Lorena Ruiz

Bile resistance mechanisms in Lactobacillus and Bifidobacterium.

Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Most of the probiotic bacteria currently available in the market belong to the genera Lactobacillus and Bifidobacterium, and specific health-promoting activities, such as treatment of diarrhea or amelioration of gastrointestinal discomfort, have been attributed to them. In order to be able to survive the gastrointestinal transit and transiently colonize our gut, these bacteria must be able to counteract the deleterious action of bile salts, which are the main components of bile. Bile salts are detergent-like biological substances synthesized in the liver from cholesterol. Host enzymes conjugate the newly synthesized free bile acids in the liver with the amino acids glycine or taurine, generating conjugated bile salts. These compounds are stored in the gall bladder and they are released into the duodenum during digestion to perform their physiological function, which is the solubilization of fat coming from diet. These bile salts possess strong antimicrobial activity, since they are able to disorganize the structure of the cell membrane, as well as trigger DNA damage. This means that bacteria inhabiting our intestinal tract must have intrinsic resistance mechanisms to cope with bile salts. To do that, Lactobacillus and Bifidobacterium display a variety of proteins devoted to the efflux of bile salts or protons, to modify sugar metabolism or to prevent protein misfolding. In this manuscript, we review and discuss specific bile resistance mechanisms, as well as the processes responsible for the adaptation of bifidobacteria and lactobacilli to bile.

How do bifidobacteria counteract environmental challenges? Mechanisms involved and physiological consequences

An effective response to stress is of paramount importance for probiotic bifidobacteria administered in foods, since it determines their performance as beneficial microorganisms. Firstly, bifidobacteria have to be resistant to the stress sources typical in manufacturing, including heating, exposure to low water activities, osmotic shock and presence of oxygen. Secondly, and once they are orally ingested, bifidobacteria have to overcome physiological barriers in order to arrive in the large intestine biologically active. These barriers are mainly the acid pH in the stomach and the presence of high bile salt concentrations in the small intestine. In addition, the large intestine is, in terms of microbial amounts, a densely populated environment in which there is an extreme variability in carbon source availability. For this reason, bifidobacteria harbours a wide molecular machinery allowing the degradation of a wide variety of otherwise non-digestible sugars. In this review, the molecular mechanisms allowing this bacterial group to favourably react to the presence of different stress sources are presented and discussed.

Inside the adaptation process of Lactobacillus delbrueckii subsp. lactis to bile

Progressive adaptation to bile might render some lactobacilli able to withstand physiological bile salt concentrations. In this work, the adaptation to bile was evaluated on previously isolated dairy strains of Lactobacillus delbrueckii subsp. lactis 200 and L. delbrueckii subsp. lactis 200+, a strain derived thereof with stable bile-resistant phenotype. The adaptation to bile was obtained by comparing cytosolic proteomes of both strains grown in the presence or absence of bile. Proteomics were complemented with physiological studies on both strains focusing on glycolytic end-products, the ability to adhere to the human intestinal epithelial cell line HT29-MTX and survival to simulated gastrointestinal conditions. Protein pattern comparison of strains grown with and without bile allowed us to identify 9 different proteins whose production was regulated by bile in both strains, and 17 proteins that showed differences in their levels between the parental and the bile-resistant derivative. These included general stress response chaperones, proteins involved in transcription and translation, in peptidoglycan/exopolysaccharide biosynthesis, in the lipid and nucleotide metabolism and several glycolytic and pyruvate catabolism enzymes. Differences in the level of metabolic end-products of the sugar catabolism were found between the strains 200 and 200+. A decrease in the adhesion of both strains to the intestinal cell line was detected in the presence of bile. In simulated gastric and intestinal juices, a protective effect was exerted by milk improving the survival of both microorganisms. These results indicate that bile tolerance in L. delbrueckii subsp. lactis involves several mechanisms responding to the deleterious impact of bile salts on bacterial physiology.

Role of extracellular transaldolase from bifidobacterium bifidum in mucin adhesion and aggregation

ABSTRACT The ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules from Bifidobacterium bifidum taxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12 B. bifidum strains. In four of them—B. bifidum LMG13195, DSM20456, DSM20239, and A8—the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation of B. bifidum A8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay of B. bifidum A8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface of B. bifidum A8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal from B. bifidum A8 was expressed in Lactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treated B. bifidum A8 cells. A recombinant L. lactis strain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface of B. bifidum, could act as an important colonization factor favoring its establishment in the gut.

The human intestinal microbiome at extreme ages of life. Dietary intervention as a way to counteract alterations

The intestinal microbiome is defined as the assembly of genomes from microorganisms inhabiting the gut. This microbial ecosystem regulates important functions of the host and its correct composition and functionality is essential for a “healthy status.” Metagenomic studies have highlighted variations of the intestinal microbiota as a function of age and diet. Colonization of the infant gut starts at birth and is influenced by feeding habits (formula vs. breast-feeding), birth mode and antibiotic exposure. The intestinal microbiota of full-term vaginally delivered breast-fed infants is considered the gold-standard, representing the reference for studies of alterations in other pediatric populations. At 2–3 years of age, the intestinal microbiota reaches a composition similar to adults, remaining without noticeable variations until senescence, when microbial instability and changes reappear. Here we summarize the current knowledge on intestinal microbiota alterations at extreme stages of life and tools for designing differentiated nutritional strategies by the use of probiotics, prebiotics and specific nutrients in order to restore a balanced microbiota and to improve immune and nutritional status.

Proteomics of stress response in Bifidobacterium.

Bifidobacteria are commensal microorganisms of the human gastrointestinal tract which are largely being used in functional foods. Some strains are considered as probiotics since they beneficially affect the composition and the metabolic activity of intestinal microbiota, as well as the health status of the host. The lack of genetic tools has hindered the development of functional genomic studies in bifidobacteria, like the identification of molecular mechanisms underlying their survival under different environmental challenges. Some of these experimental obstacles have been successfully overcome with the use of proteomics, a set of techniques that, when applied to microorganisms, are directed to the identification of all the proteins produced by the cells under a given physiological condition. The aim of this review is to discuss and summarize some of the current knowledge of the stress tolerance in bifidobacteria, mainly identified by two-dimensional electrophoresis coupled to mass spectrometry, and compare the most recent proteomic results with the currently available transcriptomic studies. The input and advantages of novel high throughput proteomic techniques are considered as well.

Factors involved in the colonization and survival of bifidobacteria in the gastrointestinal tract

The authors thank the Spanish Ministry of Science and Innovation (MICINN) and the ‘Plan Nacional I + D + i’ for the financial support of the research work through the projects AGL2010-14952 and RM2010-00012-00-00. B.S. was the recipient of a Juan de la Cierva postdoctoral contract, and I.G.-R. was the recipient of an FPI grant, from MICINN. L.R. had a JAE-Predoctoral contract, financed by CSIC.

Adaptation of bifidobacteria to the gastrointestinal tract and functional consequences

Members of the genus Bifidobacterium are considered to be important constituents of the microbiota of animals, from insects to mammals. They are gut commensals extensively used by the food industry as probiotic microorganisms, since some strains have been shown to have specific beneficial effects. However, the molecular processes underlying their functional capacities to promote a healthy status in the host, as well as those involved in survival, colonization and persistence of bifidobacteria in the gut, are far from being completely understood. This review summarizes the current knowledge on the mechanisms used by bifidobacteria to cope with gastrointestinal factors and to adapt to them, and discusses the advantages of the adaptive traits acquired by these microorganisms as a consequence of their interactions with the gastrointestinal tract environment, as well as the impact of such adaptations in the functional characteristics of bifidobacteria.

Discovering novel bile protection systems in Bifidobacterium breve UCC2003 through functional genomics.

ABSTRACT Tolerance of gut commensals to bile salt exposure is an important feature for their survival in and colonization of the intestinal environment. A transcriptomic approach was employed to study the response of Bifidobacterium breve UCC2003 to bile, allowing the identification of a number of bile-induced genes with a range of predicted functions. The potential roles of a selection of these bile-inducible genes in bile protection were analyzed following heterologous expression in Lactococcus lactis. Genes encoding three transport systems belonging to the major facilitator superfamily (MFS), Bbr_0838, Bbr_0832, and Bbr_1756, and three ABC-type transporters, Bbr_0406-0407, Bbr_1804-1805, and Bbr_1826-1827, were thus investigated and shown to provide enhanced resistance and survival to bile exposure. This work significantly improves our understanding as to how bifidobacteria respond to and survive bile exposure.

Molecular Clues To Understand the Aerotolerance Phenotype of Bifidobacterium animalis subsp. lactis

ABSTRACT Oxygen is one of the abiotic factors negatively affecting the survival of Bifidobacterium strains used as probiotics, mainly due to the induction of lethal oxidative damage. Aerobic conditions are present during the process of manufacture and storage of functional foods, and aerotolerance is a desired trait for bifidobacteria intended for use in industry. In the present study, the molecular response of Bifidobacterium animalis subsp. lactis IPLA4549 to aerobic conditions is presented. Molecular targets affected by oxygen were studied using two-dimensional electrophoresis (2DE) and quantitative reverse transcriptase (qRT) PCR. Globally, oxygen stress induced a shift in the glycolytic pathway toward the production of acetic acid with a concomitant increase in ATP formation. Several changes in the expression of genes coding for enzymes involved in redox reactions were detected, although the redox ratio remained unaltered. Interestingly, cells grown under aerobic conditions were characterized by higher activity of coproporphyrinogen III oxidase, which can directly detoxify molecular oxygen, and by higher NADH oxidase specific activity, which can oxidize NADH using hydrogen peroxide. In turn, this is in agreement with the glycolytic shift toward acetate production, in that more NADH molecules may be available due to the lower level of lactic acid formation. These findings further our ability to elucidate the mechanisms by which B. animalis copes with an oxygen-containing atmosphere.