Elsevier

Food Research International

Volume 77, Part 3, November 2015, Pages 599-607
Food Research International

Proteomic analysis of the probiotic Lactobacillus reuteri CRL1098 reveals novel tolerance biomarkers to bile acid-induced stress

https://doi.org/10.1016/j.foodres.2015.10.001Get rights and content

Highlights

  • Protein expression in L. reuteri CRL1098 under bile acid stress was evaluated.

  • 25 protein spots were differentially expressed in response to bile acids.

  • The proteome of the probiotic strain revealed novel tolerance biomarkers to bile acids.

  • Bile salt hydrolase (BSH) regulation in response to bile acids was analyzed by qRT-PCR.

  • The bsh gene was up-regulated in the presence of the conjugated bile acid.

Abstract

Lactobacillus (L.) reuteri CRL1098 is a probiotic bacterium with a proven hypocholesterolemic effect, moderate immune stimulant effect and ability to produce cobalamin. The CRL1098 strain survives the passage through the gastrointestinal tract where the exposure to bile acids (BA) causes deleterious effects. In order to characterize the molecular mechanisms through which L. reuteri CRL1098 adapts to bile, its proteomic response was evaluated in the presence of conjugated (glycodeoxycholic acid-GDCA-) and free (deoxycholic acid-DCA-) bile acids (BA). Cell growth inhibition was observed only in the presence of DCA. Two-dimensional gel electrophoresis coupled to mass spectrometry allowed us to identify 25 protein spots differentially expressed in response to both BA. The main functional categories assigned to the proteins were metabolism of nucleotides and glycerolipids, transcription and translation, pH homeostasis and stress-responses. Remarkably, cytosine triphosphate(CTP) synthetase, enzyme related to the repair of oxidative DNA, was over-expressed in the presence of GDCA and significantly repressed by DCA; also three proteins related to protein transcription and translation were over expressed in the presence of the conjugated BA and one, was repressed by the free BA. This differential expression could explain the delayed growth of the cells challenged with the free BA and the unaffected growth in the presence of GDCA. Moreover, some general stress proteins were triggered in the presence of both BA. In addition, the bile salt hydrolase (BSH) enzyme regulation in response to BA was analyzed using real time-PCR to determine its contribution to cell tolerance. An up-regulation of the bsh gene in response to BA was observed, suggesting that this enzyme could be a specific biomarker of bile adaption in L. reuteri CRL1098. The present work proposes that BA induce a complex physiological response in L. reuteri and provide new insights into the mechanisms involved in BA tolerance.

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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    Present address: Ross University School of Veterinary Medicine, PO box 334, Basseterre, St. Kitts and Nevis (West Indies).

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