1 Northern Advancement Center for Science & Technology, Kita 7 Nishi 2, Kita-ku, Sapporo 060-0807, Japan
2 Snow Brand Milk Products Co., Ltd, 1-1-2, Minamidai, Kawagoe 350-1165, Japan
3 Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands
4 Laboratory of Applied Microbiology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589, Japan
5 Laboratory of Microbial Resources and Ecology, Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589, Japan
Correspondence
Atsushi Yokota
yokota{at}chem.agr.hokudai.ac.jp
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ABSTRACT |
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Present address: Ciphergen Biosystems K.K., Yokohama 240-0005, Japan.
Present address: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK.
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INTRODUCTION |
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Within the human intestinal microbiota, the lactobacilli and bifidobacteria have attracted much attention with regard to their potential probiotic effects. Although many Lactobacillus and Bifidobacterium species have been associated with various health-promoting and beneficial properties (Ouwehand et al., 2002), their interactions with free bile acids are not well characterized. The growth inhibition of intestinal bacteria by free bile acids has been demonstrated (Binder et al., 1975
), but the effects of free bile acids on the physiology of intestinal bacteria have not been elucidated. In our previous reports (Kurdi et al., 2000
; Yokota et al., 2000
), we showed that Lactococcus lactis actively extrudes CA from the cell in an ATP-dependent manner, whereas various Lactobacillus species from the intestine, dairy products and other environments are capable of accumulating CA when they are energized by glucose. The mechanism underlying CA accumulation seems to be not transporter-mediated, but depends on the diffusion of hydrophobic CA across the bacterial cell membrane according to the transmembrane proton gradient (
pH, alkaline interior), which is formed upon energization with glucose. These findings led us to investigate the interactions of CA with bifidobacteria in the intestines of infants and healthy adults. In addition, we studied the effects on CA accumulation of short-chain fatty acids (SCFAs), which are normally present in the human large intestine as a mixture of acetate, propionate and butyrate.
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METHODS |
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When the effects of the SCFAs were examined, the sodium salts were added to a final concentration of 117 mM, i.e. 66 mM acetate, 26 mM propionate and 25 mM butyrate. These concentrations approximate the respective levels of the acids in the ascending colon (Cummings, 1997). The effect of 39 mM SCFAs with the same component ratio was also investigated, to check the dose response. The SCFA mixtures were added 8 min after energization with glucose; the addition of the SCFA mixtures did not change the pH of the medium.
Calculation of the accumulated CA.
The absolute amount of CA that was associated with the cells was expressed in nmol (mg protein)-1. The CA accumulation factor, which is defined as the ratio of the internal CA concentration to the external CA concentration, was also calculated. Calculation of the internal CA concentration was based on the assumption of an internal cell volume of 3 µl (mg protein)-1 (Poolman et al., 1987). Non-specific binding of CA to the cell surface and/or to the cell membrane was estimated from the positive deviation of the calculated internal CA concentration of the control series (no glucose added; de-energized with ionophores) from the extracellular CA concentration of 0·116 mM. The non-specific binding value estimated in this way was subtracted from the calculated internal CA concentration values in the energized series. Thus, the accumulation factor was obtained from these corrected intracellular CA concentrations, where the accumulation factor for the control series was set at 1·0.
The protein content of the cell suspensions was determined using the DC Protein Assay Kit (Bio-Rad) according to the manufacturer's instructions and BSA as the standard. The cell suspensions were boiled for 5 min in 1 M NaOH and then centrifuged; the resulting supernatants were used in the assays.
Measurement of intracellular pH.
Internal pH measurements were performed as described previously (Kurdi et al., 2000), using the internally conjugated fluorescent pH probe carboxyfluorescein succinimidyl ester (cFSE; Molecular Probes) (Breeuwer et al., 1996
). Briefly, the cells were cultured until mid-exponential phase, harvested and washed twice in Buffer 1. The cells were resuspended to an OD660 value of approximately 0·5 in Buffer 3 and incubated at 37 °C for 30 min in the presence of the precursor probe carboxyfluorescein diacetate succinimidyl ester. To eliminate unbound probe, the cells were incubated with glucose for 1 h and then washed once in Buffer 3. The cells were subsequently resuspended in Buffer 3, and the intracellular pH measurements were carried out. The effects of SCFAs on the internal pH were examined by the addition of SCFA mixtures at final total concentrations of 117 or 39 mM.
Transmembrane electrical potential () measurements.
Changes in during energization were monitored using the fluorescent dye 3,3'-dipropylthiadicarbocyanine iodide [DiSC3(5); Molecular Probes], which is a cationic probe that crosses the cell membrane, and the fluorescence of which is quenched as the membrane potential develops (negative interior). The harvested cells were washed twice with ice-cold Buffer 4 (Buffer 1 that contained 65 U catalase ml-1 instead of peroxidase), then resuspended in Buffer 5 (Buffer 3 with 65 U catalase ml-1 in place of peroxidase), to an OD660 value of approximately 10, and stored on ice. The replacement of peroxidase with catalase was important for reproducible measurements of
in the bifidobacteria because (i) peroxidase quenched the fluorescence of the DiSC3(5) probe, even before energization of the cells (see below), while catalase did not have this effect, and (ii) the addition of peroxidase or catalase was critical for bifidobacterial metabolism of glucose under experimental anaerobic conditions. The cells were added to a stirred cuvette that contained Buffer 5 (final OD660 value of 0·05) and DiSC3(5) (final concentration of 0·5 µM). Glucose (10 mM final concentration) was then added under anaerobic conditions (mixed gas was introduced into the cuvette headspace), to energize the cells. Fluorescence measurements were performed with an LS50B fluorimeter (Perkin Elmer) with excitation and emission wavelengths of 651 and 675 nm, respectively (slit widths of 4·0 nm).
CA metabolism by bifidobacteria.
The Bifidobacterium strains were cultured in 3 ml of MRS broth, as described in the transport experiment section, while the positive control, E. lentum-like strain c-25, was grown in GAM Broth Nissui (Nissui Pharmaceutical). Both of these media contained 0·15 mM sodium cholate; the cultures were incubated for 48 h under anaerobic conditions (mixed gas). The culture broths were acidified with concentrated HCl to pH 2, and the bile acids were extracted with ethyl acetate. The bile acids were separated by TLC using Silica gel 60 (Merck) and cyclohexane/ethyl acetate/acetic acid (7 : 23 : 3, v/v; Eneroth, 1963
) as the solvent. The bile acid spots on the TLC plate were visualized by spraying with the colouring reagent 5 % (w/v) phosphomolybdic acid {H3[P(Mo3O10)4].nH2O}, which was dissolved in ethanol, and then heated in an oven at 110 °C for 10 min.
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RESULTS |
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To further confirm the involvement of pH in the accumulation process, the internal pH of the JCM 1192T cells was measured with the fluorescence probe cFSE (Fig. 1b
). The internal pH started to increase (from pH 7·15 to around pH 7·5) 5 min after energization by glucose. This pH level was maintained until the addition of ionophores. The formation of the
pH coincided with CA accumulation (Fig. 1a
). The addition of valinomycin resulted in an increase in the internal pH, while the addition of nigericin abolished
pH (Fig. 1b
). These changes in internal pH and
pH may be responsible for the alteration of accumulated CA levels in the JCM 1192T cells upon the addition of ionophores (Fig. 1a
).
Monitoring of the changes of using DiSC3(5) (Fig. 1c
) revealed that the development of
upon energization with glucose and CA accumulation occurred simultaneously. However, nigericin addition increased the
, while valinomycin addition totally abolished it. These results clearly demonstrate that the
pH component and not the
component of the proton motive force is the driving force behind the CA accumulation process.
Measurements of the internal pH values of eight Bifidobacterium strains with cFSE revealed a positive correlation between the CA accumulation factors and the internal pH values of the respective strains (Table 1). The higher the pH gradient (i.e.
pH), the higher the accumulation factor in most cases, which confirms that
pH is the driving force behind CA accumulation. The theoretical accumulation factors, which were calculated from the measured internal pH values using the HendersonHasselbalch equation (pH=pKa+log[A-]/[HA], where the pKa of CA was 6·4), were lower than the actual accumulation factors (Table 1
). It is possible that active CA transporters contributed to the CA accumulation in certain strains (e.g. JCM 1192T) that had large differences between their measured and predicted accumulation factors. However, in strains with smaller differences between their predicted and measured accumulation factors, CA may have accumulated solely as the result of diffusion through the membrane, followed by
pH-dependent dissociation.
Effect of SCFAs on CA accumulation
Various mixtures of sodium acetate, sodium propionate and sodium butyrate, at final total concentrations of 117 mM (which corresponds to the concentration of these SCFAs in the ascending colon) or 39 mM, were used to test the effect of SCFAs on CA accumulation. CA accumulation in JCM 1192T cells was reduced by at least 50 % in the presence of the 117 mM SCFA mixture (Fig. 2a), as compared to cells that were incubated in the absence of SCFAs, while the 39 mM SCFA mixture produced a less pronounced reduction (
20 %) in CA accumulation. The addition of nigericin further decreased CA accumulation, which suggests that a certain
pH level was maintained in the JCM 1192T cells in the presence of 117 mM SCFAs. These SCFAs are weak acids with pKa values of 4·75, 4·87 and 4·81 for acetic, propionic and butyric acid, respectively. These weak acids, as is the case with CA, can be accumulated in bacterial cells (Russell, 1991
), and can theoretically reduce the internal pH of bacterial cells (Diez-Gonzalez & Russell, 1997
). As expected, measurements of the internal pH changes of energized JCM 1192T cells upon the addition of the SCFA mixtures revealed decreases in the internal pH (Fig. 2b, c
). The
pH levels were reduced by about 60 and 22 % by the addition of SCFA mixtures at 117 mM (Fig. 2b
) and 39 mM (Fig. 2c
), respectively. These reductions correspond to the reductions in the amounts of accumulated CA in JCM 1192T cells following treatment with the SCFA mixtures. These results indicate that in the presence of SCFAs, acidification of the intracellular environment and the subsequent decrease in
pH reduce CA accumulation.
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DISCUSSION |
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As is the case in lactobacilli (Kurdi et al., 2000), the results presented here indicate that the driving force for CA accumulation in bifidobacteria is the
pH component of the proton motive force, and not the
component (Fig. 1
). We hypothesize that CA accumulation in bifidobacteria occurs in the following way. (i) The hydrophobic CA molecules diffuse into the cytoplasm of the energized bifidobacterial cell and dissociate according to the ratio given by the HendersonHasselbalch equation under the given cytoplasmic pH value. (ii) Since the cytoplasmic pH value is higher than that outside (i.e.
pH), more dissociation takes place in the cytoplasm than in the external medium. The resulting negatively charged cholate anions cannot pass the membrane due to their polarity, and thus they are trapped inside the cell. (iii) CA influx continues until the concentration of the protonated CA molecule equilibrates on both sides of the cell membrane. In this situation, the total amount of CA (the sum of the protonated and dissociated species) is higher in the cytoplasm than in the external medium. (iv) The intracellular concentration of CA remains higher than that of the external environment, until the cells are energized and the CA concentration gradient across the membrane disappears, along with the disappearance of
pH. It is worth mentioning that substantial discrepancies were observed in some strains between the values for the measured CA accumulation factors and the values that were predicted by the above mechanism, i.e. the latter values were lower than the former values. Therefore, we cannot discount the possibility that an active CA uptake mechanism (e.g. driven by
pH) exists.
SCFA production by intestinal bacteria is a very important process in the large intestine, and provides energy for enterocytes (Cummings & Macfarlane, 1997). One of the major benefits of the fermentation of prebiotics is that it yields SCFAs (Cummings et al., 2001
), which in turn decrease the intestinal pH. CA accumulation was impaired when mixtures of SCFAs (117 or 39 mM) were added to the experimental reaction mixture (Fig. 2
), although the residual amounts of accumulated CA in these cells were clearly higher than those found in the non-energized control cells. The presence of SCFAs at the physiological concentrations found in the colon appears to exert a severe environmental stress, from the bioenergetical standpoint, on bifidobacterial cells. Even in the presence of SCFAs, CA accumulation may occur in energized Bifidobacterium cells, and this process may operate in vivo. This hypothesis was strengthened by our results which indicate that B. breve JCM 1192T accumulated CA even when external CA concentrations were 1·0 and 2·0 mM (around its physiological concentrations; estimated from Ewe & Karbach, 1989
). Moreover, CA accumulation at 1·0 mM external CA concentration was observed in JCM 1192T cells even in the presence of 117 mM SCFAs by a factor of 2·4 (data not shown).
Our experiments on CA metabolism by bifidobacteria revealed that Bifidobacterium strains were unable to chemically modify the CA molecule (Fig. 3), which is in agreement with a previous report (Takahashi & Morotomi, 1994). This absence of any chemical modification of CA concurs with our hypothetical mechanism for CA accumulation, which suggests that CA accumulation by bifidobacteria results from the co-existence of a membrane
pH and a weak acid in the same environment. The participation of a putative active CA uptake system appears to be unlikely, since the tested bifidobacteria did not utilize CA. Therefore, CA accumulation appears to be the result of energization.
The conjugated bile acid taurocholic acid is not accumulated in the BSH-negative Lactobacillus salivarius subsp. salicinius strain JCM 1044 (Kurdi et al., 2000) due to its hydrophilicity (pKa=1·4). Thus, only unconjugated free bile acids are accumulated in lactobacilli and bifidobacteria. According to the published distributions of BSH activities, most of the bifidobacterial strains are BSH-positive (Tanaka et al., 1999
). Therefore, in bifidobacteria, conjugated bile acids seem to be the source of free bile acids, which are supposed to be formed inside the cells from conjugated bile acids by BSH activities (Tanaka et al., 2000
). It is possible that the CA that is formed from taurocholic acid and glycocholic acid is kept inside bifidobacterial cells in the intestine, for as long as the bacteria are energized.
One possible consequence of CA entrapment in bifidobacterial cells would be the decreased formation of deoxycholic acid in the large intestine. The bifidobacteria do not metabolize CA (Fig. 3; Takahashi & Morotomi, 1994
), and thus are unable to produce deoxycholic acid following CA accumulation. Deoxycholic acid and lithocholic acid, which are formed via 7
-dehydroxylation from CA and chenodeoxycholic acid, respectively, by certain intestinal Clostridium and Eubacterium species (Baron & Hylemon, 1997
), are cytotoxic and possible tumour promoters (Reddy et al., 1976
; Reddy & Watanabe, 1979
). Thus, the accumulation of CA may contribute to the decreased occurrence of colon carcinogenesis. Another possible impact of CA accumulation is a decrease in recycled CA during enterohepatic circulation due to the enhanced excretion of CA from the human host via the faeces. Under these conditions, the synthesis of bile acids from blood cholesterol increases, to compensate for the lost amounts of bile acids, thereby decreasing the blood cholesterol level. Although these features appear quite attractive, experimental evidence for the probiotic relevance of CA accumulation in bifidobacterial cells is lacking. Therefore, in vivo experiments that evaluate these possibilities are urgently needed.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 29 March 2003;
accepted 28 April 2003.
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