Reverse metabolomics for the discovery of chemical structures from humans.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N-acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis1,2, we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn's disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4+ T cells3 and agonism of the pregnane X receptor4. Culture of bacteria belonging to the Bifidobacterium, Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems.

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

A vitamin-C-derived DNA modification catalysed by an algal TET homologue.

Methylation of cytosine to 5-methylcytosine (5mC) is a prevalent DNA modification found in many organisms. Sequential oxidation of 5mC by ten-eleven translocation (TET) dioxygenases results in a cascade of additional epigenetic marks and promotes demethylation of DNA in mammals1,2. However, the enzymatic activity and function of TET homologues in other eukaryotes remains largely unexplored. Here we show that the green alga Chlamydomonas reinhardtii contains a 5mC-modifying enzyme (CMD1) that is a TET homologue and catalyses the conjugation of a glyceryl moiety to the methyl group of 5mC through a carbon-carbon bond, resulting in two stereoisomeric nucleobase products. The catalytic activity of CMD1 requires Fe(II) and the integrity of its binding motif His-X-Asp, which is conserved in Fe-dependent dioxygenases3. However, unlike previously described TET enzymes, which use 2-oxoglutarate as a co-substrate4, CMD1 uses L-ascorbic acid (vitamin C) as an essential co-substrate. Vitamin C donates the glyceryl moiety to 5mC with concurrent formation of glyoxylic acid and CO2. The vitamin-C-derived DNA modification is present in the genome of wild-type C. reinhardtii but at a substantially lower level in a CMD1 mutant strain. The fitness of CMD1 mutant cells during exposure to high light levels is reduced. LHCSR3, a gene that is critical for the protection of C. reinhardtii from photo-oxidative damage under high light conditions, is hypermethylated and downregulated in CMD1 mutant cells compared to wild-type cells, causing a reduced capacity for photoprotective non-photochemical quenching. Our study thus identifies a eukaryotic DNA base modification that is catalysed by a divergent TET homologue and unexpectedly derived from vitamin C, and describes its role as a potential epigenetic mark that may counteract DNA methylation in the regulation of photosynthesis.

Complex chemical signals dictate Ah receptor activation through the gut-lung axis.

The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in rapid clearance within the intestinal tract, limiting systemic exposure and subsequent AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that functionally increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA), a gut bacterial metabolite of ellagitannins, as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). In mice, dietary exposure to UroA in a 10% broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in the liver. Thus, CYP1A1 dietary competitive substrates can lead to enhanced systemic AHR ligand distribution from the gut, likely through the lymphatic system, increasing AHR activation in key barrier tissues. Finally, this report will lead to a reassessment of the dynamics of distribution of other hydrophobic chemicals present in the diet.

Randomized Controlled-Feeding Study of Dietary Emulsifier Carboxymethylcellulose Reveals Detrimental Impacts on the Gut Microbiota and Metabolome.

BACKGROUND & AIMS: Epidemiologic and murine studies suggest that dietary emulsifiers promote development of diseases associated with microbiota dysbiosis. Although the detrimental impact of these compounds on the intestinal microbiota and intestinal health have been demonstrated in animal and in vitro models, impact of these food additives in healthy humans remains poorly characterized. METHODS: To examine this notion in humans, we performed a double-blind controlled-feeding study of the ubiquitous synthetic emulsifier carboxymethylcellulose (CMC) in which healthy adults consumed only emulsifier-free diets (n = 9) or an identical diet enriched with 15 g per day of CMC (n = 7) for 11 days. RESULTS: Relative to control subjects, CMC consumption modestly increased postprandial abdominal discomfort and perturbed gut microbiota composition in a way that reduced its diversity. Moreover, CMC-fed subjects exhibited changes in the fecal metabolome, particularly reductions in short-chain fatty acids and free amino acids. Furthermore, we identified 2 subjects consuming CMC who exhibited increased microbiota encroachment into the normally sterile inner mucus layer, a central feature of gut inflammation, as well as stark alterations in microbiota composition. CONCLUSIONS: These results support the notion that the broad use of CMC in processed foods may be contributing to increased prevalence of an array of chronic inflammatory diseases by altering the gut microbiome and metabolome (ClinicalTrials.gov, number NCT03440229).

New types of ATP-grasp ligase are associated with the novel pathway for complicated mycosporine-like amino acid production in desiccation-tolerant cyanobacteria.

Mycosporine-like amino acids (MAAs) were widespread in diverse organisms to attenuate UV radiation. We recently characterized the large, complicated MAA mycosporine-2-(4-deoxygadusolyl-ornithine) in desert cyanobacterium Nostoc flagelliforme. Synthesis of this MAA requires the five-gene cluster mysABDC2C3. Here, bioinformatic analysis indicated that mysC duplication within five-gene mys clusters is strictly limited to drought-tolerant cyanobacteria. Phylogenic analysis distinguished these duplicated MysCs into two clades that separated from canonical MysCs. Heterologous expression of N. flagelliforme mys genes in Escherichia coli showed that MysAB produces 4-deoxygadusol. The ATP-grasp ligase of MysC3 catalyses the linkage of the δ- or ε-amino group of ornithine/lysine to 4-deoxygadusol, yielding mycosporine-ornithine or mycosporine-lysine respectively. The ATP-grasp ligase of MysC2 strictly condenses the α-amino group of mycosporine-ornithine to another 4-deoxygadusol. MysD (D-Ala-D-Ala ligase) functions following MysC2 to catalyse the formation of mycosporine-2-(4-deoxygadusolyl-ornithine). High arginine content likely provides a greater pool of ornithine over other amino acids during rehydration of desiccated N. flagelliforme. Duplication of ATP-grasp ligases is specific for the use of substrates that have two amino groups (such as ornithine) for the production of complicated MAAs with multiple chromophores. This five-enzyme biosynthesis pathway for complicated MAAs is a novel adaptation of cyanobacteria for UV tolerance in drought environments.

Quantitative Metabonomic Phenotypes in Different Structures of Mung Bean (Vigna radiata) Seeds and Their Germination-Associated Dynamic Changes.

Plant seed germination involving dynamic water uptakes and biochemical changes is essential for preservation of plant germplasm resource and worldwide food supply. To understand the germination-associated compartmental biochemistry changes, we quantitatively analyzed the metabolite composition (metabonome) for embryonic axes, cotyledons, and testae of mung bean (Vigna radiata) seeds in three germination phases using the NMR-based metabonomics approach. We found that three structures of mung bean seeds had distinct metabonomic phenotypes dominated by 53 metabolites including amino acids, carbohydrates, organic acids, choline metabolites, nucleotides/nucleosides, and shikimate-mediated secondary metabolites together with calcium and magnesium cations. During germination, all three seed structures had outstanding but distinct metabonomic changes. Both embryonic axis and cotyledon showed remarkable metabolic changes related to degradation of carbohydrates and proteins, metabolism of amino acids, nucleotides/nucleosides, and choline together with energy metabolism and shikimate-mediated plant secondary metabolism. The metabonomic changes in these two structures were mostly related to multiple functions for biochemical activities in the former and nutrient mobilizations in the latter. In contrast, testa metabonomic changes mainly reflected the metabolite leakages from the other two structures. Phase 1 of germination was featured with degradation of oligosaccharides and proteins and recycling of stored nucleic acids together with anaerobic metabolisms, whereas phase 2 was dominated by energy metabolism, biosynthesis of osmolytes, and plant secondary metabolites. These provided essential metabolic information for understanding the biochemistry associated with early events of seed germination and possible metabolic functions of different seed structures for plant development.

Discovery of a non-stereoselective cytochrome P450 catalyzing either 8α- or 8ß-hydroxylation of germacrene A acid from the Chinese medicinal plant, Inula hupehensis.

Sesquiterpene lactones (STLs) are C15 terpenoid natural products with α-methylene γ-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid (GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (α- or ß-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6α-, 12,6ß-, 12,8α-, and 12,8ß-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8α and C12,8ß) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 (CYP71BL6) that can catalyze the hydroxylation of GAA in either 8α- or 8ß-configuration, resulting in the synthesis of both 8α- and 8ß-hydroxyl GAAs. Of these two products, only 8α-hydroxyl GAA spontaneously lactonizes to the C12,8α-STL while the 8ß-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8α-STL, named inunolide, and 8ß-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71BL6 displays 63-66% amino acid identity to the previously reported CYP71BL1/2 catalyzing GAA 6α- or 8ß-hydroxylation, indicating CYP71BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71BL6 transcript abundance correlates closely to the accumulation of C12,8-STLs in I. hupehensis. The identification of CYP71BL6 provides an insight into the biosynthesis of STLs in Asteraceae.

UV-B induced biosynthesis of a novel sunscreen compound in solar radiation and desiccation tolerant cyanobacteria.

The small-molecule sunscreen compounds, mycosporine-like amino acids (MAAs), have strong ultraviolet (UV) absorption and can protect cyanobacteria against UV-B damage. However, the molecular mechanism underlying UV-B signaling and MAA chemical diversity remain largely unclear. Here, we identified a five-gene cluster for MAA biosynthesis in the solar radiation and desiccation tolerant cyanobacterium Nostoc flagelliforme. A LuxR family protein OrrA was identified as a positive UV-B responsive regulator binding to the promoter region of this gene cluster. OrrA functions as an activator mediating the UV-B induced MAA biosynthesis. Overexpression of orrA strengthened its UV-B tolerance during desiccation, and enhanced the photosynthetic recovery upon rehydration. Heterologous expression of this gene cluster in Anabaena PCC 7120 produces the same MAA as that in field samples of N. flagelliforme. The MAA structure is assigned as mycosporine-2-(4-deoxygadusolyl-ornithine) with a molecular weight of 756 Da, the structurally unique MAA compound reported to date. This MAA was catalyzed by mysD-mysC2-mysC1 encoding proteins from 4-deoxygadusol, which was synthesized through the catalysis of mysA-mysB products. Thus, we elucidated the transcriptional mechanism for a novel type MAA biosynthesis in solar radiation and desiccation tolerant cyanobacteria, which shed light on the identification of other components for UV-B signaling in cyanobacteria.

Identification, Characteristics and Mechanism of 1-Deoxy-N-acetylglucosamine from Deep-Sea Virgibacillus dokdonensis MCCC 1A00493.

Xanthomonas oryzae pv. oryzae, which causes rice bacterial blight, is one of the most destructive pathogenic bacteria. Biological control against plant pathogens has recently received increasing interest. 1-Deoxy-N-acetylglucosamine (1-DGlcNAc) was extracted from the supernatant of Virgibacillus dokdonensis MCCC 1A00493 fermentation through antibacterial bioassay-guided isolation. Its structure was elucidated by LC/MS, NMR, chemical synthesis and time-dependent density functional theory (TD-DFT) calculations. 1-DGlcNAc specifically suppressed X. oryzae pv. oryzae PXO99A (MIC was 23.90 µg/mL), but not other common pathogens including Xanthomonas campestris pv. campestris str.8004 and Xanthomonas oryzae pv. oryzicola RS105. However, its diastereomer (2-acetamido-1,5-anhydro-2-deoxy-d-mannitol) also has no activity to X. oryzae pv. oryzae. This result suggested that activity of 1-DGlcNAc was related to the difference in the spatial conformation of the 2-acetamido moiety, which might be attributed to their different interactions with a receptor. Eighty-four unique proteins were found in X. oryzae pv. oryzae PXO99A compared with the genome of strains8004 and RS105 by blastp. There may be unique interactions between 1-DGlcNAc and one or more of these unique proteins in X. oryzae pv. oryzae. Quantitative real-time PCR and the pharmMapper server indicated that proteins involved in cell division could be the targets in PXO99A. This research suggested that specificity of active substance was based on the active group and spatial conformation selection, and these unique proteins could help to reveal the specific mechanism of action of 1-DGlcNAc against PXO99A.

Metabonomics uncovers a reversible proatherogenic lipid profile during infliximab therapy of inflammatory bowel disease.

BACKGROUND: One-third of inflammatory bowel disease (IBD) patients show no response to infliximab (IFX) induction therapy, and approximately half of patients responding become unresponsive over time. Thus, identification of potential treatment response biomarkers are of great clinical significance. This study employs spectroscopy-based metabolic profiling of serum from patients with IBD treated with IFX and healthy subjects (1) to substantiate the use of spectroscopy as a semi-invasive diagnostic tool, (2) to identify potential biomarkers of treatment response and (3) to characterise the metabolic changes during management of patients with tumour necrosis factor-α inhibitors. METHODS: Successive serum samples collected during IFX induction treatment (weeks 0, 2, 6 and 14) from 87 IBD patients and 37 controls were analysed by 1H nuclear magnetic resonance (NMR) spectroscopy. Data were analysed with principal components analysis and orthogonal projection to latent structures discriminant analysis using SIMCA-P+ v12 and MATLAB. RESULTS: Metabolic profiles were significantly different between active ulcerative colitis and controls, active Crohn's disease and controls, and quiescent Crohn's disease and controls. Metabolites holding differential power belonged primarily to lipids and phospholipids with proatherogenic characteristics and metabolites in the pyruvate metabolism, suggestive of an intense inflammation-driven energy demand. IBD patients not responding to IFX were identified as a potentially distinct group based on their metabolic profile, although no applicable response biomarkers could be singled out in the current setting. CONCLUSION: 1H NMR spectroscopy of serum samples is a powerful semi-invasive diagnostic tool in flaring IBD. With its use, we provide unique insights into the metabolic changes taking place during induction treatment with IFX. Of distinct clinical relevance is the identification of a reversible proatherogenic lipid profile in IBD patients with active disease, which partially explains the increased risk of cardiovascular disease associated with IBD.

Dynamic metabolic responses of brown planthoppers towards susceptible and resistant rice plants.

Brown planthopper (Nilaparvata lugens Stål, BPH) causes huge economic losses in rice-growing regions, and new strategies for combating BPH are required. To understand how BPHs respond towards BPH-resistant plants, we systematically analysed the metabolic differences between BPHs feeding on the resistant and susceptible plants using NMR and GC-FID/MS. We also measured the expression of some related genes involving glycolysis and biosyntheses of trehalose, amino acids, chitin and fatty acids using real-time PCR. BPH metabonome was dominated by more than 60 metabolites including fatty acids, amino acids, carbohydrates, nucleosides/nucleotides and TCA cycle intermediates. After initial 12 h, BPHs feeding on the resistant plants had lower levels of amino acids, glucose, fatty acids and TCA cycle intermediates than on the susceptible ones. The levels of these metabolites recovered after 24 h feeding. This accompanied with increased level in trehalose, choline metabolites and nucleosides/nucleotides compared with BPH feeding on the susceptible plants. Decreased levels of BPH metabolites at the early feeding probably resulted from less BPH uptakes of sap from resistant plants and recovery of BPH metabolites at the later stage probably resulted from their adaptation to the adverse environment with their increased hopping frequency to ingest more sap together with contributions from yeast-like symbionts in BPHs. Throughout 96 h, BPH feeding on the resistant plants showed significant up-regulation of chitin synthase catalysing biosynthesis of chitin for insect exoskeleton, peritrophic membrane lining gut and tracheae. These findings provided useful metabolic information for understanding the BPH-rice interactions and perhaps for developing new BPH-combating strategies.

Assessment of the Biological Effects of a Multifunctional Nano-Drug-Carrier and Its Encapsulated Drugs.

Polymer-nanoparticle-encapsulated doxorubicin (DOX) and paclitaxel (TAX) have the potential for novel therapeutic use against cancer in the clinic. However, the systemic biological effect of the nanoparticle material, namely, methoxypoly(ethylene glycol)-poly(lactide-co-glycolide) (mPEG-PLGA), and its encapsulated drugs have not been fully studied. We have applied NMR-based metabonomics methodology to characterize and analyze the systemic metabolic changes in mice after being exposed to mPEG-PLGA, mPEG-PLGA-encapsulated DOX and TAX (NP-D/T), and their free forms. The study revealed that mPEG-PLGA exposure only induces temporary and slight metabolic alternations and that there are detoxification effects of nanoparticle packed with D/T drugs on the heart when comparing with free-form D/T drugs. Both NP-D/T and their free forms induce a shift in energy metabolism, stimulate antioxidation pathways, and disturb the gut microbial activity of the host. However, mPEG-PLGA packaging can relieve the energy metabolism inhibition and decrease the activation of antioxidation pathways caused by D/T exposure. These findings provide a holistic insight into the biological effect of polymer nanoparticle and nanoparticle-encapsulated drugs. This study also furthers our understanding of the molecular mechanisms involved in the amelioration effects of mPEG-PLGA packaging on the toxicity of the incorporated drugs.

Developmental Changes for the Hemolymph Metabolome of Silkworm (Bombyx mori L.).

Silkworm (Bombyx mori) is a lepidopteran-holometabolic model organism. To understand its developmental biochemistry, we characterized the larval hemolymph metabonome from the third instar to prepupa stage using (1)H NMR spectroscopy whilst hemolymph fatty acid composition using GC-FID/MS. We unambiguously assigned more than 60 metabolites, among which tyrosine-o-ß-glucuronide, mesaconate, homocarnosine, and picolinate were reported for the first time from the silkworm hemolymph. Phosphorylcholine was the most abundant metabolite in all developmental stages with exception for the periods before the third and fourth molting. We also found obvious developmental dependence for the hemolymph metabonome involving multiple pathways including protein biosyntheses, glycolysis, TCA cycle, the metabolisms of choline amino acids, fatty acids, purines, and pyrimidines. Most hemolymph amino acids had two elevations during the feeding period of the fourth instar and prepupa stage. Trehalose was the major blood sugar before day 8 of the fifth instar, whereas glucose became the major blood sugar after spinning. C16:0, C18:0 and its unsaturated forms were dominant fatty acids in hemolymph. The developmental changes of hemolymph metabonome were associated with dietary nutrient intakes, biosyntheses of cell membrane, pigments, proteins, and energy metabolism. These findings offered essential biochemistry information in terms of the dynamic metabolic changes during silkworm development.

Metabonomic Changes Associated with Atherosclerosis Progression for LDLR(-/-) Mice.

Atherosclerosis resulting from hyperlipidemia causes many serious cardiovascular diseases. To understand the systems changes associated with pathogenesis and progression of atherosclerosis, we comprehensively analyzed the dynamic metabonomic changes in multiple biological matrices of LDLR(-/-) mice using NMR and GC-FID/MS with gene expression, clinical chemistry, and histopathological data as well. We found that 12 week "Western-type" diet (WD) treatment caused obvious aortic lesions, macrophage infiltration, and collagen level elevation in LDLR(-/-) mice accompanied by up-regulation of inflammatory factors including aortic ICAM-1, MCP-1, iNOS, MMP2, and hepatic TNFα and IL-1ß. The WD-induced atherosclerosis progression was accompanied by metabonomic changes in multiple matrices including biofluids (plasma, urine) and (liver, kidney, myocardial) tissues involving multiple metabolic pathways. These included disruption of cholesterol homeostasis, disturbance of biosynthesis of amino acids and proteins, altered gut microbiota functions together with metabolisms of vitamin-B3, choline, purines, and pyrimidines. WD treatment caused down-regulation of SCD1 and promoted oxidative stress reflected by urinary allantoin elevation and decreases in hepatic PUFA-to-MUFA ratio. When switching to normal diet, atherosclerotic LDLR(-/-) mice reprogrammed their metabolisms and reversed the atherosclerosis-associated metabonomic changes to a large extent, although aortic lesions, inflammation parameters, macrophage infiltration, and collagen content were only partially alleviated. We concluded that metabolisms of fatty acids and vitamin-B3 together with gut microbiota played crucially important roles in atherosclerosis development. These findings offered essential biochemistry details of the diet-induced atherosclerosis and demonstrated effectiveness of the integrated metabonomic analysis of multiple biological matrices for understanding the molecular aspects of cardiovascular diseases.

Individualized microbiotas dictate the impact of dietary fiber on colitis sensitivity.

BACKGROUND: The observation that the intestinal microbiota is  central in the development of IBD suggests that dietary fiber, the microbiota's primary source of nourishment, could play a central role in these diseases. Accordingly, enriching diets with specific soluble fibers remodels microbiota and modulates colitis sensitivity. In humans, a recent study suggests that the microbiota of select IBD patients might influence the impacts they would experience upon fiber exposure. We sought here to define the extent to which individual microbiotas varied in their responsiveness to purified soluble fiber inulin and psyllium. Moreover, the extent to which such variance might impact proneness to colitis. RESULTS: We observed a high level of inter-individual variation in microbiota responsiveness to fiber inulin and psyllium: while microbiotas from select donors exhibited stark fiber-induced modulation in composition, pro-inflammatory potential, and metabolomic profile, others were only minimally impacted. Mice transplanted with fiber-sensitive microbiomes exhibited colitis highly modulated by soluble fiber consumption, while mice receiving fiber-resistant microbiotas displayed colitis severity irrespective of fiber exposure. CONCLUSION: The extent to which select soluble fibers alter proneness to colitis is highly influenced by an individual's microbiota composition and further investigation of individual microbiota responsiveness toward specific dietary fiber could pave the way to personalized fiber-based intervention, both in IBD patients and healthy individuals. Video Abstract.

Surgery for Crohn's Disease Is Associated with a Dysbiotic Microbiome and Metabolome: Results from Two Prospective Cohorts.

BACKGROUND & AIMS: Crohn's disease is associated with alterations in the gut microbiome and metabolome described as dysbiosis. We characterized the microbial and metabolic consequences of ileal resection, the most common Crohn's disease surgery. METHODS: Patients with and without intestinal resection were identified from the Diet to Induce Remission in Crohn's Disease and Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease studies. Stool samples were analyzed with shotgun metagenomics sequencing. Fecal butyrate was measured with 1H nuclear magnetic resonance spectroscopy. Fecal bile acids and plasma 7α-hydroxy-4-cholesten-3-one (C4) was measured with mass spectrometry. RESULTS: Intestinal resection was associated with reduced alpha diversity and altered beta diversity with increased Proteobacteria and reduced Bacteroidetes and Firmicutes. Surgery was associated with higher representation of genes in the KEGG pathway for ABC transporters and reduction in genes related to bacterial metabolism. Surgery was associated with reduced concentration of the But gene but this did not translate to reduced fecal butyrate concentration. Surgery was associated with decreased abundance of bai operon genes, with increased plasma C4 concentration, increased primary bile acids and reduced secondary bile acids, including isoLCA. Additionally, Egerthella lenta, Adlercreutzia equalofaciens, and Gordonibacter pamelaeae were lower in abundance among patients with prior surgery in both cohorts. CONCLUSIONS: In 2 different populations, prior surgery in Crohn's disease is associated with altered fecal microbiome. Patients who had undergone ileal resection had reduction in the potentially beneficial bacteria E lenta and related actinobacteria and secondary bile acids, including isoLCA, suggesting that these could be biomarkers of patients at higher risk for disease progression.

Dietary carbohydrates regulate intestinal colonization and dissemination of Klebsiella pneumoniae.

Bacterial translocation from the gut microbiota is a source of sepsis in susceptible patients. Previous work suggests that overgrowth of gut pathobionts, including Klebsiella pneumoniae, increases the risk of disseminated infection. Our data from a human dietary intervention study found that, in the absence of fiber, K. pneumoniae bloomed during microbiota recovery from antibiotic treatment. We thus hypothesized that dietary nutrients directly support or suppress colonization of this gut pathobiont in the microbiota. Consistent with our study in humans, complex carbohydrates in dietary fiber suppressed the colonization of K. pneumoniae and allowed for recovery of competing commensals in mouse models. In contrast, through ex vivo and in vivo modeling, we identified simple carbohydrates as a limiting resource for K. pneumoniae in the gut. As proof of principle, supplementation with lactulose, a nonabsorbed simple carbohydrate and an FDA-approved therapy, increased colonization of K. pneumoniae. Disruption of the intestinal epithelium led to dissemination of K. pneumoniae into the bloodstream and liver, which was prevented by dietary fiber. Our results show that dietary simple and complex carbohydrates were critical not only in the regulation of pathobiont colonization but also disseminated infection, suggesting that targeted dietary interventions may offer a preventative strategy in high-risk patients.

Dietary fiber guar gum-induced shift in gut microbiota metabolism and intestinal immune activity enhances susceptibility to colonic inflammation.

With an increasing interest in dietary fibers (DFs) to promote intestinal health and the growth of beneficial gut bacteria, there is a continued rise in the incorporation of refined DFs in processed foods. It is still unclear how refined fibers, such as guar gum, affect the gut microbiota activity and pathogenesis of inflammatory bowel disease (IBD). Our study elucidated the effect and underlying mechanisms of guar gum, a fermentable DF (FDF) commonly present in a wide range of processed foods, on colitis development. We report that guar gum containing diet (GuD) increased the susceptibility to colonic inflammation. Specifically, GuD-fed group exhibited severe colitis upon dextran sulfate sodium (DSS) administration, as evidenced by reduced body weight, diarrhea, rectal bleeding, and shortening of colon length compared to cellulose-fed control mice. Elevated levels of pro-inflammatory markers in both serum [serum amyloid A (SAA), lipocalin 2 (Lcn2)] and colon (Lcn2) and extensive disruption of colonic architecture further affirmed that GuD-fed group exhibited more severe colitis than control group upon DSS intervention. Amelioration of colitis in GuD-fed group pre-treated with antibiotics suggest a vital role of intestinal microbiota in GuD-mediated exacerbation of intestinal inflammation. Gut microbiota composition and metabolite analysis in fecal and cecal contents, respectively, revealed that guar gum primarily enriches Actinobacteriota, specifically Bifidobacterium. Guar gum also altered multiple genera belonging to phyla Bacteroidota and Firmicutes. Such shift in gut microbiota composition favored luminal accumulation of intermediary metabolites succinate and lactate in the GuD-fed mice. Colonic IL-18 and tight junction markers were also decreased in the GuD-fed group. Importantly, GuD-fed mice pre-treated with recombinant IL-18 displayed attenuated colitis. Collectively, unfavorable changes in gut microbiota activity leading to luminal accumulation of lactate and succinate, reduced colonic IL-18, and compromised gut barrier function following guar gum feeding contributed to increased colitis susceptibility.

Guar gum increased susceptibility to colitisGuar gum-induced exacerbation of colitis is gut microbiota dependentGuar gum-induced shift in microbiota composition favored the accumulation of luminal intermediate metabolites succinate and lactateGuar gum-fed mice exhibited reduced colonic level of IL-18 and tight junction molecules.Exogenous IL-18 administration partly rescued mice from guar gum-induced colitis susceptibility.

Systemic responses of mice to dextran sulfate sodium-induced acute ulcerative colitis using 1H NMR spectroscopy.

The interplay between genetic mutation and environmental factors is believed to contribute to the etiology of inflammatory bowel disease (IBD). While focused attention has been paid to the aforementioned research, time-specific and organ-specific metabolic changes associated with IBD are still lacking. Here, we induced acute ulcerative colitis in mice by providing water containing 3% dextran sulfate sodium (DSS) for 7 days and investigated the metabolic changes of plasma, urine, and a range of biological tissues by employing a (1)H nuclear magnetic resonance (NMR)-based metabonomics approach with complementary information on serum clinical chemistry and histopathology. We found that DSS-induced acute ulcerative colitis leads to significant elevations in the levels of amino acids in plasma and decreased levels in the membrane-related metabolites and a range of nucleotides, nucleobases, and nucleosides in the colon. In addition, acute-colitis-induced elevations in the levels of nucleotides in the liver were observed, accompanied by reduced levels of glucose. DSS-induced acute colitis also resulted in increased levels of oxidized glutathione and attenuated levels of taurine in the spleen. Furthermore, acute colitis resulted in depletion in the levels of gut microbial cometabolites in urine along with an increase in citric acid cycle intermediates. These findings suggest that DSS-induced acute colitis causes a disturbance of lipid and energy metabolism, damage to the colon and liver, a promoted antioxidative and anti-inflammatory response, and perturbed gut microbiotal communities. The information obtained here provided details of the time-dependent and holistic metabolic changes in the development of the DSS-induced acute ulcerative colitis, which could be useful in discovery of novel therapeutic targets for management of IBD.