Project description:Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.
Project description:Much remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer. Bifidobacterium species colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, select Bifidobacterium strains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived species Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans. In silico data revealed that B. dentium lacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating that B. dentium could not use naive mucin glycans as primary carbon sources in vitro To examine B. dentium mucus modulation in vivo, Swiss Webster germfree mice were monoassociated with live or heat-killed B. dentium Live B. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markers Krüppel-like factor 4 (Klf4), Trefoil factor 3 (Tff3), Relm-?, Muc2, and several glycosyltransferases compared to both heat-killed B. dentium and germfree counterparts. Likewise, live B. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining. In vitro, B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified that B. dentium-secreted products, such as ?-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates that B. dentium is capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCE Microbe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe, Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike other Bifidobacterium species, B. dentium does not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of using B. dentium and similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.
Project description:Members of the Bifidobacterium dentium species are usually identified in the oral cavity of humans and associated with the development of plaque and dental caries. Nevertheless, they have also been detected from fecal samples, highlighting a widespread distribution among mammals. To explore the genetic variability of this species, we isolated and sequenced the genomes of 18 different B. dentium strains collected from fecal samples of several primate species and an Ursus arctos. Thus, we investigated the genomic variability and metabolic abilities of the new B. dentium isolates together with 20 public genome sequences. Comparative genomic analyses provided insights into the vast metabolic repertoire of the species, highlighting 19 glycosyl hydrolases families shared between each analyzed strain. Phylogenetic analysis of the B. dentium taxon, involving 1140 conserved genes, revealed a very close phylogenetic relatedness among members of this species. Furthermore, low genomic variability between strains was also confirmed by an average nucleotide identity analysis showing values higher than 98.2%. Investigating the genetic features of each strain, few putative functional mobile elements were identified. Besides, a consistent occurrence of defense mechanisms such as CRISPR-Cas and restriction-modification systems may be responsible for the high genome synteny identified among members of this taxon.
Project description:<h4>Background</h4>Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium.<h4>Results</h4>B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment.<h4>Conclusions</h4>Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.
Project description:Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although <i>Bifidobacterium</i> can beneficially modulate mucin production, little work has been done investigating the effects of <i>Bifidobacterium</i> on goblet cell ER stress. We hypothesized that secreted factors from <i>Bifidobacterium dentium</i> downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that <i>B. dentium</i> secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. <i>B. dentium</i> cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, <i>B. dentium</i> supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that <i>B. dentium</i> supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress <i>in vivo</i>, we first examined mono-association of <i>B. dentium</i> in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that <i>B. dentium</i> can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live <i>B. dentium</i> or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in <i>B. dentium-</i> and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both <i>B. dentium-</i> and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by <i>B. dentium-</i>secreted compounds.
Project description:<h4>Background</h4>Accumulating evidence indicates that the gut microbiota can synthesize neurotransmitters as well as impact host-derived neurotransmitter levels. In the past, it has been challenging to decipher which microbes influence neurotransmitters due to the complexity of the gut microbiota.<h4>Methods</h4>To address whether a single microbe, <i>Bifidobacterium dentium,</i> could regulate important neurotransmitters, we examined <i>Bifidobacteria</i> genomes and explored neurotransmitter pathways in secreted cell-free supernatant using LC-MS/MS. To determine if <i>B. dentium</i> could impact neurotransmitters in vivo, we mono-associated germ-free mice with <i>B. dentium</i> ATCC 27678 and examined fecal and brain neurotransmitter concentrations.<h4>Results</h4>We found that <i>B. dentium</i> possessed the enzymatic machinery to generate γ-aminobutyric acid (GABA) from glutamate, glutamine, and succinate. Consistent with the genome analysis, we found that <i>B. dentium</i> secreted GABA in a fully defined microbial media and elevated fecal GABA in <i>B. dentium</i> mono-associated mice compared to germ-free controls. We also examined the tyrosine/dopamine pathway and found that <i>B. dentium</i> could synthesize tyrosine, but could not generate L-dopa, dopamine, norepinephrine, or epinephrine. In vivo, we found that <i>B. dentium</i> mono-associated mice had elevated levels of tyrosine in the feces and brain.<h4>Conclusions</h4>These data indicate that <i>B. dentium</i> can contribute to in vivo neurotransmitter regulation.
Project description:We present the draft genome sequences of two Bifidobacterium dentium strains isolated from a fecal extract for fecal microbiota transplantation at a hospital in the Republic of Korea. Phylogenetic and functional analyses were performed to understand the physiological characteristics and functions of Bifidobacterium spp. in the human intestine.
Project description:<h4>Background</h4>Recurrent abdominal pain is a common and costly health-care problem attributed, in part, to visceral hypersensitivity. Increasing evidence suggests that gut bacteria contribute to abdominal pain perception by modulating the microbiome-gut-brain axis. However, specific microbial signals remain poorly defined. γ-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter and a key regulator of abdominal and central pain perception from peripheral afferent neurons. Although gut bacteria are reported to produce GABA, it is not known whether the microbial-derived neurotransmitter modulates abdominal pain.<h4>Methods</h4>To investigate the potential analgesic effects of microbial GABA, we performed daily oral administration of a specific Bifidobacterium strain (B. dentiumATCC 27678) in a rat fecal retention model of visceral hypersensitivity, and subsequently evaluated pain responses.<h4>Key results</h4>We demonstrate that commensal Bifidobacterium dentium produces GABA via enzymatic decarboxylation of glutamate by GadB. Daily oral administration of this specific Bifidobacterium (but not a gadB deficient) strain modulated sensory neuron activity in a rat fecal retention model of visceral hypersensitivity.<h4>Conclusions & inferences</h4>The functional significance of microbial-derived GABA was demonstrated by gadB-dependent desensitization of colonic afferents in a murine model of visceral hypersensitivity. Visceral pain modulation represents another potential health benefit attributed to bifidobacteria and other GABA-producing species of the intestinal microbiome. Targeting GABAergic signals along this microbiome-gut-brain axis represents a new approach for the treatment of abdominal pain.