Mechanism of growth inhibition by free bile acids in lactobacilli and bifidobacteria

Abstract

The effects of the free bile acids (FBAs) cholic acid (CA), deoxycholic acid (DCA), and chenodeoxycholic acid on the bioenergetics and growth of lactobacilli and bifidobacteria were investigated. It was found that these FBAs reduced the internal pH levels of these bacteria with rapid and stepwise kinetics and, at certain concentrations, dissipated DeltapH. The bile acid concentrations that dissipated DeltapH corresponded with the MICs for the selected bacteria. Unlike acetate, propionate, and butyrate, FBAs dissipated the transmembrane electrical potential (DeltaPsi). In Bifidobacterium breve JCM 1192, the synthetic proton conductor pentachlorophenol (PCP) dissipated DeltapH with a slow and continuous kinetics at a much lower concentration than FBAs did, suggesting the difference in mode of action between FBAs and true proton conductors. Membrane damage assessed by the fluorescence method and a viability decrease were also observed upon exposure to CA or DCA at the MIC but not to PCP or a short-chain fatty acid mixture. Loss of potassium ion was observed at CA concentrations more than 2 mM (0.4x MIC), while leakage of other cellular components increased at CA concentrations more than 4 mM (0.8 x MIC). Additionally, in experiments with membrane phospholipid vesicles extracted from Lactobacillus salivarius subsp. salicinius JCM 1044, CA and DCA at the MIC collapsed the DeltapH with concomitant leakage of intravesicular fluorescent pH probe, while they did not show proton conductance at a lower concentration range (e.g., 0.2x MIC). Taking these observations together, we conclude that FBAs at the MIC disturb membrane integrity and that this effect can lead to leakage of proton (membrane DeltapH and DeltaPsi dissipation), potassium ion, and other cellular components and eventually cell death.

FIG. 1.

Effects of CA (a), DCA (b), and CDCA (c) on the internal pH of B. breve JCM 1192. Cells (A₆₆₀ of ∼0.5) were preloaded with cFSE and energized with 10 mM glucose in buffer B. The respective FBAs were then added to the indicated final concentrations. Nigericin (200 nM) was added to check the dissipation of the ΔpH. The data shown in this and the following figures are representative of at least three experiments that gave similar results.

FIG. 2.

Effects of sodium acetate (a), sodium propionate (b), and sodium butyrate (c) on the membrane potential of B. breve JCM 1192. Washed cells (A₆₆₀ of ∼0.05) were added to the cuvette, which contained the DiSC₃(5) probe (0.5 μM) in buffer D. The cells were energized with glucose (10 mM), followed by the addition of the respective SCFAs at the indicated final concentrations. Valinomycin (5 nM) was added to check the dissipation of the ΔΨ. The fluorescence intensity is expressed in arbitrary units (a.u.).

FIG. 3.

Effects of CA (a), DCA (b), and CDCA (c) on the membrane potential of B. breve JCM 1192. The experimental procedures were the same as those described in the legend to Fig. 2, except that respective FBAs were added instead of SCFAs, at the indicated final concentrations.

FIG. 4.

Effects of PCP on the membrane potential (a) and internal pH (b) of L. salivarius subsp. salicinius JCM 1044. (a) The experimental procedures were the same as those described in the legend to Fig. 2, except that PCP was added instead of SCFAs at the indicated final concentrations. (b) The experimental procedures were the same as those described in the legend to Fig. 1, except that PCP was added instead of FBAs at the indicated final concentrations.

FIG. 5.

Effects of PCP (a), CA (b), and DCA (c) on the internal pH of membrane vesicles of L. salivarius subsp. salicinius JCM 1044. An artificial ΔpH was established by the addition of 28 μl of 2 N KOH to the membrane vesicle suspension in 2 ml of 150 mM potassium phosphate buffer, which altered the external pH from 6.5 to about 6.8. The chemical compounds were added at the indicated final concentrations. Nigericin (100 nM) was added to dissipate the preexisting ΔpH.

FIG. 6.

Effects of lower concentrations of CA (a) and DCA (b) on the internal pH of membrane vesicles of L. salivarius subsp. salicinius JCM 1044. Artificial ΔpH was established by the addition of 10 μl 2 N KOH to the membrane vesicle suspension in 2 ml 50 mM potassium phosphate buffer, which altered the external pH from 6.5 to about 6.8. The other experimental conditions were the same as those described in the legend to Fig. 5.

FIG. 7.

Measurement of the leakage of K⁺ (a) and other cellular materials (b) from B. breve JCM 1192 upon exposure to CA for 15 min (a) and 3 h (b). Cells (A₆₆₀ of ∼0.6) were incubated with various concentrations of CA in 150 mM sodium phosphate buffer (pH 6.5) containing 1 mM MgSO₄, 1.0 U/ml horseradish peroxidase, and 10 mM glucose. The K⁺ concentration (a) and the UV absorbance (b) at 260 nm (•) and 280 nm (○) of the cell-free supernatant were determined spectroscopically.