A GH89 human α-N-acetylglucosaminidase (hNAGLU) homologue from gut microbe Bacteroides thetaiotaomicron capable of hydrolyzing heparosan oligosaccharides.
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ABSTRACT:
SUBMITTER: Yang X
PROVIDER: S-EPMC8225759 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Thiamine (vitamin B1) is an essential cofactor for all organisms. Humans primarily acquire thiamine through their diet, and thiamine deficiencies have adverse neurological effects. However, the role gut microbes play in modulating thiamine availability is poorly understood, and little is known about how thiamine impacts the stability of microbial gut communities. To investigate thiamine's role in the gut, we utilized the model gut microbe Bacteroides thetaiotaomicron. Transcriptome sequencing (RNA-seq) revealed a global downregulation of thiamine and amino acid biosynthesis, glycolysis, and purine metabolism when thiamine was present. Using genetic mutants with thiamine biosynthesis and transport locus mutations, we determined both systems were critical for growth in thiamine-deficient medium. The defect in the double transport mutant suggests an uncharacterized feedback mechanism between thiamine transport and biosynthesis in B. thetaiotaomicron. Mutant phenotypes were recapitulated during pairwise competitions, reinforcing the importance of encoding versatile thiamine acquisition mechanisms when thiamine concentrations are variable. In addition, liquid chromatography-mass spectrometry (LC-MS) analyses corroborate that exogenous thiamine levels affect the internal thiamine pool of B. thetaiotaomicron. Furthermore, we computationally examined the ability of other gut microbes to acquire thiamine and identified lineage-specific differences in thiamine acquisition strategies. Among the Bacteroidetes, the capacities for both thiamine transport and biosynthesis are common. Together, these data show that thiamine acquisition mechanisms used by B. thetaiotaomicron not only are critical for its physiology and fitness but also provide the opportunity to model how other gut microbes may respond to the shifting availability of thiamine in the gut. IMPORTANCE Variation in the ability of gut microbes to transport, synthesize, and compete for vitamin B1 (thiamine) is expected to impact the structure and stability of the microbiota, and ultimately this variation may have both direct and indirect effects on human health. Our study identifies the diverse strategies employed by gut Bacteroidetes to acquire thiamine. We demonstrate how the presence or absence of thiamine biosynthesis or transport dramatically affects the abundance of B. thetaiotaomicron in a competitive environment. This study adds further evidence that altering the presence or concentrations of water-soluble vitamins such as thiamine may be an effective method for manipulating gut community composition. In turn, targeted thiamine delivery could be used therapeutically to alter dysbiotic communities linked to disease. Author Video: An author video summary of this article is available.
| S-EPMC5613172 | biostudies-literature
Project description:Background and aimsAs the importance of gut-brain interactions increases, understanding how specific gut microbes interact with the enteric nervous system (ENS), which is the first point of neuronal exposure becomes critical. Our aim was to understand how the dominant human gut bacterium Bacteroides thetaiotaomicron (Bt) regulates anatomical and functional characteristics of the ENS.MethodsNeuronal cell populations, as well as enteroendocrine cells, were assessed in proximal colonic sections using fluorescent immunohistochemistry in specific pathogen-free (SPF), germ-free (GF) and Bt conventionalized-germ-free mice (Bt-CONV). RNA expression of tight junction proteins and toll-like receptors (TLR) were measured using qPCR. Colonic motility was analyzed using in vitro colonic manometry.ResultsDecreased neuronal and vagal afferent innervation observed in GF mice was normalized by Bt-CONV with increased neuronal staining in mucosa and myenteric plexus. Bt-CONV also restored expression of nitric oxide synthase expressing inhibitory neurons and of choline acetyltransferase and substance P expressing excitatory motor neurons comparable to those of SPF mice. Neurite outgrowth and glial cells were upregulated by Bt-CONV. RNA expression of tight junction protein claudin 3 was downregulated while TLR2 was upregulated by Bt-CONV. The enteroendocrine cell subtypes L-cells and enterochromaffin cells were reduced in GF mice, with Bt-CONV restoring L-cell numbers. Motility as measured by colonic migrating motor complexes (CMMCs) increased in GF and Bt-CONV.ConclusionBt, common gut bacteria, is critical in regulating enteric neuronal and enteroendocrine cell populations, and neurogenic colonic activity. This highlights the potential use of this resident gut bacteria for maintaining healthy gut function.
| S-EPMC7524364 | biostudies-literature
Project description:Disruption of the human gut microbiota by antibiotics can lead to Clostridium difficile (CD)-associated diarrhea. CD overgrowth and elevated CD toxins result in gut inflammation. Herein, we report that a gut symbiont, Bacteroides thetaiotaomicron (BT), suppressed CD toxin production. The suppressive components are present in BT culture supernatant and are both heat- and proteinase K-resistant. Transposon-based mutagenesis indicated that the polysaccharide metabolism of BT is involved in the inhibitory effect. Among the genes identified, we focus on the methylerythritol 4-phosphate pathway gene gcpE, which supplies the isoprenoid backbone to produce the undecaprenyl phosphate lipid carrier that transports oligosaccharides across the membrane. Polysaccharide fractions prepared from the BT culture suppressed CD toxin production in vitro; the inhibitory effect of polysaccharide fractions was reduced in the gcpE mutant (ΔgcpE). The inhibitory effect of BT-derived polysaccharide fraction was abrogated by lysozyme treatment, indicating that cellwall-associated glycans are attributable to the inhibitory effect. BT-derived polysaccharide fraction did not affect CD toxin gene expression or intracellular toxin levels. An autolysis assay showed that CD cell autolysis was suppressed by BT-derived polysaccharide fraction, but the effect was reduced with that of ΔgcpE. These results indicate that cell wall-associated glycans of BT suppress CD toxin release by inhibiting cell autolysis.
| S-EPMC7918992 | biostudies-literature
Project description:Pectins are a major dietary nutrient source for the human gut microbiota. The prominent gut microbe Bacteroides thetaiotaomicron was recently shown to encode the founding member (BT1017) of a new family of pectin methylesterases essential for the metabolism of the complex pectin rhamnogalacturonan-II (RG-II). However, biochemical and structural knowledge of this family is lacking. Here, we showed that BT1017 is critical for the metabolism of an RG-II-derived oligosaccharide ΔBT1017oligoB generated by a BT1017 deletion mutant (ΔBT1017) during growth on carbohydrate extract from apple juice. Structural analyses of ΔBT1017oligoB using a combination of enzymatic, mass spectrometric, and NMR approaches revealed that it is a bimethylated nonaoligosaccharide (GlcA-β1,4-(2-O-Me-Xyl-α1,3)-Fuc-α1,4-(GalA-β1,3)-Rha-α1,3-Api-β1,2-(Araf-α1,3)-(GalA-α1,4)-GalA) containing components of the RG-II backbone and its side chains. We showed that the catalytic module of BT1017 adopts an α/β-hydrolase fold, consisting of a central twisted 10-stranded β-sheet sandwiched by several α-helices. This constitutes a new fold for pectin methylesterases, which are predominantly right-handed β-helical proteins. Bioinformatic analyses revealed that the family is dominated by sequences from prominent genera of the human gut microbiota, including Bacteroides and Prevotella Our re-sults not only highlight the critical role played by this family of enzymes in pectin metabolism but also provide new insights into the molecular basis of the adaptation of B. thetaiotaomicron to the human gut.
| S-EPMC7939467 | biostudies-literature
Project description:Bacteria of the genus Bacteroides are common members of the human intestinal microbiota and important degraders of polysaccharides in the gut. Among them, the species Bacteroides thetaiotaomicron has emerged as the model organism for functional microbiota research. Here, we use differential RNA sequencing (dRNA-seq) to generate a single-nucleotide resolution transcriptome map of B. thetaiotaomicron grown under defined laboratory conditions. An online browser, called 'Theta-Base' ( www.helmholtz-hiri.de/en/datasets/bacteroides ), is launched to interrogate the obtained gene expression data and annotations of ~4500 transcription start sites, untranslated regions, operon structures, and 269 noncoding RNA elements. Among the latter is GibS, a conserved, 145 nt-long small RNA that is highly expressed in the presence of N-acetyl-D-glucosamine as sole carbon source. We use computational predictions and experimental data to determine the secondary structure of GibS and identify its target genes. Our results indicate that sensing of N-acetyl-D-glucosamine induces GibS expression, which in turn modifies the transcript levels of metabolic enzymes.
| S-EPMC7366714 | biostudies-literature
Project description:Gram-negative, obligate anaerobic Bacteroides thetaiotaomicron is emerging as the model organism for gut microbiota research. Colonization of its host niche within the human colon depends to large parts on the extensive metabolic capacity encoded in the Bacteroides genome, but little is known about how its transcription is organized. Here, we transferred differential RNA-seq (dRNA-seq) to B. thetaiotaomicron type strain VPI-5482 to globally map transcription start sites (TSSs). Our screen identified ~4,500 TSSs and we globally determined untranslated regions (UTRs), operon structures, and promoter motifs. In addition, dRNA-seq led to the discovery of ~250 noncoding RNA elements, including 124 novel intergenic small RNAs (sRNAs), several of which we validated experimentally. One of them is the conserved, 145 nt-long sRNA BTnc035, that was highly expressed when B. thetaiotaomicron was grown in the presence of N-acetylglucosamine or glucuronic acid as the sole carbon source. Combining computational predictions with experimental data, we determined the secondary structure of BTnc035 and identified target genes of this sRNA. The emerging model places BTnc035 at the center of a metabolic feedback-loop, wherein sensing of N-acetyl-D-glucosamine and D-glucuronic acid induces BTnc035, which in turn may contribute to a metabolic switch from polysaccharide biosynthesis to break-down. Together, we compiled a single-nucleotide resolution transcriptome map of B. thetaiotaomicron and provide the first global analysis of noncoding RNAs in this important anaerobic model bacterium. To allow easy interrogation of our transcriptome data, we developed Theta-Base – an intuitive online browser that can be freely accessed at https://www.helmholtz-hiri.de/en/bacteroides.
2020-06-11 | GSE144492 | GEO
Project description:Glycosaminoglycans (GAGs) and GAG-degrading enzymes have wide-ranging applications in the medical and biotechnological industries. The former are also an important nutrient source for select species of the human gut microbiota (HGM), a key player in host-microbial interactions. How GAGs are metabolized by the HGM is therefore of interest and has been extensively investigated in the model human gut microbe Bacteroides thetaiotaomicron. The presence of as-yet uncharacterized GAG-inducible genes in its genome and of related species, however, is testament to our incomplete understanding of this process. Nevertheless, it presents a potential opportunity for the discovery of additional GAG-degrading enzymes. Here, we investigated a gene of unknown function (BT_3328) from the chondroitin sulfate (CS) utilization locus of B. thetaiotaomicron NMR and UV spectroscopic assays revealed that it encodes a novel polysaccharide lyase (PL), hereafter referred to as BtCDH, reflecting its source (B. thetaiotaomicron (Bt)) and its ability to degrade the GAGs CS, dermatan sulfate (DS), and hyaluronic acid (HA). When incubated with HA, BtCDH generated a series of unsaturated HA sugars, including Δ4,5UA-GlcNAc, Δ4,5UA-GlcNAc-GlcA-GlcNac, Δ4,5UA-[GlcNAc-GlcA]2-GlcNac, and Δ4,5UA-[GlcNAc-GlcA]3-GlcNac, as end products and hence was classed as endo-acting. A combination of genetic and biochemical assays revealed that BtCDH localizes to the cell surface of B. thetaiotaomicron where it enables extracellular GAG degradation. BtCDH homologs were also detected in several other HGM species, and we therefore propose that it represents the founding member of a new polysaccharide lyase family (PL29). The current discovery also contributes new insights into CS metabolism by the HGM.
| S-EPMC6240882 | biostudies-literature
Project description:To understand how a bacterium ultimately succeeds or fails in adapting to a new host, it is essential to assess the temporal dynamics of its fitness over the course of colonization. Here, we introduce a human-derived commensal organism, Bacteroides thetaiotaomicron (Bt), into the guts of germ-free mice to determine whether and how the genetic requirements for colonization shift over time. Combining a high-throughput functional genetics assay and transcriptomics, we find that gene usage changes drastically during the first days of colonization, shifting from high expression of amino acid biosynthesis genes to broad upregulation of diverse polysaccharide utilization loci. Within the first week, metabolism becomes centered around utilization of a predominant dietary oligosaccharide, and these changes are largely sustained through 6 weeks of colonization. Spontaneous mutations in wild-type Bt also evolve around this locus. These findings highlight the importance of considering temporal colonization dynamics in developing more effective microbiome-based therapies.
| S-EPMC10528517 | biostudies-literature
Project description:UnlabelledThe capsule from Bacteroides, a common gut symbiont, has long been a model system for studying the molecular mechanisms of host-symbiont interactions. The Bacteroides capsule is thought to consist of an array of phase-variable polysaccharides that give rise to subpopulations with distinct cell surface structures. Here, we report the serendipitous discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron: a surface layer composed of a protein of unknown function, BT1927. BT1927, which is expressed in a phase-variable manner by ~1:1,000 cells in a wild-type culture, forms a hexagonally tessellated surface layer. The BT1927-expressing subpopulation is profoundly resistant to complement-mediated killing, due in part to the BT1927-mediated blockade of C3b deposition. Our results show that the Bacteroides surface structure is capable of a far greater degree of structural variation than previously known, and they suggest that structural variation within a Bacteroides species is important for productive gut colonization.ImportanceMany bacterial species elaborate a capsule, a structure that resides outside the cell wall and mediates microbe-microbe and microbe-host interactions. Species of Bacteroides, the most abundant genus in the human gut, produce a capsule that consists of an array of polysaccharides, some of which are known to mediate interactions with the host immune system. Here, we report the discovery of a previously unknown surface structure in Bacteroides thetaiotaomicron. We show that this protein-based structure is expressed by a subset of cells in a population and protects Bacteroides from killing by complement, a component of the innate immune system. This novel surface layer protein is conserved across many species of the genus Bacteroides, suggesting an important role in colonization and host immune modulation.
| S-EPMC4611039 | biostudies-literature
Project description:Renal fibrosis is an inevitable outcome of various manifestations of progressive chronic kidney diseases (CKD). The need for efficacious treatment regimen against renal fibrosis can therefore not be overemphasized. Here we show a novel protective role of Bacteroides fragilis (B. fragilis) in renal fibrosis in mice. We demonstrate decreased abundance of B. fragilis in the feces of CKD patients and unilateral ureteral obstruction (UUO) mice. Oral administration of live B. fragilis attenuates renal fibrosis in UUO and adenine mice models. Increased lipopolysaccharide (LPS) levels are decreased after B. fragilis administration. Results of metabolomics and proteomics studies show decreased level of 1,5-anhydroglucitol (1,5-AG), a substrate of SGLT2, which increases after B. fragilis administration via enhancement of renal SGLT2 expression. 1,5-AG is an agonist of TGR5 that attenuates renal fibrosis by inhibiting oxidative stress and inflammation. Madecassoside, a natural product found via in vitro screening promotes B. fragilis growth and remarkably ameliorates renal fibrosis. Our findings reveal the ameliorative role of B. fragilis in renal fibrosis via decreasing LPS and increasing 1,5-AG levels.
| S-EPMC9568537 | biostudies-literature