Key bacterial taxa and metabolic pathways affecting gut short-chain fatty acid profiles in early life

Infant gut microbiota development affects the host physiology throughout life, and short-chain fatty acids (SCFAs) are promising key metabolites mediating microbiota-host relationships. Here, we investigated dense longitudinally collected faecal samples from 12 subjects during the first 2 years (n = 1048) to identify early life gut SCFA patterns and their relationships with the microbiota. Our results revealed three distinct phases of progression in the SCFA profiles: early phase characterised by low acetate and high succinate, middle-phase characterised by high lactate and formate and late-phase characterised by high propionate and butyrate. Assessment of the SCFA–microbiota relationships revealed that faecal butyrate is associated with increased Clostridiales and breastfeeding cessation, and that diverse and personalised assemblage of Clostridiales species possessing the acetyl-CoA pathway play major roles in gut butyrate production. We also found an association between gut formate and some infant-type bifidobacterial species, and that human milk oligosaccharides (HMO)-derived fucose is the substrate for formate production during breastfeeding. We identified genes upregulated in fucose and fucosylated HMO utilisation in infant-type bifidobacteria. Notably, bifidobacteria showed interspecific and intraspecific variation in the gene repertoires, and cross-feeding of fucose contributed to gut formate production. This study provides an insight into early life SCFA–microbiota relationships, which is an important step for developing strategies for modulating lifelong health.Subject terms: Microbial ecology, Microbiome, Bacterial genetics     

Fructose liquid and solid formulations differently affect gut integrity,microbiota composition and related liver toxicity: a comparative in vivo study

Despite clinical findings suggesting that the form (liquid versus solid) of the sugars may significantly affect the development of metabolic diseases, no experimental data are available on the impact of their formulations on gut microbiota, integrity and hepatic outcomes.In the present sudy, C57Bl/6j mice were fed a standard diet plus water (SD), a standard diet plus 60% fructose syrup (L-Fr) or a 60% fructose solid diet plus water (S-Fr) for 12 weeks. Gut microbiota was characterized through 16S rRNA phylogenetic profiling and shotgun sequencing of microbial genes in ileum content and related volatilome profiling.Fructose feeding led to alterations of the gut microbiota depending on the fructose formulation, with increased colonization by Clostridium, Oscillospira and Clostridiales phyla in the S-Fr group and Bacteroides, Lactobacillus, Lachnospiraceae and Dorea in the L-Fr. S-Fr evoked the highest accumulation of advanced glycation end products and barrier injury in the ileum intestinal mucosa. These effects were associated to a stronger activation of the lipopolysaccharide-dependent proinflammatory TLR4/NLRP3 inflammasome pathway in the liver of S-Fr mice than of L-Fr mice. In contrast, L-Fr intake induced higher levels of hepatosteatosis and markers of fibrosis than S-Fr. Fructose-induced ex novo lipogenesis with production of SCFA and MCFA was confirmed by metagenomic analysis.These results suggest that consumption of fructose under different forms, liquid or solid, may differently affect gut microbiota, thus leading to impairment in intestinal mucosa integrity and liver homeostasis.     

Gut microbiota in wild and captive Guizhou snub‐nosed monkeys,Rhinopithecus brelichi

Many colobine species—including the endangered Guizhou snub‐nosed monkey (Rhinopithecus brelichi) are difficult to maintain in captivity and frequently exhibit gastrointestinal (GI) problems. GI problems are commonly linked to alterations in the gut microbiota, which lead us to examine the gut microbial communities of wild and captive R. brelichi. We used high‐throughput sequencing of the 16S rRNA gene to compare the gut microbiota of wild (N = 7) and captive (N = 8) R. brelichi. Wild monkeys exhibited increased gut microbial diversity based on the Chao1 but not Shannon diversity metric and greater relative abundances of bacteria in the Lachnospiraceae and Ruminococcaceae families. Microbes in these families digest complex plant materials and produce butyrate, a short chain fatty acid critical to colonocyte health. Captive monkeys had greater relative abundances of Prevotella and Bacteroides species, which degrade simple sugars and carbohydrates, like those present in fruits and cornmeal, two staples of the captive R. brelichi diet. Captive monkeys also had a greater abundance of Akkermansia species, a microbe that can thrive in the face of host malnutrition. Taken together, these findings suggest that poor health in captive R. brelichi may be linked to diet and an altered gut microbiota.     

Pro-Inflammatory Flagellin Proteins of Prevalent Motile Commensal Bacteria Are Variably Abundant in the Intestinal Microbiome of Elderly Humans

Some Eubacterium and Roseburia species are among the most prevalent motile bacteria present in the intestinal microbiota of healthy adults. These flagellate species contribute “cell motility” category genes to the intestinal microbiome and flagellin proteins to the intestinal proteome. We reviewed and revised the annotation of motility genes in the genomes of six Eubacterium and Roseburia species that occur in the human intestinal microbiota and examined their respective locus organization by comparative genomics. Motility gene order was generally conserved across these loci. Five of these species harbored multiple genes for predicted flagellins. Flagellin proteins were isolated from R. inulinivorans strain A2-194 and from E. rectale strains A1-86 and M104/1. The amino-termini sequences of the R. inulinivorans and E. rectale A1-86 proteins were almost identical. These protein preparations stimulated secretion of interleukin-8 (IL-8) from human intestinal epithelial cell lines, suggesting that these flagellins were pro-inflammatory. Flagellins from the other four species were predicted to be pro-inflammatory on the basis of alignment to the consensus sequence of pro-inflammatory flagellins from the β- and γ- proteobacteria. Many fliC genes were deduced to be under the control of σ²⁸. The relative abundance of the target Eubacterium and Roseburia species varied across shotgun metagenomes from 27 elderly individuals. Genes involved in the flagellum biogenesis pathways of these species were variably abundant in these metagenomes, suggesting that the current depth of coverage used for metagenomic sequencing (3.13–4.79 Gb total sequence in our study) insufficiently captures the functional diversity of genomes present at low (≤1%) relative abundance. E. rectale and R. inulinivorans thus appear to synthesize complex flagella composed of flagellin proteins that stimulate IL-8 production. A greater depth of sequencing, improved evenness of sequencing and improved metagenome assembly from short reads will be required to facilitate in silico analyses of complete complex biochemical pathways for low-abundance target species from shotgun metagenomes.     

In Vitro Fermentation of NUTRIOSE? FB06, a Wheat Dextrin Soluble Fibre,in a Continuous Culture Human Colonic Model System

Wheat dextrin soluble fibre may have metabolic and health benefits, potentially acting via mechanisms governed by the selective modulation of the human gut microbiota. Our aim was to examine the impact of wheat dextrin on the composition and metabolic activity of the gut microbiota. We used a validated in vitro three-stage continuous culture human colonic model (gut model) system comprised of vessels simulating anatomical regions of the human colon. To mimic human ingestion, 7 g of wheat dextrin (NUTRIOSE^® FB06) was administered to three gut models, twice daily at 10.00 and 15.00, for a total of 18 days. Samples were collected and analysed for microbial composition and organic acid concentrations by 16S rRNA-based fluorescence in situ hybridisation and gas chromatography approaches, respectively. Wheat dextrin mediated a significant increase in total bacteria in vessels simulating the transverse and distal colon, and a significant increase in key butyrate-producing bacteria Clostridium cluster XIVa and Roseburia genus in all vessels of the gut model. The production of principal short-chain fatty acids, acetate, propionate and butyrate, which have been purported to have protective, trophic and metabolic host benefits, were increased. Specifically, wheat dextrin fermentation had a significant butyrogenic effect in all vessels of the gut model and significantly increased production of acetate (vessels 2 and 3) and propionate (vessel 3), simulating the transverse and distal regions of the human colon, respectively. In conclusion, wheat dextrin NUTRIOSE^® FB06 is selectively fermented in vitro by Clostridium cluster XIVa and Roseburia genus and beneficially alters the metabolic profile of the human gut microbiota.     

pH and Peptide Supply Can Radically Alter Bacterial Populations and Short-Chain Fatty Acid Ratios within Microbial Communities from the Human Colon

The effects of changes in the gut environment upon the human colonic microbiota are poorly understood. The response of human fecal microbial communities from two donors to alterations in pH (5.5 or 6.5) and peptides (0.6 or 0.1%) was studied here in anaerobic continuous cultures supplied with a mixed carbohydrate source. Final butyrate concentrations were markedly higher at pH 5.5 (0.6% peptide mean, 24.9 mM; 0.1% peptide mean, 13.8 mM) than at pH 6.5 (0.6% peptide mean, 5.3 mM; 0.1% peptide mean, 7.6 mM). At pH 5.5 and 0.6% peptide input, a high butyrate production coincided with decreasing acetate concentrations. The highest propionate concentrations (mean, 20.6 mM) occurred at pH 6.5 and 0.6% peptide input. In parallel, major bacterial groups were monitored by using fluorescence in situ hybridization with a panel of specific 16S rRNA probes. Bacteroides levels increased from ca. 20 to 75% of total eubacteria after a shift from pH 5.5 to 6.5, at 0.6% peptide, coinciding with high propionate formation. Conversely, populations of the butyrate-producing Roseburia group were highest (11 to 19%) at pH 5.5 but fell at pH 6.5, a finding that correlates with butyrate formation. When tested in batch culture, three Bacteroides species grew well at pH 6.7 but poorly at pH 5.5, which is consistent with the behavior observed for the mixed community. Two Roseburia isolates grew equally well at pH 6.7 and 5.5. These findings suggest that a lowering of pH resulting from substrate fermentation in the colon may boost butyrate production and populations of butyrate-producing bacteria, while at the same time curtailing the growth of Bacteroides spp.     

Gut microbiota promotes production of aromatic metabolites through degradation of barley leaf fiber

Barley leaf (BL) contains abundant plant fibers, which are important substrates for the metabolism and degradation by the gut microbiota. Here we show that mice fed a diet supplemented with BL exhibited altered gut bacterial composition characterized by the enrichment of fiber-degrading bacteria Lachnospiraceae and Prevotella. Gut microbiota-mediated BL degradation promoted butyrate and propionate production. Metabolomic analysis showed increased aromatic metabolites such as ferulic acid, 3-phenylpropanoic acid, 3-hydroxyphenylacetic acid and 3-hydroxyphenylpropionic acid in feces of mice fed with BL. Finally, antibiotic treatment and anaerobic fermentation confirmed the obligate role of gut microbiota in the production of aromatic metabolites during BL degradation. Together, these findings provide insights into a gut microbiota-mediated degradation process of BL fiber components, which results in the production of microbial-associated metabolites that may exert potential active effects on host physiology.     

Alterations in the gut microbiota and metabolite profiles of patients with Kashin-Beck disease,an endemic osteoarthritis in China

Kashin-Beck disease (KBD) is a severe osteochondral disorder that may be driven by the interaction between genetic and environmental factors. We aimed to improve our understanding of the gut microbiota structure in KBD patients of different grades and the relationship between the gut microbiota and serum metabolites. Fecal and serum samples collected from KBD patients and normal controls (NCs) were used to characterize the gut microbiota using 16S rDNA gene and metabolomic sequencing via liquid chromatography-mass spectrometry (LC/MS). To identify whether gut microbial changes at the species level are associated with the genes or functions of the gut bacteria in the KBD patients, metagenomic sequencing of fecal samples from grade I KBD, grade II KBD and NC subjects was performed. The KBD group was characterized by elevated levels of Fusobacteria and Bacteroidetes. A total of 56 genera were identified to be significantly differentially abundant between the two groups. The genera Alloprevotella, Robinsoniella, Megamonas, and Escherichia_Shigella were more abundant in the KBD group. Consistent with the 16S rDNA analysis at the genus level, most of the differentially abundant species in KBD subjects belonged to the genus Prevotella according to metagenomic sequencing. Serum metabolomic analysis identified some differentially abundant metabolites among the grade I and II KBD and NC groups that were involved in lipid metabolism metabolic networks, such as that for unsaturated fatty acids and glycerophospholipids. Furthermore, we found that these differences in metabolite levels were associated with altered abundances of specific species. Our study provides a comprehensive landscape of the gut microbiota and metabolites in KBD patients and provides substantial evidence of a novel interplay between the gut microbiome and metabolome in KBD pathogenesis.Subject terms: Metagenomics, Metabolomics     

Impact of tart cherries polyphenols on the human gut microbiota and phenolic metabolites in vitro and in vivo

Tart cherries have been reported to exert potential health benefits attributed to their specific and abundant polyphenol content. However, there is a need to study the impact and fate of tart cherries polyphenols in the gut microbiota. Here, tart cherries, pure polyphenols (and apricots) were submitted to in vitro bacterial fermentation assays and assessed through 16S rRNA gene sequence sequencing and metabolomics. A short-term (5 days, 8 oz. daily) human dietary intervention study was also conducted for microbiota analyses.Tart cherry concentrate juices were found to contain expected abundances of anthocyanins (cyanidin-glycosylrutinoside) and flavonoids (quercetin-rutinoside) and high amounts of chlorogenic and neochlorogenic acids. Targeted metabolomics confirmed that gut microbes were able to degrade those polyphenols mainly to 4-hydroxyphenylpropionic acids and to lower amounts of epicatechin and 4-hydroxybenzoic acids. Tart cherries were found to induce a large increase of Bacteroides in vitro, likely due to the input of polysaccharides, but prebiotic effect was also suggested by Bifidobacterium increase from chlorogenic acid. In the human study, two distinct and inverse responses to tart cherry consumption were associated with initial levels of Bacteroides. High-Bacteroides individuals responded with a decrease in Bacteroides and Bifidobacterium, and an increase of Lachnospiraceae, Ruminococcus and Collinsella. Low-Bacteroides individuals responded with an increase in Bacteroides or Prevotella and Bifidobacterium, and a decrease of Lachnospiraceae, Ruminococcus and Collinsella. These data confirm that gut microbiota metabolism, in particular the potential existence of different metabotypes, needs to be considered in studies attempting to link tart cherries consumption and health.     

Gene-targeted metagenomic analysis of glucan-branching enzyme gene profiles among human and animal fecal microbiota

Glycoside hydrolases (GHs), the enzymes that breakdown complex carbohydrates, are a highly diversified class of key enzymes associated with the gut microbiota and its metabolic functions. To learn more about the diversity of GHs and their potential role in a variety of gut microbiomes, we used a combination of 16S, metagenomic and targeted amplicon sequencing data to study one of these enzyme families in detail. Specifically, we employed a functional gene-targeted metagenomic approach to the 1-4-α-glucan-branching enzyme (gBE) gene in the gut microbiomes of four host species (human, chicken, cow and pig). The characteristics of operational taxonomic units (OTUs) and operational glucan-branching units (OGBUs) were distinctive in each of hosts. Human and pig were most similar in OTUs profiles while maintaining distinct OGBU profiles. Interestingly, the phylogenetic profiles identified from 16S and gBE gene sequences differed, suggesting the presence of different gBE genes in the same OTU across different vertebrate hosts. Our data suggest that gene-targeted metagenomic analysis is useful for an in-depth understanding of the diversity of a particular gene of interest. Specific carbohydrate metabolic genes appear to be carried by distinct OTUs in different individual hosts and among different vertebrate species'' microbiomes, the characteristics of which differ according to host genetic background and/or diet.     

Identification of N-Acetylhexosamine 1-Kinase in the Complete Lacto-N-Biose I/Galacto-N-Biose Metabolic Pathway in Bifidobacterium longum

We have determined the functions of the enzymes encoded by the lnpB, lnpC, and lnpD genes, located downstream of the lacto-N-biose phosphorylase gene (lnpA), in Bifidobacterium longum JCM1217. The lnpB gene encodes a novel kinase, N-acetylhexosamine 1-kinase, which produces N-acetylhexosamine 1-phosphate; the lnpC gene encodes UDP-glucose hexose 1-phosphate uridylyltransferase, which is also active on N-acetylhexosamine 1-phosphate; and the lnpD gene encodes a UDP-glucose 4-epimerase, which is active on both UDP-galactose and UDP-N-acetylgalactosamine. These results suggest that the gene operon lnpABCD encodes a previously undescribed lacto-N-biose I/galacto-N-biose metabolic pathway that is involved in the intestinal colonization of bifidobacteria and that utilizes lacto-N-biose I from human milk oligosaccharides or galacto-N-biose from mucin sugars.     

Comparative study on gut microbiota in three Anura frogs from a mountain stream

Composition and diversity in gut microbiota are impacted by a wide variety of factors. The similarity of gut microbiota in related or sympatric species has been gaining recent traction. Here, 16S rRNA gene sequencing technology was employed to study the gut microbiota of three sympatric frog species, namely Odorrana tormota, O. graminea, and Amolops wuyiensis. In these three frog species, the most abundant phylum was Proteobacteria, followed by Bacteroidetes, Verrucomicrobia, and Firmicutes. The most abundant family was Burkholderiaceae in three species. The most dominant genera were Burkholderia, Caballeronia, and Paraburkholderia with the highest relative abundance in O. tormota, O. graminea, and A. wuyiensis, respectively. No differences were observed in alpha diversity indexes among the three frog species. However, bacterial similarity of gut microbiota was significantly different between O. tormota and A. wuyiensis and between O. graminea and A. wuyiensis. Metabolism‐related gene function was predominantly enriched in the gut microbiota of the three evaluated frog species. From these findings, that the relative abundance of the gut microbiota and predicted gene functions differed in three species, we conclude that there were significant differences in the gut microbiota of the three species. Similar alpha diversity and interspecific bacterial similarity in the gut might be related to bacterial transmission among the three Anura frogs evaluated in this study.     

Cultivable butyrate-producing bacteria of elderly Japanese diagnosed with Alzheimer’s disease

The group of butyrate-producing bacteria within the human gut microbiome may be associated with positive effects on memory improvement, according to previous studies on dementia-associated diseases. Here, fecal samples of four elderly Japanese diagnosed with Alzheimer’s disease (AD) were used to isolate butyrate-producing bacteria. 226 isolates were randomly picked, their 16S rRNA genes were sequenced, and assigned into sixty OTUs (operational taxonomic units) based on BLASTn results. Four isolates with less than 97% homology to known sequences were considered as unique OTUs of potentially butyrate-producing bacteria. In addition, 12 potential butyrate-producing isolates were selected from the remaining 56 OTUs based on scan-searching against the PubMed and the ScienceDirect databases. Those belonged to the phylum Bacteroidetes and to the clostridial clusters I, IV, XI, XV, XIVa within the phylum Firmicutes. 15 out of the 16 isolates were indeed able to produce butyrate in culture as determined by high-performance liquid chromatography with UV detection. Furthermore, encoding genes for butyrate formation in these bacteria were identified by sequencing of degenerately primed PCR products and included the genes for butyrate kinase (buk), butyryl-CoA: acetate CoAtransferase (but), CoA-transferase-related, and propionate CoA-transferase. The results showed that eight isolates possessed buk, while five isolates possessed but. The CoA-transfer-related gene was identified as butyryl-CoA:4-hydroxybutyrate CoA transferase (4-hbt) in four strains. No strains contained the propionate CoA-transferase gene. The biochemical and butyrate-producing pathways analyses of butyrate producers presented in this study may help to characterize the butyrate-producing bacterial community in the gut of AD patients.     

Changes in Gut Microbiota in Rats Fed a High Fat Diet Correlate with Obesity-Associated Metabolic Parameters

The gut microbiota is emerging as a new factor in the development of obesity. Many studies have described changes in microbiota composition in response to obesity and high fat diet (HFD) at the phylum level. In this study we used 16s RNA high throughput sequencing on faecal samples from rats chronically fed HFD or control chow (n = 10 per group, 16 weeks) to investigate changes in gut microbiota composition at the species level. 53.17% dissimilarity between groups was observed at the species level. Lactobacillus intestinalis dominated the microbiota in rats under the chow diet. However this species was considerably less abundant in rats fed HFD (P<0.0001), this being compensated by an increase in abundance of propionate/acetate producing species. To further understand the influence of these species on the development of the obese phenotype, we correlated their abundance with metabolic parameters associated with obesity. Of the taxa contributing the most to dissimilarity between groups, 10 presented significant correlations with at least one of the tested parameters, three of them correlated positively with all metabolic parameters: Phascolarctobacterium, Proteus mirabilis and Veillonellaceae, all propionate/acetate producers. Lactobacillus intestinalis was the only species whose abundance was negatively correlated with change in body weight and fat mass. This species decreased drastically in response to HFD, favouring propionate/acetate producing bacterial species whose abundance was strongly correlated with adiposity and deterioration of metabolic factors. Our observations suggest that these species may play a key role in the development of obesity in response to a HFD.     

Non Digestible Oligosaccharides Modulate the Gut Microbiota to Control the Development of Leukemia and Associated Cachexia in Mice

We tested the hypothesis that changing the gut microbiota using pectic oligosaccharides (POS) or inulin (INU) differently modulates the progression of leukemia and related metabolic disorders. Mice were transplanted with Bcr-Abl-transfected proB lymphocytes mimicking leukemia and received either POS or INU in their diet (5%) for 2 weeks. Combination of pyrosequencing, PCR-DGGE and qPCR analyses of the 16S rRNA gene revealed that POS decreased microbial diversity and richness of caecal microbiota whereas it increased Bifidobacterium spp., Roseburia spp. and Bacteroides spp. (affecting specifically B. dorei) to a higher extent than INU. INU supplementation increased the portal SCFA propionate and butyrate, and decreased cancer cell invasion in the liver. POS treatment did not affect hepatic cancer cell invasion, but was more efficient than INU to decrease the metabolic alterations. Indeed, POS better than INU delayed anorexia linked to cancer progression. In addition, POS treatment increased acetate in the caecal content, changed the fatty acid profile inside adipose tissue and counteracted the induction of markers controlling β-oxidation, thereby hampering fat mass loss. Non digestible carbohydrates with prebiotic properties may constitute a new nutritional strategy to modulate gut microbiota with positive consequences on cancer progression and associated cachexia.     

The gut microbiome and microbial metabolites in acute myocardial infarction

Emerging evidence has highlighted the role of gut microbiome in human health. However, the integrative role of gut microbiome and microbial metabolites in acute myocardial infarction (AMI) remains unclear. The current study profiles the microbial community through 16S rRNA gene sequencing and shotgun metagenomic sequencing and measures fecal short-chain fatty acids and circulating choline pathway metabolites among 117 new-onset AMI cases and 78 controls. Significant microbial alternations are observed in AMI patients compared with controls (P = 0.001). The abundances of nine species (e.g., Streptococcus salivarius and Klebsiella pneumoniae) are positively associated, and one species (Roseburia hominis) is inversely associated with AMI status and severity. A gut microbial score at disease onset is associated with the risk of major adverse cardiovascular events in 3.2 years (hazard ratio [95% CI]: 2.01 [1.04–4.24]) in AMI patients. The molar proportions of fecal acetate and butyrate are higher, and the circulating levels of choline and carnitine are lower in AMI patients than in controls. In addition, disease classifiers show that AMI cases and controls have a more distinct pattern in taxonomical composition than in pathways or metabolites. Our findings suggest that microbial composition and functional potentials are associated with AMI status and severity.     

Formation of propionate and butyrate by the human colonic microbiota

The human gut microbiota ferments dietary non‐digestible carbohydrates into short‐chain fatty acids (SCFA). These microbial products are utilized by the host and propionate and butyrate in particular exert a range of health‐promoting functions. Here an overview of the metabolic pathways utilized by gut microbes to produce these two SCFA from dietary carbohydrates and from amino acids resulting from protein breakdown is provided. This overview emphasizes the important role played by cross‐feeding of intermediary metabolites (in particular lactate, succinate and 1,2‐propanediol) between different gut bacteria. The ecophysiology, including growth requirements and responses to environmental factors, of major propionate and butyrate producing bacteria are discussed in relation to dietary modulation of these metabolites. A detailed understanding of SCFA metabolism by the gut microbiota is necessary to underpin effective strategies to optimize SCFA supply to the host.     

Discovery and Characterization of FMN-Binding β-Glucuronidases in the Human Gut Microbiome

The human gut microbiota encodes β-glucuronidases (GUSs) that play key roles in health and disease via the metabolism of glucuronate-containing carbohydrates and drugs. Hundreds of putative bacterial GUS enzymes have been identified by metagenomic analysis of the human gut microbiome, but less than 10% have characterized structures and functions. Here we describe a set of unique gut microbial GUS enzymes that bind flavin mononucleotide (FMN). First, we show using mass spectrometry, isothermal titration calorimetry, and x-ray crystallography that a purified GUS from the gut commensal microbe Faecalibacterium prausnitzii binds to FMN on a surface groove located 30 Å away from the active site. Second, utilizing structural and functional data from this FMN-binding GUS, we analyzed the 279 unique GUS sequences from the Human Microbiome Project database and identified 14 putative FMN-binding GUSs. We characterized four of these hits and solved the structure of two, the GUSs from Ruminococcus gnavus and Roseburia hominis, which confirmed that these are FMN binders. Third, binding and kinetic analysis of the FMN-binding site mutants of these five GUSs show that they utilize a conserved site to bind FMN that is not essential for GUS activity, but can affect K_M. Lastly, a comprehensive structural review of the PDB reveals that the FMN-binding site employed by these enzymes is unlike any structurally characterized FMN binders to date. These findings reveal the first instance of an FMN-binding glycoside hydrolase and suggest a potential link between FMN and carbohydrate metabolism in the human gut microbiota.     

Increased Expression of Colonic Mucosal Melatonin in Patients with Irritable Bowel Syndrome Correlated with Gut Dysbiosis

Dysregulation of the gut microbiota/gut hormone axis contributes to the pathogenesis of irritable bowel syndrome (IBS). Melatonin plays a beneficial role in gut motility and immunity. However, altered expression of local mucosal melatonin in IBS and its relationship with the gut microbiota remain unclear. Therefore, we aimed to detect the colonic melatonin levels and microbiota profiles in patients with diarrhea-predominant IBS (IBS-D) and explore their relationship in germ-free (GF) rats and BON-1 cells. Thirty-two IBS-D patients and twenty-eight healthy controls (HCs) were recruited. Fecal specimens from IBS-D patients and HCs were separately transplanted into GF rats by gavage. The levels of colon mucosal melatonin were assessed by immunohistochemical methods, and fecal microbiota communities were analyzed using 16S rDNA sequencing. The effect of butyrate on melatonin synthesis in BON-1 cells was evaluated by ELISA. Melatonin levels were significantly increased and negatively correlated with visceral hypersensitivity in IBS-D patients. GF rats inoculated with fecal microbiota from IBS-D patients had high colonic melatonin levels. Butyrate-producing Clostridium cluster XIVa species, such as Roseburia species and Lachnospira species, were positively related to colonic mucosal melatonin expression. Butyrate significantly increased melatonin secretion in BON-1 cells. Increased melatonin expression may be an adaptive protective mechanism in the development of IBS-D. Moreover, some Clostridium cluster XIVa species could increase melatonin expression via butyrate production. Modulation of the gut hormone/gut microbiota axis offers a promising target of interest for IBS in the future.     

Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.