Biotechnol Lett (2014) 36:975–983 DOI 10.1007/s10529-013-1434-9
ORIGINAL RESEARCH PAPER
Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198 Xiang-Chao Gu • Xue-Gang Luo • Chong-Xi Wang • De-Yun Ma • Yan Wang • Ying-Ying He • Wen Li • Hao Zhou • Tong-Cun Zhang
Received: 16 October 2013 / Accepted: 12 December 2013 / Published online: 29 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs
Xiang-Chao Gu and Xue-Gang Luo contributed equally to this project.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-013-1434-9) contains supplementary material, which is available to authorized users. X.-C. Gu X.-G. Luo (&) C.-X. Wang D.-Y. Ma Y. Wang Y.-Y. He W. Li H. Zhou T.-C. Zhang (&) Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Street, Tianjin Economic and Technological Development Area, Tianjin 300457, China e-mail: [email protected] T.-C. Zhang e-mail: [email protected] X.-C. Gu X.-G. Luo C.-X. Wang D.-Y. Ma Y.-Y. He H. Zhou T.-C. Zhang Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China
had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids. Keywords Bile salts Bile salt hydrolase Glycodeoxycholic acid Hypercholesterolemia Lactobacillus plantarum Phylogenetic tree Taurodeoxycholic acid
Introduction Hypercholesterolemia is a major cause of atherosclerosis in coronary arteries (Ross 1993). Serum cholesterol can be lowered by decreasing hepatic cholesterol biosynthesis, increasing serum cholesterol removal and decreasing dietary cholesterol uptake (Jeun et al. 2010). Currently, drug therapy (such as 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors, bile acid-binding agents and fibrates) for treatment of hyperlipemia is widely used. However, some of these drugs are expensive and may have harmful sideeffects (Kumar et al. 2011). Therefore, safe and costeffective alternatives for controlling the cholesterolrelated diseases are still needed to be explored. The relationship between probiotics and reduction of serum cholesterol levels has been widely reported. Soybean products fermented by Lactobacillus jugurti and Bacillus spp. can reduce cholesterolemia, decrease total lipids and triglycerides in serum and liver and control the adipocyte circumference in hypercholesterolemic rats (Lim et al. 2011; Cheik et al. 2008).
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Lactobacillus plantarum KCTC 3928 was also has hypocholesterolemic effects achieved by increasing hepatic bile acid synthesis and fecal bile acid excretion (Jeun et al. 2010). Therefore, it is widely accepted that probiotics may be a good choice for the prevention and treatment of cholesterol-related diseases. Bile salts are synthesized from cholesterol and conjugated with glycine or taurine in the liver. They are then secreted into the small intestine (Kim and Lee 2005). Bile salts play an essential role in lipid digestion and act as a biological detergent to show their potent antimicrobial activity (Hofmann and Eckmann 2006). The gastrointestinal microflora has evolved the ability to transform bile salts to a great extent (Kim and Lee 2005), and one of these biotransformation reactions is bile salt deconjugation. In this reaction, bile salt hydrolases (BSHs) play an essential role in catalyzing the release of free amino acids from conjugated bile salts (Grill et al. 2000). BSHs belong to the N-terminal nucleophilic (Ntn) hydrolase superfamily that also contains pencillin V acylase (also called choloylglycine hydrolases) (Kumar et al. 2006; Lambert et al. 2008a, b). The deconjugation of bile salts is functional in bile detoxification (Grill et al. 2000). In addition, probiotics strains with BSHs activity can reduce serum cholesterol levels (Jeun et al. 2010; Takemura et al. 2010). BSHs activity has been reported in Bifidobacterium (Kumar et al. 2006; Kim and Lee 2008), Lactobacillus (Elkins et al. 2001), Enterococcus (Franz et al. 2001), Bacteroides (Jones et al. 2008) and many other bacteria. However, data on the BSH enzymes from L. plantarum are limited and the precise mechanism underlying the hypocholesterolemic effect of BSHs is still not clear. In the present study, the tolerance to bile salts and ability for removing cholesterol of L. plantarum CGMCC 8198, a novel Lactobacillus strain isolated in our previous study (Tian et al. 2012), were investigated for the first time. Furthermore, the genes of BSHs in this strain were also cloned, characterized and expressed in Escherichia coli Rosetta (DE3), and then the hydrolysis activity of the purified recombinant BSHs was examined.
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Lactobacillus acidophilus 1.1859, 1.2686, Lactobacillus casei 1.2435, 1.539, Lactobacillus sake, were cultured anaerobically at 37 °C in MRS broth. E. coli DH5a and Rosetta (DE3) cells were propagated at 37 °C in LB broth with vigorous shaking or on LB medium solidified with 1.5 % (w/v) agar. pET22b vector was used as the cloning and expressing plasmid. Glycodeoxycholic acids (GDCA), taurocholic acids (TCA) and taurodeoxycholic acids (TDCA) were purchased from Sigma-Aldrich. Kunming mice (4 weeks old, 20 ± 2 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science of PLA, and housed in standard plastic cages in a temperature-controlled room under a natural light/dark cycle. Bile salts tolerance test For assessing the tolerance to bile salts, Lactobacillus cells were adjusted to 108 c.f.u./ml with MRS which contained 0.1–0.3 % (w/v) bile salts and incubated at 37 °C for 4 h. Cells incubated in MRS without bile salts were used as control. The survival rate was tested with the MTT assay. The survival rate was calculated as follows: Survival rateð%Þ ¼
ODbile salts 100: ODcontrol
Animal tests
Materials and methods
Mice were randomly allocated into three groups (n = 10). Those in group A were fed with a normal fat diet and served as the control. The other two groups were fed with a high fat diet (HFD, normal diet 78.8 %, egg yolk powder 10 %, lard oil 10 %, cholesterol 1 % and deoxycholate 0.2 %). Mice in groups A and B were administered intragastrically with 0.3 ml phosphate-buffered solution (PBS, 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4 and 8 mM K2HPO4, pH 7.2), while group C were administered intragastrically with 0.3 ml PBS containing fresh L. plantarum CGMCC 8198 (5 9 108 c.f.u./ mouse). After 3 weeks of feeding, the blood was collected and the total cholesterol was measured by cholesterol kit (ZKTeco, China).
Strains, reagents and animals
RT-PCR
Lactobacillus plantarum CGMCC 8198 (previously named as strain TH1—see Tian et al. 2012),
Cells of L. plantarum were cultured anaerobically in MRS with up to 0.3 % bile salts. After 12 h
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stimulation, cells were harvested. Total RNA was extracted using TRIzol and lysozyme, and then reverse-transcribed into cDNA (Bron et al. 2004). mRNA expression levels of bsh and 16S rRNA were quantified by RT-PCR analysis. The primers were listed in Supplementary Table 1. PCR products were electrophoretically separated in 1.5 % agarose gels and the densities of the bands were analyzed with Quantity One software.
Mixtures were centrifuged at 15,0009g for 10 min to obtain the reaction samples (Ren et al. 2011). The production of glycine or taurine in the reaction samples was detected using HPLC with a pre-column derivatization by 2,4-dinitrofluorobenzene and gradient elution (Chen et al. 2009). One unit of BSH activity was defined as the amount of enzyme that liberated 1 lmol amino acids from the substrate per min (Nguyen et al. 2007).
Cloning, analysis of bsh genes and construction of expression plasmids
Results
bsh genes were PCR-amplified from the genomic DNA of L. plantarum CGMCC 8198 using the primers (Supplementary Table 1), which were designed based on the published genome sequence of L. plantarum WCFS1 (NC_004567.2). PCR products were inserted into pET22b vector and then transformed into E. coli DH5a. BLASTX analysis and protein homology assay of the obtained sequences were performed at the website of NCBI. Multiple amino acid sequence alignments and phylogenetic tree were carried out using ClustalX2.1 and MEGA5.10 software package. Expression and purification of the BSH enzymes Recombinant plasmids were transformed into E. coli Rosetta (DE3). Positive transformants were cultured in LB liquid medium supplemented with ampicillin (100 lg/ml) and chloramphenicol (34 lg/ml) at 30 °C until OD600 reached 0.6 to 0.8. Expression of BSHs was induced by 0.1 mM IPTG and detected using 12 % (v/v) SDS-PAGE. Recombinant proteins were purified by a Ni2?-chelating affinity chromatography and identified by western blotting using specific antibodies against His-Tag (1:1000, Abcam) and HRP-conjugated anti-mouse secondary antibodies (1:10,000, Abcam). The protein concentration was measured with the Bradford method.
Lactobacillus plantarum CGMCC 8198 has high bile salts resistance and hypocholesterolemic effects As there is a linear relationship between OD570 and viable cell counts using the MTT assay, a simpler method was used to test the survival rate of Lactobacillus instead of traditional method of cell counting (Supplementary Fig. 1). In addition, MRS medium rather than a buffer was used to avoid death of cells due to lack nutrition. As shown in Fig. 1, of the six bacteria tested, L. plantarum CGMCC 8198 had the highest ability to resist bile salts. The survival rates of L. plantarum were 85 to 68 and 48 % after being treated with bile salts at 0.1 0.2 and 0.3 %, respectively. In contrast, L. casei 1.539 was the weakest one to resist the bile salts, and its survival was only 8 % when cultured in MRS broth with 0.3 % bile salts for 4 h.
HPLC assay of BSHs activity Approx. 750 ll reaction buffer (0.1 M sodium phosphate, pH 6.0) was mixed with a 100 ll purified BSH enzyme, 100 ll (10 mM final) of bile salts (GDCA, TCA or TDCA), and 100 ll liquid paraffin. The reaction mixtures were incubated at 37 °C for 30 min, and then the reaction was stopped by 200 ll 15 % (w/v) trichloroacetic acid.
Fig. 1 The ability of Lactobacillus spp. to resist different concentration of bile salts. Six strains of Lactobacillus were cultured in MRS with different concentration of bile salts for 4 h, respectively, then the survival rate were assessed with MTT assay. The survival rate of control was defined as 100 %. Results represented mean ± S.D. obtained in triplicate in three independent experiments
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Biotechnol Lett (2014) 36:975–983 Fig. 4 Multiple sequence alignment of BSH proteins of L. c plantarum CGMCC 8198 with BSHs from other bacteria. LpWCFS, L. plantarum WCFS1; LpST, L. plantarum ST-III; Lp8198, L. plantarum CGMCC 8198; LpenMP, L. pentosus MP-10; Lbre, L. brevis ATCC 367; LparaF0439, L. parafarraginis F0439; Ljohn33200, L. johnsonii ATCC 33200; Lacid, L. acidophilus; pva-B.spha, PVA from B. sphaericus. Five proposed active sites (C, D, Y, N, and R) are highlighted by arrows. Asterisk conserved residues, colon conservative substitutions, dot less conservative substitutions
Fig. 2 The effect of L. plantarum CGMCC 8198 on serum total cholesterol (TC) in vivo. Mice were fed with high fat diet (HFD) and administered with L. plantarum CGMCC 8198 (HFD ? Lp8198, 5 9 108 c.f.u./mouse) or PBS (HFD ? PBS) daily for 3 weeks. Subsequently, the TC of serum was measured. Mice fed normal diet were used as control. Values were expressed as mean ± S.D. **p \ 0.01
shown in Fig. 2, L. plantarum-administered mice had lower concentrations of serum total cholesterol (TC) in comparison to PBS-administered mice (p \ 0.01), indicating that L. plantarum might have valuable therapeutic effects in lowering the concentrations of serum TC in hypercholesterolemia. Bile salts upregulate BSH mRNA levels of L. plantarum The results of RT-PCR showed that bsh2, bsh3 and bsh4 were all upregulated by bile salts in a dosedependent manner, suggesting that BSHs might play important roles in the bile salts resistance and hypocholesterolemic effects of L. plantarum CGMCC 8198 (Fig. 3). In addition, bsh1 could not be detected even when 3 pairs of different conserved primers were used to amplify either cDNA or genomic DNA (data not shown), indicating that homologous bsh1 might be absence or less conserved in this strain. Cloning and analysis of the bsh genes
Fig. 3 The effect of bile salts on the mRNA levels of BSHs of L. plantarum CGMCC 8198. a The mRNA of L. plantarum CGMCC 8198 was isolated and RT-PCR was performed to examine the mRNA levels of BSHs. b The densities of the bands were analyzed with Quantity One software and relative mRNA levels were deduced from the ratio of the mean values of BSHs to that of 16S rRNA
Furthermore, to detect whether L. plantarum had hypocholesterolemic effects in vivo, a hypercholesterolemia model was established using mice. As
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Using PCR, the full-length of bsh genes in L. plantarum were cloned and deposited in the GenBank database under accession numbers KC197710, KC197711 and KC197712. The results of sequencing showed that bsh2–4 contained single ORFs of 1017, 987 and 954 nucleotides, respectively. Using the online software ExPASY (http://web.expasy.org/ compute_pi/), it showed that the deduced proteins of BSH2, BSH3 and BSH4 had theoretical molecular weights of 37.53, 36.14 and 35.65 kDa and pI values of 5.93, 5.12 and 7.82, respectively. Aligned with the reported BSHs or penicillin V acylase (PVA), we found that BSHs of L. plantarum CGMCC 8198 had highest sequence identities of 98– 99 % to the BSHs of L. plantarum ST-III (GenBank Accession. no. YP_003923374, YP_003925427, YP_
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Fig. 5 Phylogenetic tree illustrating the relationship among BSHs. The phylogenetic relationship among BSHs from Lactobacillus and PVA from B. sphaericus was illustrated by the phylogenetic tree. The data on the branches standard for the percent of the reliability in bootrap test
003926069) and L. plantarum WCFS1 (YP_004888 146, YP_004890864, YP_004890239), and shared 82– 92, 46–60, 27–30, 27–31, 26–60 and 28–33 % identities with BSHs from L. pentosus MP-10 (CCB81545, CCB81988, CCB82175), L. brevis ATCC 367 (YP_79 6052, YP_796216), Lactobacillus acidophilus (AAD0 3709), L. johnsonii ATCC 33200 (ZP_04006912), Lactobacillus parafarraginis F0439 (ZP_09392500) and PVA from Bacillus. sphaericus (M15660), respectively (Fig. 4). Furthermore, five putative active sites, Cys1, Asp20, Tyr78, Asn171 and Arg224, were also found in all 3 bsh genes of L. plantarum CGMCC 8198. However, these active sites were as same as PVA but were a little different with BSHs in Lactobacillus. For example, the catalytic residue of Tyr78 was replaced by Asn78 in BSHs of L. johnsonii ATCC 33200 and L. acidophilus. These results indicated that BSHs in our strain might have individual mechanisms for the hydrolysis of bile salts. In addition, phylogenetic tree analysis revealed that the three bsh genes in L. plantarum had longer genetic distances to each other but were highly conserved in evolution (Fig. 5). Furthermore, bsh2 and bsh3 are more homologous than bsh4 among strains, implying that evolutionary bsh4 might appear earlier than bsh2 and bsh3.
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Expression, purification and enzymatic activity of BSH proteins As shown in Fig. 6a, three monomeric proteins with molecular mass of approx. 35–39 kDa were observed by SDS-PAGE. After affinity chromatography, the results of SDS-PAGE and western blotting showed that the final products have sufficient purity for the measurement of their enzymatic activity (Fig. 6b, c). The hydrolytic activities of the purified BSHs were then examined: BSH2 and BSH3 had greater hydrolyzing activity with GDCA, whereas BSH4 had greater hydrolyzing activity with TDCA (Fig. 6d). The hydrolyzing activity of all the three BSHs on TCA was too low to be detected by HPLC (data not shown). These results suggested that all the three BSHs of L. plantarum had bile salt hydrolysis activity. Furthermore, the ability of BSH2 was higher than that of the other two enzymes.
Discussion Many previous studies have revealed that Lactobacillus has a natural advantage of resisting bile salts and
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Fig. 6 Expression, purification and enzymatic activity of BSH proteins of L. plantarum CGMCC 8198. a Expression of BSH proteins after IPTG induction. The predicted proteins BSH2, BSH3, and BSH4 are marked by arrows. M Marker, N no induction, I induction. b SDS-PAGE analysis of purified proteins, Lane M: protein marker; 1: BSH2; 2: BSH3; 3: BSH4. c The corresponding proteins were detected by western blotting with specific antibodies against His-Tag. d The activity of purified recombinant BSHs to different substrates: GDCA glycodeoxycholic acids, TDCA taurocholic acids, there was no activity with (TCA). Relative activity was defined as the amount of units of BSH activity per mg purified recombinant BSH. Data were expressed as mean ± SD
removing cholesterol (Kumar et al. 2012). In mice with hyperlipemia, L. plantarum strain No. 14 can significantly reduce the mean size of adipocytes, the weight of white adipose tissue, the leptin concentrations and serum total cholesterol (Takemura et al. 2010). In this study, we described the growth of L. plantarum CGMCC 8198 in media containing bile salts and its ability to reduce serum total cholesterol. It showed a 48 % survival in MRS broth with 0.3 % bile salts and decreased the serum cholesterol level by 20 % in mice with hyperlipemia. These results indicated that L. plantarum CGMCC 8198 might have the potential advantage in preventing cardiovascular disease. Bile acids and bile salts facilitate digestion and absorption of lipids in the small intestine. They are also important modulators of cholesterol homeostasis. BSHs catalyze hydrolysis of conjugated bile salts thereby lowering serum cholesterol levels (Hofmann and Eckmann 2006; Kumar et al. 2006). A study on the evolutionary conservation of bsh homologues has shown that bsh2, bsh3 and bsh4, but not bsh1, appear
to be conserved among L. plantarum strains (Molenaar et al. 2005). Coincidently, here, only bsh2-4 genes could be cloned from L. plantarum CGMCC 8198, and they all had high similarity with L. plantarum WCFS1 and ST-III. In contrast, bsh1 seemed to be absent or the sequence of the bsh1 gene was less conserved in L. plantarum CGMCC 8198, although this enzyme exhibited high hydrolysis activities in L. plantarum WCFS1 and ST-III (Lambert et al. 2008a, b; Ren et al. 2011). To confirm this issue, the whole-genome sequencing of L. plantarum CGMCC 8198 will be performing in the near future. The three recombinant BSHs had similar activities for the hydrolysis of TDCA, while the hydrolyzing activity of BSH2 against GDCA was 2.3-fold and 5.5-fold greater than those of BSH3 and BSH4, respectively. Therefore, BSH2 might play a major role in the bile salt hydrolysis activity in L. plantarum CGMCC 8198. In addition, the recombinant BSH2 exhibited a high ability for the hydrolysis of GDCA, implying that it has a higher affinity toward deoxy-conjugates and might be used as a potential food-grade selection
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marker in Lactobacilli expression vectors (Yin et al. 2011; de Vos 1999). The diversity of bile salt hydrolases from a number of bacterial strains makes the understanding of substrate-hydrolysing capabilities difficult. In the present study, BLAST analysis and multiple sequence alignment showed that BSH2-4 in L. plantarum CGMCC 8198 belong to the Ntn hydrolase superfamily which contain a conserved N-terminal cysteine residue (Cys1) in the active center (Rossocha et al. 2005) and serve as a nucleophile and a proton donor in the process of catalysis (Kumar et al. 2006). The other four predicted catalysis sites (Asp20, Tyr78, Asn171 and Arg224) are also conserved with most BSHs or PVA from other bacteria. However, among these active sites, Tyr78 is sometimes replaced by Asn78 in some Lactobacillus spp. such as L. johnsonii ATCC 33200 and L. acidophilus. Replacement of Asn78 in BSH2-4 by Tyr78 does not, though, alter the nature of the active site in the catalytic process because the peptide NH atoms of Tyr78 or Asn78 residues play an important role in forming the oxyanion hole together with Asn171 Nd2 by providing an electron acceptor (Lambert et al. 2008a, b; Ren et al. 2011; Rossocha et al. 2005). All three recombinant BSHs in our strain had high relative activities for hydrolysis of both GDCA and TDCA; therefore they might recognize substrates predominantly at the amino acid moieties but not at the cholate moieties and this predication is consistent with the study of Rossocha et al. (2005). Although the BSHs of L. plantarum CGMCC 8198 have the same catalytic sites to L. plantarum WCFS1, the capabilities of substrate-hydrolysing are different. The recombinant BSH4 in this study exhibits a high ability for the hydrolysis of TDCA but the BSH4 of L. plantarum WCFS1 tends to GDCA (Lambert et al. 2008a, b). In our opinion, this difference might be due to the change of spatial protein structure, which was caused by other mutations. In order to further confirm the structure–activity relationship and the substrate binding pocket of BSHs in L. plantarum CGMCC 8198, the structural studies may be necessary. In summary, L. plantarum CGMCC 8198 may escape from cell death by bile salts in the intestine and also reduce plasma cholesterol in vivo through the hydrolysis activity of its BSHs. This work indicates that this novel probiotic stain could be used in the food or drug system to improve the health of patients suffering from cholesterol-related diseases.
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Biotechnol Lett (2014) 36:975–983 Acknowledgments We thank Dr. Hong-Peng He and Dr. Tao Wang for the assistance in editing the language. This study was financially supported by the 863 (Hi-tech research and development program of China) program under contract No. 2012AA022108 and No. 2008AA10Z336, and the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1166).
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ORIGINAL RESEARCH PAPER
Cloning and analysis of bile salt hydrolase genes from Lactobacillus plantarum CGMCC No. 8198 Xiang-Chao Gu • Xue-Gang Luo • Chong-Xi Wang • De-Yun Ma • Yan Wang • Ying-Ying He • Wen Li • Hao Zhou • Tong-Cun Zhang
Received: 16 October 2013 / Accepted: 12 December 2013 / Published online: 29 December 2013 Ó Springer Science+Business Media Dordrecht 2013
Abstract Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs
Xiang-Chao Gu and Xue-Gang Luo contributed equally to this project.
Electronic supplementary material The online version of this article (doi:10.1007/s10529-013-1434-9) contains supplementary material, which is available to authorized users. X.-C. Gu X.-G. Luo (&) C.-X. Wang D.-Y. Ma Y. Wang Y.-Y. He W. Li H. Zhou T.-C. Zhang (&) Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, College of Biotechnology, Tianjin University of Science and Technology, No. 29, 13th Street, Tianjin Economic and Technological Development Area, Tianjin 300457, China e-mail: [email protected] T.-C. Zhang e-mail: [email protected] X.-C. Gu X.-G. Luo C.-X. Wang D.-Y. Ma Y.-Y. He H. Zhou T.-C. Zhang Tianjin Key Laboratory of Industrial Microbiology, Tianjin 300457, China
had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids. Keywords Bile salts Bile salt hydrolase Glycodeoxycholic acid Hypercholesterolemia Lactobacillus plantarum Phylogenetic tree Taurodeoxycholic acid
Introduction Hypercholesterolemia is a major cause of atherosclerosis in coronary arteries (Ross 1993). Serum cholesterol can be lowered by decreasing hepatic cholesterol biosynthesis, increasing serum cholesterol removal and decreasing dietary cholesterol uptake (Jeun et al. 2010). Currently, drug therapy (such as 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors, bile acid-binding agents and fibrates) for treatment of hyperlipemia is widely used. However, some of these drugs are expensive and may have harmful sideeffects (Kumar et al. 2011). Therefore, safe and costeffective alternatives for controlling the cholesterolrelated diseases are still needed to be explored. The relationship between probiotics and reduction of serum cholesterol levels has been widely reported. Soybean products fermented by Lactobacillus jugurti and Bacillus spp. can reduce cholesterolemia, decrease total lipids and triglycerides in serum and liver and control the adipocyte circumference in hypercholesterolemic rats (Lim et al. 2011; Cheik et al. 2008).
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Lactobacillus plantarum KCTC 3928 was also has hypocholesterolemic effects achieved by increasing hepatic bile acid synthesis and fecal bile acid excretion (Jeun et al. 2010). Therefore, it is widely accepted that probiotics may be a good choice for the prevention and treatment of cholesterol-related diseases. Bile salts are synthesized from cholesterol and conjugated with glycine or taurine in the liver. They are then secreted into the small intestine (Kim and Lee 2005). Bile salts play an essential role in lipid digestion and act as a biological detergent to show their potent antimicrobial activity (Hofmann and Eckmann 2006). The gastrointestinal microflora has evolved the ability to transform bile salts to a great extent (Kim and Lee 2005), and one of these biotransformation reactions is bile salt deconjugation. In this reaction, bile salt hydrolases (BSHs) play an essential role in catalyzing the release of free amino acids from conjugated bile salts (Grill et al. 2000). BSHs belong to the N-terminal nucleophilic (Ntn) hydrolase superfamily that also contains pencillin V acylase (also called choloylglycine hydrolases) (Kumar et al. 2006; Lambert et al. 2008a, b). The deconjugation of bile salts is functional in bile detoxification (Grill et al. 2000). In addition, probiotics strains with BSHs activity can reduce serum cholesterol levels (Jeun et al. 2010; Takemura et al. 2010). BSHs activity has been reported in Bifidobacterium (Kumar et al. 2006; Kim and Lee 2008), Lactobacillus (Elkins et al. 2001), Enterococcus (Franz et al. 2001), Bacteroides (Jones et al. 2008) and many other bacteria. However, data on the BSH enzymes from L. plantarum are limited and the precise mechanism underlying the hypocholesterolemic effect of BSHs is still not clear. In the present study, the tolerance to bile salts and ability for removing cholesterol of L. plantarum CGMCC 8198, a novel Lactobacillus strain isolated in our previous study (Tian et al. 2012), were investigated for the first time. Furthermore, the genes of BSHs in this strain were also cloned, characterized and expressed in Escherichia coli Rosetta (DE3), and then the hydrolysis activity of the purified recombinant BSHs was examined.
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Lactobacillus acidophilus 1.1859, 1.2686, Lactobacillus casei 1.2435, 1.539, Lactobacillus sake, were cultured anaerobically at 37 °C in MRS broth. E. coli DH5a and Rosetta (DE3) cells were propagated at 37 °C in LB broth with vigorous shaking or on LB medium solidified with 1.5 % (w/v) agar. pET22b vector was used as the cloning and expressing plasmid. Glycodeoxycholic acids (GDCA), taurocholic acids (TCA) and taurodeoxycholic acids (TDCA) were purchased from Sigma-Aldrich. Kunming mice (4 weeks old, 20 ± 2 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Science of PLA, and housed in standard plastic cages in a temperature-controlled room under a natural light/dark cycle. Bile salts tolerance test For assessing the tolerance to bile salts, Lactobacillus cells were adjusted to 108 c.f.u./ml with MRS which contained 0.1–0.3 % (w/v) bile salts and incubated at 37 °C for 4 h. Cells incubated in MRS without bile salts were used as control. The survival rate was tested with the MTT assay. The survival rate was calculated as follows: Survival rateð%Þ ¼
ODbile salts 100: ODcontrol
Animal tests
Materials and methods
Mice were randomly allocated into three groups (n = 10). Those in group A were fed with a normal fat diet and served as the control. The other two groups were fed with a high fat diet (HFD, normal diet 78.8 %, egg yolk powder 10 %, lard oil 10 %, cholesterol 1 % and deoxycholate 0.2 %). Mice in groups A and B were administered intragastrically with 0.3 ml phosphate-buffered solution (PBS, 135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4 and 8 mM K2HPO4, pH 7.2), while group C were administered intragastrically with 0.3 ml PBS containing fresh L. plantarum CGMCC 8198 (5 9 108 c.f.u./ mouse). After 3 weeks of feeding, the blood was collected and the total cholesterol was measured by cholesterol kit (ZKTeco, China).
Strains, reagents and animals
RT-PCR
Lactobacillus plantarum CGMCC 8198 (previously named as strain TH1—see Tian et al. 2012),
Cells of L. plantarum were cultured anaerobically in MRS with up to 0.3 % bile salts. After 12 h
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stimulation, cells were harvested. Total RNA was extracted using TRIzol and lysozyme, and then reverse-transcribed into cDNA (Bron et al. 2004). mRNA expression levels of bsh and 16S rRNA were quantified by RT-PCR analysis. The primers were listed in Supplementary Table 1. PCR products were electrophoretically separated in 1.5 % agarose gels and the densities of the bands were analyzed with Quantity One software.
Mixtures were centrifuged at 15,0009g for 10 min to obtain the reaction samples (Ren et al. 2011). The production of glycine or taurine in the reaction samples was detected using HPLC with a pre-column derivatization by 2,4-dinitrofluorobenzene and gradient elution (Chen et al. 2009). One unit of BSH activity was defined as the amount of enzyme that liberated 1 lmol amino acids from the substrate per min (Nguyen et al. 2007).
Cloning, analysis of bsh genes and construction of expression plasmids
Results
bsh genes were PCR-amplified from the genomic DNA of L. plantarum CGMCC 8198 using the primers (Supplementary Table 1), which were designed based on the published genome sequence of L. plantarum WCFS1 (NC_004567.2). PCR products were inserted into pET22b vector and then transformed into E. coli DH5a. BLASTX analysis and protein homology assay of the obtained sequences were performed at the website of NCBI. Multiple amino acid sequence alignments and phylogenetic tree were carried out using ClustalX2.1 and MEGA5.10 software package. Expression and purification of the BSH enzymes Recombinant plasmids were transformed into E. coli Rosetta (DE3). Positive transformants were cultured in LB liquid medium supplemented with ampicillin (100 lg/ml) and chloramphenicol (34 lg/ml) at 30 °C until OD600 reached 0.6 to 0.8. Expression of BSHs was induced by 0.1 mM IPTG and detected using 12 % (v/v) SDS-PAGE. Recombinant proteins were purified by a Ni2?-chelating affinity chromatography and identified by western blotting using specific antibodies against His-Tag (1:1000, Abcam) and HRP-conjugated anti-mouse secondary antibodies (1:10,000, Abcam). The protein concentration was measured with the Bradford method.
Lactobacillus plantarum CGMCC 8198 has high bile salts resistance and hypocholesterolemic effects As there is a linear relationship between OD570 and viable cell counts using the MTT assay, a simpler method was used to test the survival rate of Lactobacillus instead of traditional method of cell counting (Supplementary Fig. 1). In addition, MRS medium rather than a buffer was used to avoid death of cells due to lack nutrition. As shown in Fig. 1, of the six bacteria tested, L. plantarum CGMCC 8198 had the highest ability to resist bile salts. The survival rates of L. plantarum were 85 to 68 and 48 % after being treated with bile salts at 0.1 0.2 and 0.3 %, respectively. In contrast, L. casei 1.539 was the weakest one to resist the bile salts, and its survival was only 8 % when cultured in MRS broth with 0.3 % bile salts for 4 h.
HPLC assay of BSHs activity Approx. 750 ll reaction buffer (0.1 M sodium phosphate, pH 6.0) was mixed with a 100 ll purified BSH enzyme, 100 ll (10 mM final) of bile salts (GDCA, TCA or TDCA), and 100 ll liquid paraffin. The reaction mixtures were incubated at 37 °C for 30 min, and then the reaction was stopped by 200 ll 15 % (w/v) trichloroacetic acid.
Fig. 1 The ability of Lactobacillus spp. to resist different concentration of bile salts. Six strains of Lactobacillus were cultured in MRS with different concentration of bile salts for 4 h, respectively, then the survival rate were assessed with MTT assay. The survival rate of control was defined as 100 %. Results represented mean ± S.D. obtained in triplicate in three independent experiments
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Biotechnol Lett (2014) 36:975–983 Fig. 4 Multiple sequence alignment of BSH proteins of L. c plantarum CGMCC 8198 with BSHs from other bacteria. LpWCFS, L. plantarum WCFS1; LpST, L. plantarum ST-III; Lp8198, L. plantarum CGMCC 8198; LpenMP, L. pentosus MP-10; Lbre, L. brevis ATCC 367; LparaF0439, L. parafarraginis F0439; Ljohn33200, L. johnsonii ATCC 33200; Lacid, L. acidophilus; pva-B.spha, PVA from B. sphaericus. Five proposed active sites (C, D, Y, N, and R) are highlighted by arrows. Asterisk conserved residues, colon conservative substitutions, dot less conservative substitutions
Fig. 2 The effect of L. plantarum CGMCC 8198 on serum total cholesterol (TC) in vivo. Mice were fed with high fat diet (HFD) and administered with L. plantarum CGMCC 8198 (HFD ? Lp8198, 5 9 108 c.f.u./mouse) or PBS (HFD ? PBS) daily for 3 weeks. Subsequently, the TC of serum was measured. Mice fed normal diet were used as control. Values were expressed as mean ± S.D. **p \ 0.01
shown in Fig. 2, L. plantarum-administered mice had lower concentrations of serum total cholesterol (TC) in comparison to PBS-administered mice (p \ 0.01), indicating that L. plantarum might have valuable therapeutic effects in lowering the concentrations of serum TC in hypercholesterolemia. Bile salts upregulate BSH mRNA levels of L. plantarum The results of RT-PCR showed that bsh2, bsh3 and bsh4 were all upregulated by bile salts in a dosedependent manner, suggesting that BSHs might play important roles in the bile salts resistance and hypocholesterolemic effects of L. plantarum CGMCC 8198 (Fig. 3). In addition, bsh1 could not be detected even when 3 pairs of different conserved primers were used to amplify either cDNA or genomic DNA (data not shown), indicating that homologous bsh1 might be absence or less conserved in this strain. Cloning and analysis of the bsh genes
Fig. 3 The effect of bile salts on the mRNA levels of BSHs of L. plantarum CGMCC 8198. a The mRNA of L. plantarum CGMCC 8198 was isolated and RT-PCR was performed to examine the mRNA levels of BSHs. b The densities of the bands were analyzed with Quantity One software and relative mRNA levels were deduced from the ratio of the mean values of BSHs to that of 16S rRNA
Furthermore, to detect whether L. plantarum had hypocholesterolemic effects in vivo, a hypercholesterolemia model was established using mice. As
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Using PCR, the full-length of bsh genes in L. plantarum were cloned and deposited in the GenBank database under accession numbers KC197710, KC197711 and KC197712. The results of sequencing showed that bsh2–4 contained single ORFs of 1017, 987 and 954 nucleotides, respectively. Using the online software ExPASY (http://web.expasy.org/ compute_pi/), it showed that the deduced proteins of BSH2, BSH3 and BSH4 had theoretical molecular weights of 37.53, 36.14 and 35.65 kDa and pI values of 5.93, 5.12 and 7.82, respectively. Aligned with the reported BSHs or penicillin V acylase (PVA), we found that BSHs of L. plantarum CGMCC 8198 had highest sequence identities of 98– 99 % to the BSHs of L. plantarum ST-III (GenBank Accession. no. YP_003923374, YP_003925427, YP_
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Fig. 5 Phylogenetic tree illustrating the relationship among BSHs. The phylogenetic relationship among BSHs from Lactobacillus and PVA from B. sphaericus was illustrated by the phylogenetic tree. The data on the branches standard for the percent of the reliability in bootrap test
003926069) and L. plantarum WCFS1 (YP_004888 146, YP_004890864, YP_004890239), and shared 82– 92, 46–60, 27–30, 27–31, 26–60 and 28–33 % identities with BSHs from L. pentosus MP-10 (CCB81545, CCB81988, CCB82175), L. brevis ATCC 367 (YP_79 6052, YP_796216), Lactobacillus acidophilus (AAD0 3709), L. johnsonii ATCC 33200 (ZP_04006912), Lactobacillus parafarraginis F0439 (ZP_09392500) and PVA from Bacillus. sphaericus (M15660), respectively (Fig. 4). Furthermore, five putative active sites, Cys1, Asp20, Tyr78, Asn171 and Arg224, were also found in all 3 bsh genes of L. plantarum CGMCC 8198. However, these active sites were as same as PVA but were a little different with BSHs in Lactobacillus. For example, the catalytic residue of Tyr78 was replaced by Asn78 in BSHs of L. johnsonii ATCC 33200 and L. acidophilus. These results indicated that BSHs in our strain might have individual mechanisms for the hydrolysis of bile salts. In addition, phylogenetic tree analysis revealed that the three bsh genes in L. plantarum had longer genetic distances to each other but were highly conserved in evolution (Fig. 5). Furthermore, bsh2 and bsh3 are more homologous than bsh4 among strains, implying that evolutionary bsh4 might appear earlier than bsh2 and bsh3.
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Expression, purification and enzymatic activity of BSH proteins As shown in Fig. 6a, three monomeric proteins with molecular mass of approx. 35–39 kDa were observed by SDS-PAGE. After affinity chromatography, the results of SDS-PAGE and western blotting showed that the final products have sufficient purity for the measurement of their enzymatic activity (Fig. 6b, c). The hydrolytic activities of the purified BSHs were then examined: BSH2 and BSH3 had greater hydrolyzing activity with GDCA, whereas BSH4 had greater hydrolyzing activity with TDCA (Fig. 6d). The hydrolyzing activity of all the three BSHs on TCA was too low to be detected by HPLC (data not shown). These results suggested that all the three BSHs of L. plantarum had bile salt hydrolysis activity. Furthermore, the ability of BSH2 was higher than that of the other two enzymes.
Discussion Many previous studies have revealed that Lactobacillus has a natural advantage of resisting bile salts and
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Fig. 6 Expression, purification and enzymatic activity of BSH proteins of L. plantarum CGMCC 8198. a Expression of BSH proteins after IPTG induction. The predicted proteins BSH2, BSH3, and BSH4 are marked by arrows. M Marker, N no induction, I induction. b SDS-PAGE analysis of purified proteins, Lane M: protein marker; 1: BSH2; 2: BSH3; 3: BSH4. c The corresponding proteins were detected by western blotting with specific antibodies against His-Tag. d The activity of purified recombinant BSHs to different substrates: GDCA glycodeoxycholic acids, TDCA taurocholic acids, there was no activity with (TCA). Relative activity was defined as the amount of units of BSH activity per mg purified recombinant BSH. Data were expressed as mean ± SD
removing cholesterol (Kumar et al. 2012). In mice with hyperlipemia, L. plantarum strain No. 14 can significantly reduce the mean size of adipocytes, the weight of white adipose tissue, the leptin concentrations and serum total cholesterol (Takemura et al. 2010). In this study, we described the growth of L. plantarum CGMCC 8198 in media containing bile salts and its ability to reduce serum total cholesterol. It showed a 48 % survival in MRS broth with 0.3 % bile salts and decreased the serum cholesterol level by 20 % in mice with hyperlipemia. These results indicated that L. plantarum CGMCC 8198 might have the potential advantage in preventing cardiovascular disease. Bile acids and bile salts facilitate digestion and absorption of lipids in the small intestine. They are also important modulators of cholesterol homeostasis. BSHs catalyze hydrolysis of conjugated bile salts thereby lowering serum cholesterol levels (Hofmann and Eckmann 2006; Kumar et al. 2006). A study on the evolutionary conservation of bsh homologues has shown that bsh2, bsh3 and bsh4, but not bsh1, appear
to be conserved among L. plantarum strains (Molenaar et al. 2005). Coincidently, here, only bsh2-4 genes could be cloned from L. plantarum CGMCC 8198, and they all had high similarity with L. plantarum WCFS1 and ST-III. In contrast, bsh1 seemed to be absent or the sequence of the bsh1 gene was less conserved in L. plantarum CGMCC 8198, although this enzyme exhibited high hydrolysis activities in L. plantarum WCFS1 and ST-III (Lambert et al. 2008a, b; Ren et al. 2011). To confirm this issue, the whole-genome sequencing of L. plantarum CGMCC 8198 will be performing in the near future. The three recombinant BSHs had similar activities for the hydrolysis of TDCA, while the hydrolyzing activity of BSH2 against GDCA was 2.3-fold and 5.5-fold greater than those of BSH3 and BSH4, respectively. Therefore, BSH2 might play a major role in the bile salt hydrolysis activity in L. plantarum CGMCC 8198. In addition, the recombinant BSH2 exhibited a high ability for the hydrolysis of GDCA, implying that it has a higher affinity toward deoxy-conjugates and might be used as a potential food-grade selection
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marker in Lactobacilli expression vectors (Yin et al. 2011; de Vos 1999). The diversity of bile salt hydrolases from a number of bacterial strains makes the understanding of substrate-hydrolysing capabilities difficult. In the present study, BLAST analysis and multiple sequence alignment showed that BSH2-4 in L. plantarum CGMCC 8198 belong to the Ntn hydrolase superfamily which contain a conserved N-terminal cysteine residue (Cys1) in the active center (Rossocha et al. 2005) and serve as a nucleophile and a proton donor in the process of catalysis (Kumar et al. 2006). The other four predicted catalysis sites (Asp20, Tyr78, Asn171 and Arg224) are also conserved with most BSHs or PVA from other bacteria. However, among these active sites, Tyr78 is sometimes replaced by Asn78 in some Lactobacillus spp. such as L. johnsonii ATCC 33200 and L. acidophilus. Replacement of Asn78 in BSH2-4 by Tyr78 does not, though, alter the nature of the active site in the catalytic process because the peptide NH atoms of Tyr78 or Asn78 residues play an important role in forming the oxyanion hole together with Asn171 Nd2 by providing an electron acceptor (Lambert et al. 2008a, b; Ren et al. 2011; Rossocha et al. 2005). All three recombinant BSHs in our strain had high relative activities for hydrolysis of both GDCA and TDCA; therefore they might recognize substrates predominantly at the amino acid moieties but not at the cholate moieties and this predication is consistent with the study of Rossocha et al. (2005). Although the BSHs of L. plantarum CGMCC 8198 have the same catalytic sites to L. plantarum WCFS1, the capabilities of substrate-hydrolysing are different. The recombinant BSH4 in this study exhibits a high ability for the hydrolysis of TDCA but the BSH4 of L. plantarum WCFS1 tends to GDCA (Lambert et al. 2008a, b). In our opinion, this difference might be due to the change of spatial protein structure, which was caused by other mutations. In order to further confirm the structure–activity relationship and the substrate binding pocket of BSHs in L. plantarum CGMCC 8198, the structural studies may be necessary. In summary, L. plantarum CGMCC 8198 may escape from cell death by bile salts in the intestine and also reduce plasma cholesterol in vivo through the hydrolysis activity of its BSHs. This work indicates that this novel probiotic stain could be used in the food or drug system to improve the health of patients suffering from cholesterol-related diseases.
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Biotechnol Lett (2014) 36:975–983 Acknowledgments We thank Dr. Hong-Peng He and Dr. Tao Wang for the assistance in editing the language. This study was financially supported by the 863 (Hi-tech research and development program of China) program under contract No. 2012AA022108 and No. 2008AA10Z336, and the Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (IRT1166).
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