Proteomic analysis of the probiotic Lactobacillus reuteri CRL1098 reveals novel tolerance biomarkers to bile acid-induced stress
Introduction
The successful development of functional foods depends on the careful selection of probiotic strains, whose bile resistance is an important selection criterion. Bile is secreted into the intestine, during the digestion process, where it plays a major role in lipid emulsification and absorption. It is an aqueous solution mainly composed of conjugated bile acids (BA) (approximately 50%), phospholipids, cholesterol and biliverdin (Begley, Gahan, & Hill, 2005). The concentration of bile usually varies between 0.2 and 2% after food ingestion, being higher after an intake of lipids (Begley et al., 2005). The hydrolysis of conjugated BA releases free BA and taurine or glycine. This phenomenon takes place due to the bile salt hydrolase (BSH) activity, an enzyme present exclusively in certain species of the intestinal microbiota (Chae, J., et al., 2013, Jayashree, S., et al., 2014, Joyce, S. A., et al., 2014a). Some researchers have suggested a possible relationship between BSH activity and BA resistance in many lactic acid bacteria (LAB) (Bustos, A. Y., et al., 2012, Joyce, S. A., et al., 2014b). In addition, BA are biological detergents that could disrupt the lipid bilayer structure of bacterial cellular membranes, induce protein misfolding, and cause oxidative damage to DNA and RNA and intracellular acidification(Begley, M., et al., 2005, Taranto, M. P., et al., 2006, Bustos, A. Y., et al., 2012, Lebeer, S., et al., 2010).
Lactobacillus (L.) reuteri is the most widely distributed Lactobacillus species in mammals and usually proposed for the design of functional food. L. reuteri CRL1098 is a probiotic bacterium with a proven hypocholesterolemic effect (Taranto, Medici, Perdigon, Ruiz Holgado, & Valdez, 2000) and ability to produce corrinoids with cobalamin activity (Molina, V., et al., 2012, Taranto, M. P., et al., 2003). Its ability to survive the passage of the intestinal tract, and hence resisting the toxic effect of BA, is a key factor specifically related to its probiotic function. The mechanisms of tolerance to bile stress are not fully understood and little is known about the protein expression profiles of L. reuteri strains in response to stress produced by BA. The aim of this study is to characterize, by means of two-dimensional gel electrophoresis (2DE) and mass spectrometry, the intracellular proteins differentially expressed by L. reuteri CRL1098 when this bacterium adapts to conjugated- and free-BA. In addition, the molecular regulation of the BSH enzyme in response to BA was evaluated to determine its contribution to cell tolerance. These data will improve our understanding of L. reuteri adaption to the intestinal tract passage considering the importance of evaluating resistance/response to bile in a probiotic strain as a prelude to its successful application in functional foods.
Section snippets
Bacterial strain and growth conditions
L. reuteri CRL1098 was grown on 200 mL MRS broth in the absence of BA (control culture) or in the presence of 1 mM of glycodeoxycholic acid (GDCA) or 1 mM deoxycholic acid (DCA) (both BA from Sigma Aldrich, St. Louis, MO, USA), equivalent to 0.05% (w/w) of GDCA and 0.04% (w/w) DCA.
Cells were harvested at the early-exponential phase (OD560 nm ∼ 0.9) by centrifugation at 10,000 × g for 10 min at 4 °C. The cells were then washed with 100 mM Tris–HCl buffer, pH 7.5 and the resulting cell pellets were stored at
Physiological response of L. reuteri CRL1098 to bile acids
Probiotics as natural members of intestinal microbiota must tolerate high stressful conditions prevailing throughout the gastrointestinal tract (GIT) such as different concentrations of chloride acid, pancreatic enzymes or bile, among others. The growth of L. reuteri CRL1098 in MRS containing 1 mM of DCA or 1 mM GDCA was compared with bacterial growth in BA-free medium. The results demonstrated that even when the DCA affected L. reuteri CRL1098 cell viability, this strain showed considerable
Conclusions
The present work reveals that conjugated (GDCA) and free (DCA) BA induce a deep metabolic reorganization in L. reuteri CRL1098 in order to achieve adaption and survival to stressful conditions such as those herein induced. This is reflected by differences in growth parameters as well as by differential expression of many proteins related to different metabolic categories. DCA clearly affected cell viability while in the presence of GDCA growth parameters were similar to the control conditions
Acknowledgments
This research has been supported by grants from CONICET(PIP2011-0100406) and SECyT (PICT2011 0175). Authors AM Almeida and S Fadda are indebted to COST action FA1002 — Proteomics in Farm Animals (http://www.cost-faproteomics.org/) for the network funding that made possible this collaboration between Argentinean and European researchers. Author AM Almeida finally acknowledges a RSTSM (Reciprocal Short Term Scientific Mission) from the COST office (COST-RSTSM-RA — Argentina—06463).
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