Showing papers by "Michiel Kleerebezem published in 2016"

Stress Physiology of Lactic Acid Bacteria

Abstract: Lactic acid bacteria (LAB) are important starter, commensal, or pathogenic microorganisms. The stress physiology of LAB has been studied in depth for over 2 decades, fueled mostly by the technological implications of LAB robustness in the food industry. Survival of probiotic LAB in the host and the potential relatedness of LAB virulence to their stress resilience have intensified interest in the field. Thus, a wealth of information concerning stress responses exists today for strains as diverse as starter (e.g., Lactococcus lactis), probiotic (e.g., several Lactobacillus spp.), and pathogenic (e.g., Enterococcus and Streptococcus spp.) LAB. Here we present the state of the art for LAB stress behavior. We describe the multitude of stresses that LAB are confronted with, and we present the experimental context used to study the stress responses of LAB, focusing on adaptation, habituation, and cross-protection as well as on self-induced multistress resistance in stationary phase, biofilms, and dormancy. We also consider stress responses at the population and single-cell levels. Subsequently, we concentrate on the stress defense mechanisms that have been reported to date, grouping them according to their direct participation in preserving cell energy, defending macromolecules, and protecting the cell envelope. Stress-induced responses of probiotic LAB and commensal/pathogenic LAB are highlighted separately due to the complexity of the peculiar multistress conditions to which these bacteria are subjected in their hosts. Induction of prophages under environmental stresses is then discussed. Finally, we present systems-based strategies to characterize the "stressome" of LAB and to engineer new food-related and probiotic LAB with improved stress tolerance.

Exopolysaccharides produced by lactic acid bacteria: from health-promoting benefits to stress tolerance mechanisms

Abstract: A wide range of lactic acid bacteria (LAB) is able to produce capsular or extracellular polysaccharides, with various chemical compositions and properties. Polysaccharides produced by LAB alter the rheological properties of the matrix in which they are dispersed, leading to typically viscous and “ropy” products. Polysaccharides are involved in several mechanisms such as prebiosis and probiosis, tolerance to stress associated to food process, and technological properties of food. In this paper, we summarize the beneficial properties of exopolysaccharides (EPS) produced by LAB with particular attention to prebiotic properties and to the effect of exopolysaccharides on the LAB-host interaction mechanisms, such as bacterial tolerance to gastrointestinal tract conditions, ability of ESP-producing probiotics to adhere to intestinal epithelium, their immune-modulatory activity, and their role in biofilm formation. The pro-technological aspect of exopolysaccharides is discussed, focusing on advantageous applications of EPS in the food industry, i.e., yogurt and gluten-free bakery products, since it was found that these microbial biopolymers positively affect the texture of foods. Finally, the involvement of EPS in tolerance to stress conditions that are commonly encountered in fermented beverages such as wine is discussed.

Strain-Specific Features of Extracellular Polysaccharides and Their Impact on Lactobacillus plantarum-Host Interactions.

Abstract: Lactobacilli are found in diverse environments and are widely applied as probiotic, health-promoting food supplements. Polysaccharides are ubiquitously present on the cell surface of lactobacilli and are considered to contribute to the species- and strain-specific probiotic effects that are typically observed. Two Lactobacillus plantarum strains, SF2A35B and Lp90, have an obvious ropy phenotype, implying high extracellular polysaccharide (EPS) production levels. In this work, we set out to identify the genes involved in EPS production in these L. plantarum strains and to demonstrate their role in EPS production by gene deletion analysis. A model L. plantarum strain, WCFS1, and its previously constructed derivative that produced reduced levels of EPS were included as reference strains. The constructed EPS-reduced derivatives were analyzed for the abundance and sugar compositions of their EPS, revealing cps2-like gene clusters in SF2A35B and Lp90 responsible for major EPS production. Moreover, these mutant strains were tested for phenotypic characteristics that are of relevance for their capacity to interact with the host epithelium in the intestinal tract, including bacterial surface properties as well as survival under the stress conditions encountered in the gastrointestinal tract (acid and bile stress). In addition, the Toll-like receptor 2 (TLR2) signaling and immunomodulatory capacities of the EPS-negative derivatives and their respective wild-type strains were compared, revealing strain-specific impacts of EPS on the immunomodulatory properties. Taken together, these experiments illustrate the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. IMPORTANCE This study evaluates the role of extracellular polysaccharides that are produced by different strains of Lactobacillus plantarum in the determination of the cell surface properties of these bacteria and their capacity to interact with their environment, including their signaling to human host cells. The results clearly show that the consequences of removal of these polysaccharides are very strain specific, illustrating the diverse and unpredictable roles of these polysaccharides in the environmental interactions of these bacterial strains. In the context of the use of lactobacilli as health-promoting probiotic organisms, this study exemplifies the importance of strain specificity.

The Variable Regions of Lactobacillus rhamnosus Genomes Reveal the Dynamic Evolution of Metabolic and Host-Adaptation Repertoires

Abstract: Lactobacillus rhamnosus is a diverse Gram-positive species with strains isolated from different ecological niches. Here, we report the genome sequence analysis of 40 diverse strains of L. rhamnosus and their genomic comparison, with a focus on the variable genome. Genomic comparison of 40 L. rhamnosus strains discriminated the conserved genes (core genome) and regions of plasticity involving frequent rearrangements and horizontal transfer (variome). The L. rhamnosus core genome encompasses 2,164 genes, out of 4,711 genes in total (the pan-genome). The accessory genome is dominated by genes encoding carbohydrate transport and metabolism, extracellular polysaccharides (EPS) biosynthesis, bacteriocin production, pili production, the cas system, and the associated clustered regularly interspaced short palindromic repeat (CRISPR) loci, and more than 100 transporter functions and mobile genetic elements like phages, plasmid genes, and transposons. A clade distribution based on amino acid differences between core (shared) proteins matched with the clade distribution obtained from the presence-absence of variable genes. The phylogenetic and variome tree overlap indicated that frequent events of gene acquisition and loss dominated the evolutionary segregation of the strains within this species, which is paralleled by evolutionary diversification of core gene functions. The CRISPR-Cas system could have contributed to this evolutionary segregation. Lactobacillus rhamnosus strains contain the genetic and metabolic machinery with strain-specific gene functions required to adapt to a large range of environments. A remarkable congruency of the evolutionary relatedness of the strains' core and variome functions, possibly favoring interspecies genetic exchanges, underlines the importance of gene-acquisition and loss within the L. rhamnosus strain diversification.

Effect of a long-term high-protein diet on survival, obesity development, and gut microbiota in mice

Abstract: Female C57BL/6J mice were fed a regular low-fat diet or high-fat diets combined with either high or low protein-to-sucrose ratios during their entire lifespan to examine the long-term effects on obesity development, gut microbiota, and survival. Intake of a high-fat diet with a low protein/sucrose ratio precipitated obesity and reduced survival relative to mice fed a low-fat diet. By contrast, intake of a high-fat diet with a high protein/sucrose ratio attenuated lifelong weight gain and adipose tissue expansion, and survival was not significantly altered relative to low-fat-fed mice. Our findings support the notion that reduced survival in response to high-fat/high-sucrose feeding is linked to obesity development. Digital gene expression analyses, further validated by qPCR, demonstrated that the protein/sucrose ratio modulated global gene expression over time in liver and adipose tissue, affecting pathways related to metabolism and inflammation. Analysis of fecal bacterial DNA using the Mouse Intestinal Tract Chip revealed significant changes in the composition of the gut microbiota in relation to host age and dietary fat content, but not the protein/sucrose ratio. Accordingly, dietary fat rather than the protein/sucrose ratio or adiposity is a major driver shaping the gut microbiota, whereas the effect of a high-fat diet on survival is dependent on the protein/sucrose ratio.

Identification of Commensal Species Positively Correlated with Early Stress Responses to a Compromised Mucus Barrier.

Abstract: Background: Our aims were (1) to correlate changes in the microbiota to intestinal gene expression before and during the development of colitis in Muc2(-/-) mice and (2) to investigate whether the heterozygote Muc2(+/-) mouse would reveal host markers of gut barrier stress. Methods: Colon histology, transcriptomics, and microbiota profiling of faecal samples was performed on wild type, Muc2(+/-), and Muc2(-/-) mice at 2, 4, and 8 weeks of age. Results: Muc2(-/-) mice develop colitis in proximal colon after weaning, resulting in inflammatory and adaptive immune responses, and expression of genes associated with human inflammatory bowel disease. Muc2(+/-) mice do not develop colitis, but produce a thinner mucus layer. The transcriptome of Muc2(+/-) mice revealed differential expression of genes participating in mucosal stress responses and exacerbation of a transient inflammatory state around the time of weaning. Young wild type and Muc2(+/-) mice have a more constrained group of bacteria as compared with the Muc2(-/-) mice, but at 8 weeks the microbiota composition is more similar in all mice. At all ages, microbiota composition discriminated the groups of mice according to their genotype. Specific bacterial clusters correlated with altered gene expression responses to stress and bacteria, before colitis development, including colitogenic members of the genus Bacteroides. Conclusions: The abundance of Bacteroides pathobionts increased before histological signs of pathology suggesting they may play a role in triggering the development of colitis. The Muc2(+/-) mouse produces a thinner mucus layer and can be used to study mucus barrier stress in the absence of colitis.

Functional Profiling of Unfamiliar Microbial Communities Using a Validated De Novo Assembly Metatranscriptome Pipeline.

Abstract: BACKGROUND: Metatranscriptomic landscapes can provide insights in functional relationships within natural microbial communities. Analysis of complex metatranscriptome datasets of these communities poses a considerable bioinformatic challenge since they are non-restricted with a varying number of participating strains and species. For RNA-Seq data a standard approach is to align the generated reads to a set of closely related reference genomes. This only works well for microbial communities for which a near complete catalogue of reference genomes is available at a small evolutionary distance. In this study, we focus on the design of a validated de novo metatranscriptome assembly pipeline for single-end Illumina RNA-Seq data to obtain functional and taxonomic profiles of murine microbial communities. RESULTS: The here developed de novo assembly metatranscriptome pipeline combined rRNA removal, IDBA-UD assembler, functional annotation and taxonomic classification. Different assemblers were tested and validated using RNA-Seq data from an in silico generated mock community and in vivo RNA-Seq data from a restricted microbial community taken from a mouse model colonized with Altered Schaedler Flora (ASF). Precision and recall of resulting gene expression, functional and taxonomic profiles were compared to those obtained with a standard alignment method. The validated pipeline was subsequently used to generate expression profiles from non-restricted cecal communities of four C57BL/6J mice fed on a high-fat high-protein diet spiked with an RNA-Seq data set from a well-characterized human sample. The spike in control was used to estimate precision and recall at assembly, functional and taxonomic level of non-restricted communities. CONCLUSIONS: A generic de novo assembly pipeline for metatranscriptome data analysis was designed for microbial ecosystems, which can be applied for microbial metatranscriptome analysis in any chosen niche

Complete Genome Sequence of Streptococcus salivarius HSISS4, a Human Commensal Bacterium Highly Prevalent in the Digestive Tract

Abstract: The human commensal bacterium Streptococcus salivarius plays a major role in the equilibrium of microbial communities of the digestive tract. Here, we report the first complete genome sequence of a Streptococcus salivarius strain isolated from the small intestine, namely, HSISS4. Its circular chromosome comprises 1,903 coding sequences and 2,100,988 nucleotides.

Characterization of the transcriptional regulation of the tarIJKL locus involved in ribitol-containing wall teichoic acid biosynthesis in Lactobacillus plantarum.

Abstract: Lactobacillus plantarum strains produce either glycerol (Gro)- or ribitol (Rbo)-backbone wall teichoic acid (WTA) (Gro-WTA and Rbo-WTA, respectively). The strain WCFS1 has been shown to be able to activate the tarIJKL locus involved in Rbo-WTA synthesis when the tagD1F1F2 locus for Gro-WTA synthesis was mutated, resulting in switching of the native Gro-WTA into Rbo-WTA. Here, we identify a regulator involved in the WTA backbone alditol switching and activation of the tarIJKL locus. Promoter reporter assays of the tarI promoter (Ptar) demonstrated its activity in the Rbo-WTA-producing mutant derivative (ΔtagF1-2) but not in the parental strain WCFS1. An electrophoresis mobility shift assay using a Ptar nucleotide fragment showed that this fragment bound to Ptar-binding protein(s) in a cell-free extract of WCFS1. Three proteins were subsequently isolated using Ptar bound to magnetic beads. These proteins were isolated efficiently from the lysate of WCFS1 but not from the lysate of its ΔtagF1-2 derivative, and were identified as redox-sensitive transcription regulator (Lp_0725), catabolite control protein A (Lp_2256) and TetR family transcriptional regulator (Lp_1153). The role of these proteins in Ptar regulation was investigated by knockout mutagenesis, showing that the Δlp_1153 mutant expressed the tarI gene at a significantly higher level, supporting its role as a repressor of the tarIJKL locus. Notably, the Δlp_1153 mutation also led to reduced expression of the tagF1 gene. These results show that Lp_1153 is a regulatory factor that plays a role in WTA alditol switching in Lb. plantarum WCFS1 and we propose to rename this gene/protein wasR/WasR, for WTA alditol switch regulator.

Draft Genome Sequence of Lactobacillus plantarum SF2A35B.

Abstract: The lactic acid bacterium Lactobacillus plantarum is intensively studied as a model probiotic species. Here, we present the draft genome sequence of the exopolysaccharide-producing strain SF2A35B.