Active efflux of bile acids in Lactobacillus reuteri CRL 1098

Abstract

Bile acids are powerful detergents which disorganizes the lipid bilayer structure of cellular membranes. One of the main factors involved in bile tolerance in Gram-negative bacteria, which are inherently more resistant to bile than, are efflux pumps that remove bile that gets through the outer membrane. The defenses mechanism of Gram-positive bacteria against bile, however, is still poorly understood. The lactic acid bacteria Lactobacillus (L.) reuteri, a member of the intestinal microbiota currently used as probiotic, showed a significant degree of resistance to the toxic action of bile acids (BA). We show in this work the presence of an active efflux of BA in the L. reuteri CRL 1098 strain and study whether the exporting mechanism is a primary transport mediated by ATP or by proton motive force (PMF). Everted vesicles of CRL 1098, prepared from stationary cells by disruption with several passages through a French pressure, were incubated for 20 min at 37 ºC in the presence of 2 mM conjugated and free BA [taurocholic (TCA) and cholic (CA) acids, respectively]. The experiments were carryied out at pH 5.2 and 6.5, using not energized vesicles, energized vesicles with 5 mM ATP, or deenergized vesicles (5 mM ATP plus 1 mM sodium vanadate or nigericine). In independent experiments, a proton gradient was generated trough the cell membrane (value of inside pH of 5.2 and outside pH of 6.5; and inversely). Remaining BA in the supernatant of samples removed after several times of incubation were determined by HPLC. Results showed that there was an ATP-dependent accumulation of BA in L. reuteri CRL 1098: both acids (CA and TCA) were incorporated by active transport and reached the maximum accumulation at 15 min (about 3 mM/mg protein). However, a small fraction of the CA was also able to diffuse passively through the vesicular membranes, even in the absence of ATP. These observations could be explained by differences in the pKa of both acids. The unconjugated BA, CA, has a pKa value of 5.5, so a significant portion of these compounds exists in lipophilic, uncharged forms, and should be able to traverse the lipid bilayer domains of the outer membrane. On the contrary, TCA, with a pKa value of 1.5, is a strong acid and is completely decoupled at the pHs tested, and, therefore, cannot diffuse freely across membranes. To our knowledge, our results are the first evidence of an active efflux of bile acid in probiotic bacteria L. reuteri CRL 1098.