was achieved by the addition of 200 ll of diazomethane as previously indicated for TLC analysis. After evaporation to dryness, the samples were silylated by addition of N,O-bis(trimethylsilyl)trifluoroacetamide/trimethylchlorosilane (BSTFA/TMCS; 99/1, v/v) trimethylsilylimidazole/pyridine (TMSI/pyridine; 1/4, v/v). Trimethylsilyl derivates were analyzed using a gas chromatograph (Peri 2000; Perichrom, Saulx-les-Chartreux, France) equipped with a Ros injector, a flame ionization detector and an OV-1701 column 30 m 0.32 mm 0.25 lm (Perichrom). The chromatograph was used under isothermal conditions at 250 C. Helium was used as the carrier gas. Individual GLC peaks were identified by relative retention time in relation to the external standard 5a-cholestane. A pool of standards at different concentrations was chromatographed at the same time for bile acid identification and quantification Identification of epimerizing strains by biochemical properties and 16S rdna sequences Biochemical testing was performed from 24-h-anaerobic cultures in BHI-YE at 37 C using the Api 20A kit (BioMerieux, Marcy-l Etoile, France). For 16S rdna sequence analyses, total bacterial DNA was extracted from 24-h cultures in BHI-YE medium using a QIAamp DNA stool mini kit (Qiagen, Courtaboeuf, France), as specified by the manufacturer. The forward primer S-D-Bact-0008-a-S-20 (5 0 -AGAGTTTGATCCTGGC- TCAG-3 0 ), which targets the domain Bacteria, and the reverse primer S-*-Univ-1492-b-A-21 (5 0 -AC- GGCTACCTTGTTACGACTT-3 0 ), which targets all living organisms, were used to amplify bacterial 16S rdnas by PCR [32]. Reaction tubes contained 5 ng (1 ll) of studied strain genomic DNA, 1.25 U of Taq DNA polymerase (AmpliTaq Gold; Perkin Elmer Corporation, Foster City, CA, USA), 1X AmpliTaq Gold reaction buffer, 2.5 mm MgCl 2, 200 lm of each deoxyribonucleotide triphosphate and 0.40 lm of each primer in a final volume of 50 ll. Initial DNA denaturation and enzyme activation steps were performed at 94 C for 10 min in a PTC 150 thermocycler (MJ Research, Inc., Watertown, MA, USA), followed by 35 cycles of denaturation at 92 C for 1 min, annealing at 52 C for 1 min, and elongation at 72 C for 1 min 30 s, which were followed by a final elongation at 72 C for 15 min. PCR product was purified and concentrated with a QIAquick spin PCR purification kit (Qiagen). Purified PCR product was sequenced using the drhodamine Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS (fluorescent sequencing) (Perkin Elmer Corporation) and an automated ABI Prism 377 DNA sequencer (Applied Biosystems, Perkin Elmer Corporation). Internal primers for sequencing were Univ-536R (5 0 -GWATTACCGC- GGCKGCTG-3 0 ), which targets all living organisms, and Bact-1115R (5 0 -AGGGTTGCGCTCGTTR-3 0 )and Bact 930F (5 0 -AGGAATTGRCGGGGGC-3 0 ), which target the domain Bacteria. Blast homology searches were performed using the Genbank database. The 16S rdna sequences of strains C3, C7, C9, D12 and I11 were deposited in the Genbank database under Accession Numbers AY , AY341241, AY341239, AY and AY341242, respectively. 3. Results Five of the 108 strains isolated from human feces on 8H agar, identified as C3, C7, C9, D12 and I11, were able to epimerize unconjugated CDCA to UDCA in unstirred anaerobic cultures. The colony forming unit (cfu) and ph final values of bile salt-enriched cultures were comparable whether CDCA, TCDCA or GCDCA were used as the substrate. Mean respective values were (SD ) cfu ml 1 and 6.3 (SD 0.2) ph units. Fig. 2 shows the chromatogram of CDCA transformation by strain C9. All of these five strains epimerized CDCA to UDCA with formation of the intermediate 7keto-LCA but without formation of LCA or other bile acids. The percentages of CDCA and its derivative products are summarized in Table 1, showing that all strains mainly transformed CDCA to UDCA with low yields of 7-keto-LCA. All 7-epimerizing strains, including C. absonum, also exhibited a bile salt hydrolase activity towards TCDCA, and partially

4 68 P. Lepercq et al. / FEMS Microbiology Letters 235 (2004) Fig. 2. Gas chromatograms of standard mixture (A) and strain C9 incubated with CDCA (chenodeoxycholic acid) (B). Peaks are 1, 5a-cholestane; 2, LCA (lithocholic acid); 3, CDCA; 4, UDCA (ursodeoxycholic acid); 5, 7keto-LCA (7-keto-lithocholic acid). epimerized the released unconjugated homologue to UDCA (Table 1). For all strains however, the final UDCA yields, whether expressed in molar percentage of total bile acids (Table 1) or of deconjugated TCDCA (not represented), were lower when starting from the substrate TCDCA as compared to CDCA. Conversely, no strain could hydrolyze GCDCA, which remained the only detected bile acid in the cultures. The five bacteria were anaerobic rods, Gram-positive and spore forming. Their biochemical profiles, as inferred from the Api 20A system, were all similar with the exception of C7 which did not metabolize lactose and glycerol. The five biochemical profiles were highly similar to that of C. baratii (Table 2). Almost complete 16S rdna gene sequences (between 1466 and 1468 nucleotides) were determined for strains C7, D12 and I11. Sequence analyses revealed that these isolates were part of the cluster I of the genus Clostridium [33]. The sequence similarity values indicated that all these strains were most similar to C. baratii ATCC Levels of sequence similarity were 98.9% for C7, 98.8% for D12, and 99.0% for I11. Partial 16S rdna gene sequences were determined for strains C3 and C9 (474 and 473 nucleotides, respectively), using Univ-536R internal primer. Their respective sequence similarities to C. baratii ATCC were 99.4% and 99.2%. 4. Discussion Knowledge of bacteria which generate UDCA in the gut is clinically relevant, due to the multiple beneficial effects of this bile acid on human health. We chose 8H agar for the isolation procedure since clostridia were the only anaerobic 7-epimerizing bacteria known hitherto [19,20,27], and previous attempts to isolate epimerizing

5 P. Lepercq et al. / FEMS Microbiology Letters 235 (2004) Table 1 Molar percentages of bile acids recovered from 48-h pure cultures in BHY-YE enriched with either CDCA a or TCDCA b (10 4 M) as the substrate Substrates CDCA a TCDCA b Recovered bile acids CDCA a 7-keto-LCA c UDCA d TCDCA b CDCA a 7-keto-LCA c UDCA d Microorganisms Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE C C C D I C. absonum ATCC Values are means (SE) from three independent cultures. a CDCA, chenodeoxycholic acid. b TCDCA, taurochenodeoxycholic acid. c 7-keto-LCA, 7-keto-lithocholic acid. d UDCA, ursodeoxycholic acid. organisms from the intestinal microbiota of healthy individuals using other media have failed [19,20,34]. Using this medium, we were able to isolate for the first time five strains capable of epimerizing CDCA to UDCA, from the stool of a healthy volunteer. All five strains occurred at P 10 7 organisms g 1 wet stool, as inferred from the isolation procedure. Biochemical testing and 16S rdna analysis converged to show that they belong to the C. baratii species, which has been previously shown to support b-glucosidase and b-glucuronidase activities in the intestine of gnotobiotic mice, mono-associated with this bacterium [35]. C. baratii belongs to the Clostridium 16S rdna phylogenic cluster I, and is most closely related to C. absonum [33]. Interestingly, under our test conditions, the ability of the new C. baratii strains to epimerize CDCA to UDCA was as efficient as that of C. absonum, a soil isolate which could never be found in human feces [19,20,34]. Members of the Clostridium cluster I have been found in the predominant human microbiota using 16S rdna sequence analysis [36,37], whereas in another study including nine healthy human volunteers, counts of the Clostridium histolyticum group (Clostridium clusters I and II) with the Chis150 probe, were estimated to be in the range of to organisms g 1 dry feces [38], i.e., to organisms g 1 wet feces. We can therefore argue that the new C. baratii strains isolated in our study are normal members of the human intestinal microbiota. Whether there is a relationship between counts of these organisms and the overall epimerizing activity in the human gut, and what contribution these organisms make to the overall epimerizing activity in whole human microbiota, could be further investigated with new specifically designed probes and in vivo experiments using gnotobiotic animals harboring reconstituted human intestinal microbiota. The five strains epimerized CDCA to UDCA via 7keto-LCA as inferred from the detection of this intermediate product in all of the cultures starting from CDCA. This is in agreement with previous studies on epimerizing bacteria [19]. Clinical studies using radiolabeled bile salts [24,39] strongly suggested that CDCA to UDCA epimerization proceeds via 7keto-LCA in vivo as well. However, in the human gut, 7a-dehydroxylation is usually the major bacterial transformation of CDCA, generating LCA (Fig. 1), as inferred from bile acid profiles in feces, blood and bile [1,2,40] and in vitro incubation of total human fecal flora with CDCA [22]. Under our test conditions, none of the 7-epimerizing strains were found to generate LCA. All five strains also generated unconjugated CDCA, UDCA and 7keto-LCA starting from TCDCA. Conversely, whole GCDCA remained in its conjugated form and no metabolite of this bile acid could be detected in the cultures. These results indicate that 7-epimerization, just like 7-dehydroxylation [41,42], may operate only on

6 70 P. Lepercq et al. / FEMS Microbiology Letters 235 (2004) Table 2 Biochemical profile of the five epimerizing strains compared to that of Clostridium baratii, using the Api 20A system Reaction C3 C7 C9 D12 I11 Description of Clostridium baratii according to the Api A20 system Catalase ) ) ) ) ) 0 a Urease ) ) ) ) ) 0 Indole production ) ) ) ) ) 0 Gelatin hydrolysis ) ) ) ) ) 0 Esculin hydrolysis Oxidation/fermentation of: Arabinose ) ) ) ) ) 0 Cellobiose Glucose Glycerol + ) Lactose + ) Maltose Mannitol ) ) ) ) ) 8 Mannose Melezitose ) ) ) ) ) 0 Raffinose ) ) ) ) ) 0 Rhamnose ) ) ) ) ) 8 Salicin Sorbitol ) ) ) ) ) 8 Sucrose Trehalose Xylose ) ) ) ) ) 0 a Percentage of strains positive for the relevant character. unconjugated bile acids, which is however unlikely based on the studies with partially purified enzymes [20,43]. It is more likely that conjugated bile acids are not able to cross the cell membrane and that deconjugation outside the cell or on the cell surface is a prerequisite to the entry of bile acids into the cell where oxidoreduction would take place in the presence of cytoplasmic NAD(P)/NAD(P)H cofactors [44,45]. The presence of an extracytoplasmic taurine specific bile salt hydrolase in C. baratii and C. absonum would allow only free bile acids derived from taurine conjugates to enter the bacterial cell. There are at least three previous examples of specific hydrolysis of tauro-conjugated bile acids by whole cell cultures of Lactobacillus acidophilus [46] and Peptostreptococcus intermedius [47], by crude protein extracts of P. intermedius [47], or by bile salt hydrolase purified to homogeneity from Bacteroides vulgatus [48]. However, in the human intestinal microbiota, bile salt hydrolase activity towards glyco-conjugates is widespread and should hardly represent a limiting factor for bile salt epimerization. In conclusion, this work reports the first isolation of CDCA to UDCA epimerizing bacteria from the intestinal microbiota of a healthy human volunteer. Our findings demonstrated that all five strains belong to the C. baratii species (99% sequence similarity), which was itself most closely related to the epimerizing organism C. absonum. This, together with the previous failure to isolate active organisms on other media, suggest that the 7-epimerizing activity would not be widespread in the human microbiota, and that these new C. baratii strains might be important actors of this bioconversion in the human gut. This work therefore contributes to our knowledge of 7-epimerizing organisms of human intestinal origin, and encourages further investigations in this field, not only to propose new effective bacteria for the industrial production of UDCA, but also to try to enhance in vivo formation of UDCA from CDCA in order to benefit from its protective effects on health. Acknowledgements This work was co-supported by the French Ministry of Education, Research and Technology (MENRT, France), the National Institute for Agricultural Research (INRA, France), and the society Danone-Vitapole Company (Project no. AQS 99-N08). We thank Christine Young for her advice on American translation, and Lionel Rigottier-Gois for comments on the manuscript. References [1] Woollett, L.A., Buckley, D.D., Yao, L., Jones, P.J.H., Granholm, N.A., Tolley, E.A. and Heubi, J.E. (2003) Effect of ursodeoxycholic acid on cholesterol absorption and metabolism in humans. J. Lipid. Res. 44, [2] van Gorkom, B.A., van der Meer, R., Boersma-van Ek, W., Termont, D.S., de Vries, E.G. and Kleibeuker, J.H. (2002) Changes in bile acid composition and effect on cytolytic activity of fecal water by ursodeoxycholic acid administration: a placebocontrolled cross-over intervention trial in healthy volunteers. Scand. J. Gastroenterol. 37,

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