RESUMEN
Dairy industries apply selected lactococcal strains and mixed cultures to produce diverse fermented products with distinctive flavor and texture properties. Innovation of the starter culture functionality in cheese applications embraces natural biodiversity of the Lactococcus species to identify novel strains with alternative flavor or texture forming capacities and/or increased processing robustness and phage resistance. Mobile genetic elements (MGE), like integrative conjugative elements (ICEs) play an important role in shaping the biodiversity of bacteria. Besides the genes involved in the conjugation of ICEs from donor to recipient strains, these elements also harbor cargo genes that encode a wide range of functions. The definition of such cargo genes can only be achieved by accurate identification of the ICE boundaries (delimiting). Here, we delimited 25 ICEs in lactococcal genome sequences with low contig numbers using insertion-sites flanking single-copy core-genome genes as markers for each of the distinct ICE-integrases we identified previously within the conserved ICE-core genes. For ICEs in strains for which genome information with large numbers of contigs is available, we exemplify that CRISPR-Cas9 driven ICE-curing, followed by resequencing, allows accurate delimitation and cargo definition of ICEs. Finally, we compare and contrast the cargo gene repertoire of the 26 delimited lactococcal ICEs, identifying high plasticity among the cargo of lactococccal ICEs and a range of encoded functions that is of apparent industrial interest, including restriction modification, abortive infection, and stress adaptation genes.
Asunto(s)
Genoma Bacteriano , Lactococcus/genética , Secuencias Repetitivas Esparcidas/genética , Sistemas CRISPR-Cas , Conjugación GenéticaRESUMEN
Lactococcus lactis is widely applied by the dairy industry for the fermentation of milk into products such as cheese. Adaptation of L. lactis to the dairy environment often depends on functions encoded by mobile genetic elements (MGEs) such as plasmids. Other L. lactis MGEs that contribute to industrially relevant traits like antimicrobial production and carbohydrate utilization capacities belong to the integrative conjugative elements (ICE). Here we investigate the prevalence of ICEs in L. lactis using an automated search engine that detects colocalized, ICE-associated core-functions (involved in conjugation or mobilization) in lactococcal genomes. This approach enabled the detection of 36 candidate-ICEs in 69 L. lactis genomes. By phylogenetic analysis of conserved protein functions encoded in all lactococcal ICEs, these 36 ICEs could be classified in three main ICE-families that encompass 7 distinguishable ICE-integrases and are characterized by apparent modular-exchangeability and plasticity. Finally, we demonstrate that phylogenetic analysis of the conjugation-associated VirB4 ATPase function differentiates ICE- and plasmid-derived conjugation systems, indicating that conjugal transfer of lactococcal ICEs and plasmids involves genetically distinct machineries. Our genomic analysis and sequence-based classification of lactococcal ICEs creates a comprehensive overview of the conserved functional repertoires encoded by this family of MGEs in L. lactis, which can facilitate the future exploitation of the functional traits they encode by ICE mobilization to appropriate starter culture strains.
Asunto(s)
Lactococcus lactis , Lactococcus lactis/genética , Filogenia , Plásmidos/genética , Proteínas/metabolismo , Genoma , Conjugación Genética , Elementos Transponibles de ADNRESUMEN
The enzymatic repertoire of starter cultures belonging to the Lactococcus genus determines various important characteristics of fermented dairy products but might change in response to the substantial environmental changes in the manufacturing process. Assessing bacterial proteome adaptation in dairy and other food environments is challenging due to the high matrix-protein concentration and is even further complicated in particularly cheese by the high fat concentrations, the semi-solid state of that matrix, and the non-growing state of the bacteria. Here, we present bacterial harvesting and processing procedures that enable reproducible, high-resolution proteome determination in lactococcal cultures harvested from laboratory media, milk, and miniature Gouda cheese. Comparative proteome analysis of Lactococcus cremoris NCDO712 grown in laboratory medium and milk revealed proteome adaptations that predominantly reflect the differential (micro-)nutrient availability in these two environments. Additionally, the drastic environmental changes during cheese manufacturing only elicited subtle changes in the L. cremoris NCDO712 proteome, including modified expression levels of enzymes involved in flavour formation. The technical advances we describe offer novel opportunities to evaluate bacterial proteomes in relation to their performance in complex, protein- and/or fat-rich food matrices and highlight the potential of steering starter culture performance by preculture condition adjustments.
Asunto(s)
Queso , Productos Lácteos Cultivados , Lactococcus lactis , Animales , Proteoma/metabolismo , Fermentación , Queso/microbiología , Leche/microbiología , Lactococcus lactis/genética , Lactococcus lactis/metabolismoRESUMEN
Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit health benefits in the consumer. Lactiplantibacillus plantarum strains display high survival during transit through the mammalian gastrointestinal tract and were shown to have health-promoting properties. Growth on the fructose polysaccharide inulin is relatively uncommon in L. plantarum, and in this study we describe FosE, a plasmid-encoded ß-fructosidase of L. plantarum strain Lp900 which has inulin-hydrolyzing properties. FosE contains an LPxTG-like motif involved in sortase-dependent cell wall anchoring but is also (partially) released in the culture supernatant. In addition, we examined the effect of diet supplementation with inulin on the intestinal persistence of Lp900 in adult male Wistar rats in diets with distinct calcium levels. Inulin supplementation in high-dietary-calcium diets significantly increased the intestinal persistence of L. plantarum Lp900, whereas this effect was not observed upon inulin supplementation of the low-calcium diet. Moreover, intestinal persistence of L. plantarum Lp900 was determined when provided as a probiotic (by itself) or as a synbiotic (i.e., in an inulin suspension) in rats that were fed unsupplemented diets containing the different calcium levels, revealing that the synbiotic administration increased bacterial survival and led to higher abundance of L. plantarum Lp900 in rats, particularly in a low-calcium-diet context. Our findings demonstrate that inulin supplementation can significantly enhance the intestinal delivery of L. plantarum Lp900 but that this effect strongly depends on calcium levels in the diet.IMPORTANCE Synbiotics combine probiotics with prebiotics to synergistically elicit a health benefit in the consumer. Previous studies have shown that prebiotics can selectively stimulate the growth in the intestine of specific bacterial strains. In synbiotic supplementations the prebiotics constituent could increase the intestinal persistence and survival of accompanying probiotic strain(s) and/or modulate the endogenous host microbiota to contribute to the synergistic enhancement of the health-promoting effects of the synbiotic constituents. Our study establishes a profound effect of dietary-calcium-dependent inulin supplementation on the intestinal persistence of inulin-utilizing L. plantarum Lp900 in rats. We also show that in rats on a low-dietary-calcium regime, the survival and intestinal abundance of L. plantarum Lp900 are significantly increased by administering it as an inulin-containing synbiotic. This study demonstrates that prebiotics can enhance the intestinal delivery of specific probiotics and that the prebiotic effect is profoundly influenced by the calcium content of the diet.
Asunto(s)
Calcio de la Dieta/farmacología , Intestinos/microbiología , Inulina/farmacología , Lactobacillus plantarum , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dieta , Lactobacillus plantarum/efectos de los fármacos , Lactobacillus plantarum/enzimología , Lactobacillus plantarum/crecimiento & desarrollo , Masculino , Ratas Wistar , Simbióticos , beta-Fructofuranosidasa/química , beta-Fructofuranosidasa/genética , beta-Fructofuranosidasa/metabolismoRESUMEN
Synbiotics are food supplements that combine probiotics and prebiotics to synergistically elicit a health effect in humans. Lactobacillus plantarum exhibits remarkable genetic and phenotypic diversity, in particular in strain-specific carbohydrate utilization capacities, and several strains are marketed as probiotics. We have screened 77 L. plantarum strains for their abilities to utilize specific prebiotic fibers, revealing variable and strain-specific growth efficiencies on isomalto- and galactooligosaccharides. We identified a single strain within the screening panel that was able to effectively utilize inulin and fructooligosaccharides (FOS), which did not support efficient growth of the rest of the strains. In the panel we tested, we did not find strains that could utilize arabinoxylooligosaccharides or sulfated fucoidan. The strain-specific growth phenotype on isomaltooligosaccharides was further analyzed using high-performance anion-exchange chromatography, which revealed distinct substrate utilization phenotypes within the strain panel. The strain-specific phenotypes could be linked to the strains' genotypes by identifying gene clusters coding for carbohydrate membrane transport systems that are predicted to be involved in the utilization of isomaltose and other (unidentified) oligosaccharides in the isomaltooligosaccharide substrate.IMPORTANCE Synbiotics combine prebiotics and probiotics to synergistically enhance the health benefits associated with these ingredients. Lactobacillus plantarum is encountered as a natural inhabitant of the gastrointestinal tract, and specific strains are marketed as probiotics based on their strain-specific health-promoting activities. Strain-specific stimulation of growth through prebiotic substrates could enhance the persistence and/or activity of L. plantarumin situ Our study establishes a high-throughput screening model for prebiotic substrate utilization by individual strains of bacteria, which can be readily employed for synbiotic matchmaking approaches that aim to enhance the intestinal delivery of probiotics through strain-specific, selective growth stimulation.
Asunto(s)
Genes Bacterianos , Lactobacillus plantarum/genética , Lactobacillus plantarum/metabolismo , Oligosacáridos/metabolismo , Simbióticos , Fenotipo , PrebióticosRESUMEN
Lactic acid bacteria (LAB) are frequently used in food fermentation and are invaluable for the taste and nutritional value of the fermentation end-product. To gain a better understanding of underlying biochemical and microbiological mechanisms and cell-to-cell variability in LABs, single-molecule techniques such as single-particle tracking photo-activation localization microscopy (sptPALM) hold great promises but are not yet employed due to the lack of detailed protocols and suitable assays. Here, we qualitatively test various fluorescent proteins including variants that are photoactivatable and therefore suitable for sptPALM measurements in Lactococcus lactis, a key LAB for the dairy industry. In particular, we fused PAmCherry2 to dCas9 allowing the successful tracking of single dCas9 proteins, whilst the dCas9 chimeras bound to specific guide RNAs retained their gene silencing ability in vivo. The diffusional information of the dCas9 without any targets showed different mechanistic states of dCas9: freely diffusing, bound to DNA, or transiently interacting with DNA. The capability of performing sptPALM with dCas9 in L. lactis can lead to a better, general understanding of CRISPR-Cas systems as well as paving the way for CRISPR-Cas based interrogations of cellular functions in LABs.
Asunto(s)
Lactococcus lactis/aislamiento & purificación , Proteínas Luminiscentes/análisis , Sistemas CRISPR-Cas , Microscopía Fluorescente , Procesos FotoquímicosRESUMEN
We are facing a global metabolic health crisis provoked by an obesity epidemic. Here we report the human gut microbial composition in a population sample of 123 non-obese and 169 obese Danish individuals. We find two groups of individuals that differ by the number of gut microbial genes and thus gut bacterial richness. They contain known and previously unknown bacterial species at different proportions; individuals with a low bacterial richness (23% of the population) are characterized by more marked overall adiposity, insulin resistance and dyslipidaemia and a more pronounced inflammatory phenotype when compared with high bacterial richness individuals. The obese individuals among the lower bacterial richness group also gain more weight over time. Only a few bacterial species are sufficient to distinguish between individuals with high and low bacterial richness, and even between lean and obese participants. Our classifications based on variation in the gut microbiome identify subsets of individuals in the general white adult population who may be at increased risk of progressing to adiposity-associated co-morbidities.
Asunto(s)
Bacterias/aislamiento & purificación , Biomarcadores/metabolismo , Tracto Gastrointestinal/microbiología , Metagenoma , Adiposidad , Adulto , Bacterias/clasificación , Bacterias/genética , Índice de Masa Corporal , Estudios de Casos y Controles , Dieta , Dislipidemias/microbiología , Metabolismo Energético , Europa (Continente)/etnología , Femenino , Genes Bacterianos , Humanos , Inflamación/microbiología , Resistencia a la Insulina , Masculino , Metagenoma/genética , Obesidad/metabolismo , Obesidad/microbiología , Sobrepeso/metabolismo , Sobrepeso/microbiología , Filogenia , Delgadez/microbiología , Aumento de Peso , Pérdida de Peso , Población BlancaRESUMEN
CRISPR-Cas9 technology has been exploited for the removal or replacement of genetic elements in a wide range of prokaryotes and eukaryotes. Here, we describe the extension of the Cas9 application toolbox to the industrially important dairy species Lactococcus lactis The Cas9 expression vector pLABTarget, encoding the Streptocccus pyogenes Cas9 under the control of a constitutive promoter, was constructed, allowing plug and play introduction of short guide RNA (sgRNA) sequences to target specific genetic loci. Introduction of a pepN-targeting derivative of pLABTarget into L. lactis strain MG1363 led to a strong reduction in the number of transformants obtained, which did not occur in a pepN deletion derivative of the same strain, demonstrating the specificity and lethality of the Cas9-mediated double-strand breaks in the lactococcal chromosome. Moreover, the same pLABTarget derivative allowed the selection of a pepN deletion subpopulation from its corresponding single-crossover plasmid integrant precursor, accelerating the construction and selection of gene-specific deletion derivatives in L. lactis Finally, pLABTarget, which contained sgRNAs designed to target mobile genetic elements, allowed the effective curing of plasmids, prophages, and integrative conjugative elements (ICEs). These results establish that pLABTarget enables the effective exploitation of Cas9 targeting in L. lactis, while the broad-host-range vector used suggests that this toolbox could readily be expanded to other Gram-positive bacteria.IMPORTANCE Mobile genetic elements in Lactococcus lactis and other lactic acid bacteria (LAB) play an important role in dairy fermentation, having both positive and detrimental effects during the production of fermented dairy products. The pLABTarget vector offers an efficient cloning platform for Cas9 application in lactic acid bacteria. Targeting Cas9 toward mobile genetic elements enabled their effective curing, which is of particular interest in the context of potentially problematic prophages present in a strain. Moreover, Cas9 targeting of other mobile genetic elements enables the deciphering of their contribution to dairy fermentation processes and further establishment of their importance for product characteristics.
Asunto(s)
Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas/genética , Lactococcus lactis/genética , ARN Bacteriano/metabolismo , ARN Guía de Kinetoplastida/metabolismo , Proteínas Bacterianas/metabolismo , Proteína 9 Asociada a CRISPR/metabolismo , Fermentación , Lactobacillales/genética , Lactobacillales/metabolismo , Lactococcus lactis/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Streptococcus pyogenes/genéticaRESUMEN
Colorectal cancer risk is associated with diets high in red meat. Heme, the pigment of red meat, induces cytotoxicity of colonic contents and elicits epithelial damage and compensatory hyperproliferation, leading to hyperplasia. Here we explore the possible causal role of the gut microbiota in heme-induced hyperproliferation. To this end, mice were fed a purified control or heme diet (0.5 µmol/g heme) with or without broad-spectrum antibiotics for 14 d. Heme-induced hyperproliferation was shown to depend on the presence of the gut microbiota, because hyperproliferation was completely eliminated by antibiotics, although heme-induced luminal cytotoxicity was sustained in these mice. Colon mucosa transcriptomics revealed that antibiotics block heme-induced differential expression of oncogenes, tumor suppressors, and cell turnover genes, implying that antibiotic treatment prevented the heme-dependent cytotoxic micelles to reach the epithelium. Our results indicate that this occurs because antibiotics reinforce the mucus barrier by eliminating sulfide-producing bacteria and mucin-degrading bacteria (e.g., Akkermansia). Sulfide potently reduces disulfide bonds and can drive mucin denaturation and microbial access to the mucus layer. This reduction results in formation of trisulfides that can be detected in vitro and in vivo. Therefore, trisulfides can serve as a novel marker of colonic mucolysis and thus as a proxy for mucus barrier reduction. In feces, antibiotics drastically decreased trisulfides but increased mucin polymers that can be lysed by sulfide. We conclude that the gut microbiota is required for heme-induced epithelial hyperproliferation and hyperplasia because of the capacity to reduce mucus barrier function.
Asunto(s)
Colon/microbiología , Colon/patología , Dieta , Células Epiteliales/patología , Hemo/farmacología , Microbiota/efectos de los fármacos , Moco/metabolismo , Animales , Antibacterianos/farmacología , Antioxidantes/farmacología , Biomarcadores/metabolismo , Peso Corporal/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Colon/efectos de los fármacos , Recuento de Colonia Microbiana , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Heces/microbiología , Inmunohistoquímica , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Antígeno Ki-67/metabolismo , Masculino , Ratones Endogámicos C57BL , Modelos Biológicos , Moco/efectos de los fármacos , Sulfuros/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
In biotechnological workhorses like Streptococcus thermophilus and Bacillus subtilis, natural competence can be induced, which facilitates genetic manipulation of these microbes. However, in strains of the important dairy starter Lactococcus lactis, natural competence has not been established to date. However, in silico analysis of the complete genome sequences of 43 L. lactis strains revealed complete late competence gene sets in 2 L. lactis subsp. cremoris strains (KW2 and KW10) and at least 10 L. lactis subsp. lactis strains, including the model strain IL1403 and the plant-derived strain KF147. The remainder of the strains, including all dairy isolates, displayed genomic decay in one or more of the late competence genes. Nisin-controlled expression of the competence regulator comX in L. lactis subsp. lactis KF147 resulted in the induction of expression of the canonical competence regulon and elicited a state of natural competence in this strain. In contrast, comX expression in L. lactis NZ9000, which was predicted to encode an incomplete competence gene set, failed to induce natural competence. Moreover, mutagenesis of the comEA-EC operon in strain KF147 abolished the comX-driven natural competence, underlining the involvement of the competence machinery. Finally, introduction of nisin-inducible comX expression into nisRK-harboring derivatives of strains IL1403 and KW2 allowed the induction of natural competence in these strains also, expanding this phenotype to other L. lactis strains of both subspecies.IMPORTANCE Specific bacterial species are able to enter a state of natural competence in which DNA is taken up from the environment, allowing the introduction of novel traits. Strains of the species Lactococcus lactis are very important starter cultures for the fermentation of milk in the cheese production process, where these bacteria contribute to the flavor and texture of the end product. The activation of natural competence in this industrially relevant organism can accelerate research aiming to understand industrially relevant traits of these bacteria and can facilitate engineering strategies to harness the natural biodiversity of the species in optimized starter strains.
RESUMEN
Intestinal barrier integrity is a prerequisite for homeostasis of mucosal function, which is balanced to maximise absorptive capacity, while maintaining efficient defensive reactions against chemical and microbial challenges. Evidence is mounting that disruption of epithelial barrier integrity is one of the major aetiological factors associated with several gastrointestinal diseases, including infection by pathogens, obesity and diabetes, necrotising enterocolitis, irritable bowel syndrome and inflammatory bowel disease. The notion that specific probiotic bacterial strains can affect barrier integrity fuelled research in which in vitro cell lines, animal models and clinical trials are used to assess whether probiotics can revert the diseased state back to homeostasis and health. This review catalogues and categorises the lines of evidence available in literature for the role of probiotics in epithelial integrity and, consequently, their beneficial effect for the reduction of gastrointestinal disease symptoms.
Asunto(s)
Enfermedades Intestinales/prevención & control , Intestinos/efectos de los fármacos , Intestinos/fisiología , Probióticos/farmacología , Animales , HumanosRESUMEN
Our knowledge of species and functional composition of the human gut microbiome is rapidly increasing, but it is still based on very few cohorts and little is known about variation across the world. By combining 22 newly sequenced faecal metagenomes of individuals from four countries with previously published data sets, here we identify three robust clusters (referred to as enterotypes hereafter) that are not nation or continent specific. We also confirmed the enterotypes in two published, larger cohorts, indicating that intestinal microbiota variation is generally stratified, not continuous. This indicates further the existence of a limited number of well-balanced host-microbial symbiotic states that might respond differently to diet and drug intake. The enterotypes are mostly driven by species composition, but abundant molecular functions are not necessarily provided by abundant species, highlighting the importance of a functional analysis to understand microbial communities. Although individual host properties such as body mass index, age, or gender cannot explain the observed enterotypes, data-driven marker genes or functional modules can be identified for each of these host properties. For example, twelve genes significantly correlate with age and three functional modules with the body mass index, hinting at a diagnostic potential of microbial markers.
Asunto(s)
Bacterias/clasificación , Intestinos/microbiología , Metagenoma , Bacterias/genética , Técnicas de Tipificación Bacteriana , Biodiversidad , Biomarcadores/análisis , Europa (Continente) , Heces/microbiología , Femenino , Humanos , Masculino , Metagenómica , FilogeniaRESUMEN
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.
Asunto(s)
Dieta con Restricción de Grasas , Proteínas en la Dieta/farmacocinética , Sacarosa en la Dieta/farmacocinética , Microbioma Gastrointestinal/fisiología , Obesidad/metabolismo , Tasa de Supervivencia , Animales , Proteínas en la Dieta/administración & dosificación , Proteínas en la Dieta/efectos adversos , Sacarosa en la Dieta/efectos adversos , Femenino , Estudios Longitudinales , Ratones , Ratones Endogámicos C57BL , Obesidad/etiologíaRESUMEN
The ability of bacteria to adapt to diverse environmental conditions is well-known. The process of bacterial adaptation to a niche has been linked to large changes in the genome content, showing that many bacterial genomes reflect the constraints imposed by their habitat. However, some highly versatile bacteria are found in diverse habitats that almost share nothing in common. Lactobacillus plantarum is a lactic acid bacterium that is found in a large variety of habitat. With the aim of unravelling the link between evolution and ecological versatility of L. plantarum, we analysed the genomes of 54 L. plantarum strains isolated from different environments. Comparative genome analysis identified a high level of genomic diversity and plasticity among the strains analysed. Phylogenomic and functional divergence studies coupled with gene-trait matching analyses revealed a mixed distribution of the strains, which was uncoupled from their environmental origin. Our findings revealed the absence of specific genomic signatures marking adaptations of L. plantarum towards the diverse habitats it is associated with. This suggests fundamentally similar trends of genome evolution in L. plantarum, which occur in a manner that is apparently uncoupled from ecological constraint and reflects the nomadic lifestyle of this species.
Asunto(s)
Adaptación Fisiológica/genética , Genoma Bacteriano/genética , Lactobacillus plantarum/genética , Lactobacillus plantarum/aislamiento & purificación , Secuencia de Bases , Evolución Biológica , Hibridación Genómica Comparativa , ADN Bacteriano/genética , Ecosistema , Ambiente , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Lactobacillus plantarum/fisiología , Fenotipo , Análisis de Secuencia de ADNRESUMEN
UNLABELLED: 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.
Asunto(s)
Genes Bacterianos , Lactobacillus plantarum/metabolismo , Redes y Vías Metabólicas/genética , Polisacáridos Bacterianos/metabolismo , Células Cultivadas , Análisis Mutacional de ADN , Tracto Gastrointestinal/microbiología , Eliminación de Gen , Humanos , Factores Inmunológicos/metabolismo , Lactobacillus plantarum/genética , Lactobacillus plantarum/inmunología , Lactobacillus plantarum/fisiología , Leucocitos Mononucleares/inmunología , Viabilidad Microbiana , Polisacáridos Bacterianos/genética , Probióticos/metabolismoRESUMEN
The gut microbiota provide important stimuli to the human innate and adaptive immune system and co-mediate metabolic and immune homeostasis. Probiotic bacteria can be regarded as part of the natural human microbiota, and have been associated with improving homeostasis, albeit with different levels of success. Composition of microbiota, probiotic strain identity, and host genetic differences may account for differential modulation of immune responses by probiotics. Here, we review the mechanisms of immunomodulating capacities of specific probiotic strains, the responses they can induce in the host, and how microbiota and genetic differences between individuals may co-influence host responses and immune homeostasis.
Asunto(s)
Enfermedades Inflamatorias del Intestino/inmunología , Enfermedades Inflamatorias del Intestino/microbiología , Intestinos/inmunología , Lactobacillus/inmunología , Microbiota/inmunología , Probióticos , Animales , Interacción Gen-Ambiente , Homeostasis/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunidad Mucosa , Inmunomodulación , Enfermedades Inflamatorias del Intestino/terapia , Intestinos/microbiologíaRESUMEN
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.
Asunto(s)
Lactobacillales/metabolismo , Polisacáridos/metabolismo , Prebióticos , Adhesión Bacteriana , Biopelículas/crecimiento & desarrollo , Células Epiteliales/microbiología , Factores Inmunológicos/metabolismo , Lactobacillales/fisiologíaRESUMEN
OBJECTIVE: Repetitive interaction with microbial stimuli renders epithelial cells (ECs) hyporesponsive to microbial stimulation. Previously, we have reported that buccal ECs from a subset of paediatric patients with Crohn's disease are not hyporesponsive and spontaneously released chemokines. We now aimed to identify kinetics and mechanisms of acquisition of hyporesponsiveness to microbial stimulation using primary human buccal epithelium. DESIGN: Buccal ECs collected directly after birth and in later stages of life were investigated. Chemokine release and regulatory signalling pathways were studied using primary buccal ECs and the buccal EC line TR146. Findings were extended to the intestinal mucosa using murine model systems. RESULTS: Directly after birth, primary human buccal ECs spontaneously produced the chemokine CXCL-8 and were responsive to microbial stimuli. Within the first weeks of life, these ECs attained hyporesponsiveness, associated with inactivation of the NF-κB pathway and upregulation of the novel NF-κB inhibitor SLPI but no other known NF-κB inhibitors. SLPI protein was abundant in the cytoplasm and the nucleus of hyporesponsive buccal ECs. Knock-down of SLPI in TR146-buccal ECs induced loss of hyporesponsiveness with increased NF-κB activation and subsequent chemokine release. This regulatory mechanism extended to the intestine, as colonisation of germfree mice elicited SLPI expression in small intestine and colon. Moreover, SLPI-deficient mice had increased chemokine expression in small intestinal and colonic ECs. CONCLUSIONS: We identify SLPI as a new player in acquisition of microbial hyporesponsiveness by buccal and intestinal epithelium in the first weeks after microbial colonisation.
Asunto(s)
Envejecimiento/inmunología , Epitelio/inmunología , Epitelio/microbiología , Mucosa Bucal/citología , Mucosa Bucal/microbiología , Inhibidor Secretorio de Peptidasas Leucocitarias/metabolismo , Adulto , Animales , Células Cultivadas , Quimiocina CXCL2/metabolismo , Regulación hacia Abajo , Epitelio/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Tolerancia Inmunológica , Lactante , Recién Nacido , Interleucina-8/metabolismo , Mucosa Intestinal/citología , Mucosa Intestinal/microbiología , Ratones , Persona de Mediana Edad , FN-kappa B/metabolismo , Peptidoglicano/farmacologíaRESUMEN
Nutrient scarcity is a common condition in nature, but the resulting extremely low growth rates (below 0.025 h(-1) ) are an unexplored research area in Bacillus subtilis. To understand microbial life in natural environments, studying the adaptation of B. subtilis to near-zero growth conditions is relevant. To this end, a chemostat modified for culturing an asporogenous B. subtilisâ sigF mutant strain at extremely low growth rates (also named a retentostat) was set up, and biomass accumulation, culture viability, metabolite production and cell morphology were analysed. During retentostat culturing, the specific growth rate decreased to a minimum of 0.00006 h(-1) , corresponding to a doubling time of 470 days. The energy distribution between growth and maintenance-related processes showed that a state of near-zero growth was reached. Remarkably, a filamentous cell morphology emerged, suggesting that cell separation is impaired under near-zero growth conditions. To evaluate the corresponding molecular adaptations to extremely low specific growth, transcriptome changes were analysed. These revealed that cellular responses to near-zero growth conditions share several similarities with those of cells during the stationary phase of batch growth. However, fundamental differences between these two non-growing states are apparent by their high viability and absence of stationary phase mutagenesis under near-zero growth conditions.
Asunto(s)
Aclimatación/fisiología , Bacillus subtilis/crecimiento & desarrollo , Transcriptoma/genética , Aclimatación/genética , Bacillus subtilis/citología , Bacillus subtilis/genética , Biomasa , Perfilación de la Expresión Génica , Glucosa/metabolismo , Transcripción GenéticaRESUMEN
Experimental evolution is a powerful approach to unravel how selective forces shape microbial genotypes and phenotypes. To this date, the available examples focus on the adaptation to conditions specific to the laboratory. The lactic acid bacterium Lactococcus lactis naturally occurs on plants and in dairy environments, and it is proposed that dairy strains originate from the plant niche. Here we investigate the adaptation of a L. lactis strain isolated from a plant to a dairy niche by propagating it for 1000 generations in milk. Two out of three independently evolved strains displayed significantly increased acidification rates and biomass yields in milk. Genome resequencing, revealed six, seven, and 28 mutations in the three strains, including point mutations in loci related to amino acid biosynthesis and transport and in the gene encoding MutL, which is involved in DNA mismatch repair. Two strains lost a conjugative transposon containing genes important in the plant niche but dispensable in milk. A plasmid carrying an extracellular protease was introduced by transformation. Although improving growth rate and growth yield significantly, the plasmid was rapidly lost. Comparative transcriptome and phenotypic analyses confirmed that major physiological changes associated with improved growth in milk relate to nitrogen metabolism and the loss or down-regulation of several pathways involved in the utilization of complex plant polymers. Reproducing the transition from the plant to the dairy niche through experimental evolution revealed several genome, transcriptome, and phenotype signatures that resemble those seen in strains isolated from either niche.