Exploring the sequence-function space of microbial fucosidases.

Microbial α-L-fucosidases catalyse the hydrolysis of terminal α-L-fucosidic linkages and can perform transglycosylation reactions. Based on sequence identity, α-L-fucosidases are classified in glycoside hydrolases (GHs) families of the carbohydrate-active enzyme database. Here we explored the sequence-function space of GH29 fucosidases. Based on sequence similarity network (SSN) analyses, 15 GH29 α-L-fucosidases were selected for functional characterisation. HPAEC-PAD and LC-FD-MS/MS analyses revealed substrate and linkage specificities for α1,2, α1,3, α1,4 and α1,6 linked fucosylated oligosaccharides and glycoconjugates, consistent with their SSN clustering. The structural basis for the substrate specificity of GH29 fucosidase from Bifidobacterium asteroides towards α1,6 linkages and FA2G2 N-glycan was determined by X-ray crystallography and STD NMR. The capacity of GH29 fucosidases to carry out transfucosylation reactions with GlcNAc and 3FN as acceptors was evaluated by TLC combined with ESI-MS and NMR. These experimental data supported the use of SSN to further explore the GH29 sequence-function space through machine-learning models. Our lightweight protein language models could accurately allocate test sequences in their respective SSN clusters and assign 34,258 non-redundant GH29 sequences into SSN clusters. It is expected that the combination of these computational approaches will be used in the future for the identification of novel GHs with desired specificities.

A novel Mediterranean diet-inspired supplement ameliorates cognitive, microbial, and metabolic deficits in a mouse model of low-grade inflammation.

The Mediterranean diet (MD) and its bioactive constituents have been advocated for their neuroprotective properties along with their capacity to affect gut microbiota speciation and metabolism. Mediated through the gut brain axis, this modulation of the microbiota may partly contribute to the neuroprotective properties of the MD. To explore this potential interaction, we evaluated the neuroprotective properties of a novel bioactive blend (Neurosyn240) resembling the Mediterranean diet in a rodent model of chronic low-grade inflammation. Behavioral tests of cognition, brain proteomic analysis, 16S rRNA sequencing, and 1H NMR metabolomic analyses were employed to develop an understanding of the gut-brain axis interactions involved. Recognition memory, as assessed by the novel object recognition task (NOR), decreased in response to LPS insult and was restored with Neurosyn240 supplementation. Although the open field task performance did not reach significance, it correlated with NOR performance indicating an element of anxiety related to this cognitive change. Behavioral changes associated with Neurosyn240 were accompanied by a shift in the microbiota composition which included the restoration of the Firmicutes: Bacteroidota ratio and an increase in Muribaculum, Rikenellaceae Alloprevotella, and most notably Akkermansia which significantly correlated with NOR performance. Akkermansia also correlated with the metabolites 5-aminovalerate, threonine, valine, uridine monophosphate, and adenosine monophosphate, which in turn significantly correlated with NOR performance. The proteomic profile within the brain was dramatically influenced by both interventions, with KEGG analysis highlighting oxidative phosphorylation and neurodegenerative disease-related pathways to be modulated. Intriguingly, a subset of these proteomic changes simultaneously correlated with Akkermansia abundance and predominantly related to oxidative phosphorylation, perhaps alluding to a protective gut-brain axis interaction. Collectively, our results suggest that the bioactive blend Neurosyn240 conferred cognitive and microbiota resilience in response to the deleterious effects of low-grade inflammation.

Refined diet consumption increases neuroinflammatory signalling through bile acid dysmetabolism.

Over recent decades, dietary patterns have changed significantly due to the increasing availability of convenient, ultra-processed refined foods. Refined foods are commonly depleted of key bioactive compounds, which have been associated with several deleterious health conditions. As the gut microbiome can influence the brain through a bidirectional communication system known as the 'microbiota-gut-brain axis', the consumption of refined foods has the potential to affect cognitive health. In this study, multi-omics approaches were employed to assess the effect of a refined diet on the microbiota-gut-brain axis, with a particular focus on bile acid metabolism. Mice maintained on a refined low-fat diet (rLFD), consisting of high sucrose, processed carbohydrates and low fibre content, for eight weeks displayed significant gut microbial dysbiosis, as indicated by diminished alpha diversity metrics (p < 0.05) and altered beta diversity (p < 0.05) when compared to mice receiving a chow diet. Changes in gut microbiota composition paralleled modulation of the metabolome, including a significant reduction in short-chain fatty acids (acetate, propionate and n-butyrate; p < 0.001) and alterations in bile acid concentrations. Interestingly, the rLFD led to dysregulated bile acid concentrations across both the colon (p < 0.05) and the brain (p < 0.05) which coincided with altered neuroinflammatory gene expression. In particular, the concentration of TCA, TDCA and T-α-MCA was inversely correlated with the expression of NF-κB1, a key transcription factor in neuroinflammation. Overall, our results suggest a novel link between a refined low-fat diet and detrimental neuronal processes, likely in part through modulation of the microbiota-gut-brain axis and bile acid dysmetabolism.

Saffron extract (Safr'Inside™) improves anxiety related behaviour in a mouse model of low-grade inflammation through the modulation of the microbiota and gut derived metabolites.

Treatment of anxiety and depression predominantly centres around pharmacological interventions, which have faced criticism for their associated side effects, lack of efficacy and low tolerability. Saffron, which is reportedly well tolerated in humans, has been recognised for its antidepressant and anti-anxiety properties. Indeed, we previously reported upon the efficacy of saffron extract supplementation in healthy adults with subclinical anxiety. However, the molecular aetiology remains unclear. In a rodent model of low-grade chronic inflammation, we explored the impact of a saffron extract (Safr'Inside™) supplemented at a physiological dose, which equated to 22 ± 1.2 mg per day human equivalent dose for a person of 60 kg. Behavioural tests (Open Field task, Y maze, Novel object recognition), caecal 16S rRNA microbial sequencing, caecal 1H NMR metabolomic analysis and 2DE brain proteomic analyses were completed to probe gut-brain axis interactions. Time occupying the centre of the Open Field maze (OF) was increased by 62% in saffron supplemented animals. This improvement in anxiety-related behaviour coincided with gut microbial shifts, notably Akkermansia, Muribaculaceae, Christensenellacae and Alloprevotella which significantly increased in response to saffron supplementation. Akkermansia and Muribaculaceae abundance negatively correlated with the neurotoxic metabolite dimethylamine which was reduced in saffron supplemented animals. Brain proteomic analysis highlighted several significantly altered proteins including ketimine reductase mu-crystallin which also correlated with dimethylamine concentration. Both dimethylamine and ketimine reductase mu-crystallin were associated with OF performance. This may be indicative of a novel interaction across the gut-brain axis which contributes to anxiety-related disorders.

(-)-Epicatechin mitigates anxiety-related behavior in a mouse model of high fat diet-induced obesity.

Mounting evidence demonstrates that consumption of high fat diet (HFD) and subsequent development of obesity leads to alterations in cognition and mood. While obesity can affect brain function, consumption of select dietary bioactives may help prevent obesity-related cognitive decline. This study investigated the capacity of the dietary flavonoid (-)-epicatechin (EC) to mitigate HFD-induced obesity-associated alterations in memory and mood. Healthy 8-week old male C57BL/6J mice were maintained on either a control diet (10 kCal% from fat) or a HFD (45 kCal% from fat) and were supplemented with EC at 2 or 20 mg/kg body weight (B.W.) for a 24 week period. Between week 20 and 22, anxiety-related behavior, recognition memory, and spatial memory were measured. Underlying mechanisms were assessed by measuring the expression of selected genes in the hippocampus and by 16S rRNA sequencing and metabolomic analysis of the gut microbiota. 24 weeks of HFD feeding resulted in obesity, which was not affected by EC supplementation. HFD-associated increase in anxiety-related behavior was mitigated by EC in a dose-response manner and was accompanied by increased hippocampal brain-derived neurotrophic factor (BDNF), as well as partial or full restoration of glucocorticoid receptor, mineralocorticoid receptor and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) expression. Higher EC dosage (20 mg/kg B.W.) also restored aberrant Lactobacillus and Enterobacter abundance altered by HFD and/or the associated obesity. Together, these results demonstrate how EC mitigates anxiety-related behaviors, revealing a connection between BDNF- and glucocorticoids-mediated signaling. Our findings link changes in the hippocampus and the gut microbiota in a context of HFD-induced obesity and anxiety.

Maternal gut microbiota Bifidobacterium promotes placental morphogenesis, nutrient transport and fetal growth in mice.

The gut microbiota plays a central role in regulating host metabolism. While substantial progress has been made in discerning how the microbiota influences host functions post birth and beyond, little is known about how key members of the maternal gut microbiota can influence feto-placental growth. Notably, in pregnant women, Bifidobacterium represents a key beneficial microbiota genus, with levels observed to increase across pregnancy. Here, using germ-free and specific-pathogen-free mice, we demonstrate that the bacterium Bifidobacterium breve UCC2003 modulates maternal body adaptations, placental structure and nutrient transporter capacity, with implications for fetal metabolism and growth. Maternal and placental metabolome were affected by maternal gut microbiota (i.e. acetate, formate and carnitine). Histological analysis of the placenta confirmed that Bifidobacterium modifies placental structure via changes in Igf2P0, Dlk1, Mapk1 and Mapk14 expression. Additionally, B. breve UCC2003, acting through Slc2a1 and Fatp1-4 transporters, was shown to restore fetal glycaemia and fetal growth in association with changes in the fetal hepatic transcriptome. Our work emphasizes the importance of the maternal gut microbiota on feto-placental development and sets a foundation for future research towards the use of probiotics during pregnancy.

Microbial-derived metabolites as a risk factor of age-related cognitive decline and dementia.

A consequence of our progressively ageing global population is the increasing prevalence of worldwide age-related cognitive decline and dementia. In the absence of effective therapeutic interventions, identifying risk factors associated with cognitive decline becomes increasingly vital. Novel perspectives suggest that a dynamic bidirectional communication system between the gut, its microbiome, and the central nervous system, commonly referred to as the microbiota-gut-brain axis, may be a contributing factor for cognitive health and disease. However, the exact mechanisms remain undefined. Microbial-derived metabolites produced in the gut can cross the intestinal epithelial barrier, enter systemic circulation and trigger physiological responses both directly and indirectly affecting the central nervous system and its functions. Dysregulation of this system (i.e., dysbiosis) can modulate cytotoxic metabolite production, promote neuroinflammation and negatively impact cognition. In this review, we explore critical connections between microbial-derived metabolites (secondary bile acids, trimethylamine-N-oxide (TMAO), tryptophan derivatives and others) and their influence upon cognitive function and neurodegenerative disorders, with a particular interest in their less-explored role as risk factors of cognitive decline.

Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain.

BACKGROUND: Altered intestinal microbiota composition in later life is associated with inflammaging, declining tissue function, and increased susceptibility to age-associated chronic diseases, including neurodegenerative dementias. Here, we tested the hypothesis that manipulating the intestinal microbiota influences the development of major comorbidities associated with aging and, in particular, inflammation affecting the brain and retina. METHODS: Using fecal microbiota transplantation, we exchanged the intestinal microbiota of young (3 months), old (18 months), and aged (24 months) mice. Whole metagenomic shotgun sequencing and metabolomics were used to develop a custom analysis workflow, to analyze the changes in gut microbiota composition and metabolic potential. Effects of age and microbiota transfer on the gut barrier, retina, and brain were assessed using protein assays, immunohistology, and behavioral testing. RESULTS: We show that microbiota composition profiles and key species enriched in young or aged mice are successfully transferred by FMT between young and aged mice and that FMT modulates resulting metabolic pathway profiles. The transfer of aged donor microbiota into young mice accelerates age-associated central nervous system (CNS) inflammation, retinal inflammation, and cytokine signaling and promotes loss of key functional protein in the eye, effects which are coincident with increased intestinal barrier permeability. Conversely, these detrimental effects can be reversed by the transfer of young donor microbiota. CONCLUSIONS: These findings demonstrate that the aging gut microbiota drives detrimental changes in the gut-brain and gut-retina axes suggesting that microbial modulation may be of therapeutic benefit in preventing inflammation-related tissue decline in later life. Video abstract.

Macrophage metabolism in the intestine is compartment specific and regulated by the microbiota.

Intestinal macrophages play a vital role in the maintenance of gut homeostasis through signals derived from the microbiota. We previously demonstrated that microbial-derived metabolites can shape the metabolic functions of macrophages. Here, we show that antibiotic-induced disruption of the intestinal microbiota dramatically alters both the local metabolite environment and the metabolic functions of macrophages in the colon. Broad-spectrum antibiotic administration in mice increased the expression of the large neutral amino acid transporter LAT1 and accordingly, amino acid uptake. Subsequently, antibiotic administration enhanced the metabolic functions of colonic macrophages, increasing phosphorylation of components of mammalian/mechanistic target of rapamycin signalling pathways, with increased expression of genes involved in glycolysis and oxidative phosphorylation (OXPHOS), increased mitochondrial function, increased rate of extracellular acidification (ECAR; measure of glycolysis) and increased rate of oxygen consumption (OCR; measure of OXPHOS). Small bowel macrophages were less metabolically active than their colonic counterparts, with macrophage metabolism in the small intestine being independent of the microbiota. Finally, we reveal tissue-resident Tim4+  CD4+ macrophages exhibit enhanced fatty acid uptake alongside reduced fatty acid synthesis compared to recruited macrophages. Thus, the microbiota shapes gut macrophage metabolism in a compartment-specific manner, with important implications for monocyte recruitment and macrophage differentiation.

Using a multi-omic approach to investigate the mechanism of 12-bis-THA activity against Burkholderia thailandensis.

Burkholderia pseudomallei is the causative agent of the tropical disease, melioidosis. It is intrinsically resistant to many antimicrobials and treatment requires an onerous regimen of intravenous and orally administered drugs. Relapse of disease and high rates of mortality following treatment are common, demonstrating the need for new anti-Burkholderia agents. The cationic bola-amphiphile, 12,12'-(dodecane-1,12-diyl) bis (9-amino-1,2,3,4-tetrahydroacridinium), referred to as 12-bis-THA, is a molecule with the potential to treat Burkholderia infections. 12-bis-THA spontaneously forms cationic nanoparticles that bind anionic phospholipids in the prokaryotic membrane and are readily internalized. In this study, we examine the antimicrobial activity of 12-bis-THA against strains of Burkholderia thailandensis. As B. pseudomallei produces a polysaccharide capsule we first examined if this extra barrier influenced the activity of 12-bis-THA which is known to act on the bacterial envelope. Therefore two strains of B. thailandensis were selected for further testing, strain E264 which does not produce a capsule and strain E555 which does produce a capsule that is chemically similar to that found in B. pseudomallei. In this study no difference in the minimum inhibitory concentration (MIC) was observed when capsulated (E555) and unencapsulated (E264) strains of B. thailandensis were compared, however time-kill analysis showed that the unencapsulated strain was more susceptible to 12-bis-THA. The presence of the capsule did not affect the membrane permeation of 12-bis-THA at MIC concentrations. Proteomic and metabolomic analyses showed that 12-bis-THA causes a shift in central metabolism away from glycolysis and glyoxylate cycle, and suppressed the production of the F1 domain of ATP synthase. In summary, we provide insight into the molecular mechanisms underpinning the activity of 12-bis-THA against B. thailandensis and discuss its potential for further development.

Lipopolysaccharide associated with ß-2,6 fructan mediates TLR4-dependent immunomodulatory activity in vitro.

Levan, a ß-2,6 fructofuranose polymer produced by microbial species, has been reported for its immunomodulatory properties via interaction with toll-like receptor 4 (TLR4) which recognises lipopolysaccharide (LPS). However, the molecular mechanisms underlying these interactions remain elusive. Here, we investigated the immunomodulatory properties of levan using thoroughly-purified and characterised samples from Erwinia herbicola and other sources. E. herbicola levan was purified by gel-permeation chromatography and LPS was removed from the levan following a novel alkali treatment developed in this study. E. herbicola levan was then characterised by gas chromatography-mass spectrometry and NMR. We found that levan containing LPS, but not LPS-depleted levan, induced TLR4-mediated cytokine production by bone marrow-derived dendritic cells and/or activated TLR4 reporter cells. These data indicated that the immunomodulatory properties of the levan toward TLR4-expressing immune cells were mediated by the LPS. This work also demonstrates the importance of LPS removal when assessing the immunomodulatory activity of polysaccharides.

Differential Influence of Soluble Dietary Fibres on Intestinal and Hepatic Carbohydrate Response.

Refined foods are commonly depleted in certain bioactive components that are abundant in 'natural' (plant) foods. Identification and addition of these 'missing' bioactives in the diet is, therefore, necessary to counteract the deleterious impact of convenience food. In this study, multiomics approaches were employed to assess the addition of the popular supplementary soluble dietary fibers inulin and psyllium, both in isolation and in combination with a refined animal feed. A 16S rRNA sequencing and 1H NMR metabolomic investigation revealed that, whilst inulin mediated an increase in Bifidobacteria, psyllium elicited a broader microbial shift, with Parasutterella and Akkermansia being increased and Enterorhabdus and Odoribacter decreased. Interestingly, the combination diet benefited from both inulin and psyllium related microbial changes. Psyllium mediated microbial changes correlated with a reduction of glucose (R -0.67, -0.73, respectively, p < 0.05) and type 2 diabetes associated metabolites: 3-methyl-2-oxovaleric acid (R -0.72, -0.78, respectively, p < 0.05), and citrulline (R -0.77, -0.71, respectively, p < 0.05). This was in line with intestinal and hepatic carbohydrate response (e.g., Slc2a2, Slc2a5, Khk and Fbp1) and hepatic lipogenesis (e.g., Srebf1 and Fasn), which were significantly reduced under psyllium addition. Although established in the liver, the intestinal response associated with psyllium was absent in the combination diet, placing greater significance upon the established microbial, and subsequent metabolomic, shift. Our results therefore highlight the heterogeneity that exists between distinct dietary fibers in the context of carbohydrate uptake and metabolism, and supports psyllium containing combination diets, for their ability to negate the impact of a refined diet.

Antibiotic-induced disturbances of the gut microbiota result in accelerated breast tumor growth.

The gut microbiota's function in regulating health has seen it linked to disease progression in several cancers. However, there is limited research detailing its influence in breast cancer (BrCa). This study found that antibiotic-induced perturbation of the gut microbiota significantly increases tumor progression in multiple BrCa mouse models. Metagenomics highlights the common loss of several bacterial species following antibiotic administration. One such bacteria, Faecalibaculum rodentium, rescued this increased tumor growth. Single-cell transcriptomics identified an increased number of cells with a stromal signature in tumors, and subsequent histology revealed an increased abundance of mast cells in the tumor stromal regions. We show that administration of a mast cell stabilizer, cromolyn, rescues increased tumor growth in antibiotic treated animals but has no influence on tumors from control cohorts. These findings highlight that BrCa-microbiota interactions are different from other cancers studied to date and suggest new research avenues for therapy development.

Anti-Inflammatory Effects of Quercetin on High-Glucose and Pro-Inflammatory Cytokine Challenged Vascular Endothelial Cell Metabolism.

SCOPE: Pro-inflammatory stimuli such as hyperglycemia and cytokines have been shown to negatively affect endothelial cell functions. The aim of this study is to assess the potential of quercetin and its human metabolites to overcome the deleterious effects of hyperglycemic or inflammatory conditions on the vascular endothelium by modulating endothelial cell metabolism. METHODS AND RESULTS: A metabolomics approach enabled identification and quantification of 27 human umbilical vein endothelial cell (HUVEC) metabolites. Treatment of HUVECs with high-glucose concentrations causes significant increases in lactate and glutamate concentrations. Quercetin inhibits glucose-induced increases in lactate and adenosine 5'-triphosphate (ATP) and also increased inosine concentrations. Tumor necrosis factor α-treatment (TNFα) of HUVECs causes increases in asparagine and decreases in aspartate concentrations. Co-treatment with quercetin reduces pyruvate concentrations compared to TNFα-only treated controls. Subsequently, it was shown that quercetin and its HUVEC phase-2 conjugates inhibit adenosine deaminase, xanthine oxidase and 5'nucleotidase (CD73) but not ectonucleoside triphosphate diphosphohydrolase-1 (CD39) or purine nucleoside phosphorylase activities. CONCLUSION: Quercetin was shown to alter the balance of HUVEC metabolites towards a less inflamed phenotype, both alone and in the presence of pro-inflammatory stimuli. These changes are consistent with the inhibition of particular enzymes involved in purine metabolism by quercetin and its HUVEC metabolites.

Effects of in vitro metabolism of a broccoli leachate, glucosinolates and S-methylcysteine sulphoxide on the human faecal microbiome.

PURPOSE: Brassica are an important food source worldwide and are characterised by the presence of compounds called glucosinolates. Studies indicate that the glucosinolate derived bioactive metabolite sulphoraphane can elicit chemoprotective benefits on human cells. Glucosinolates can be metabolised in vivo by members of the human gut microbiome, although the prevalence of this activity is unclear. Brassica and Allium plants also contain S-methylcysteine sulphoxide (SMCSO), that may provide additional health benefits but its metabolism by gut bacteria is not fully understood. METHODS: We examined the effects of a broccoli leachate (BL) on the composition and function of human faecal microbiomes of five different participants under in vitro conditions. Bacterial isolates from these communities were then tested for their ability to metabolise glucosinolates and SMCSO. RESULTS: Microbial communities cultured in vitro in BL media were observed to have enhanced growth of lactic acid bacteria, such as lactobacilli, with a corresponding increase in the levels of lactate and short-chain fatty acids. Members of Escherichia isolated from these faecal communities were found to bioconvert glucosinolates and SMCSO to their reduced analogues. CONCLUSION: This study uses a broccoli leachate to investigate the bacterial-mediated bioconversion of glucosinolates and SMCSO, which may lead to further products with additional health benefits to the host. We believe that this is the first study that shows the reduction of the dietary compound S-methylcysteine sulphoxide by bacteria isolated from human faeces.

Faecal microbiota transplant from aged donor mice affects spatial learning and memory via modulating hippocampal synaptic plasticity- and neurotransmission-related proteins in young recipients.

BACKGROUND: The gut-brain axis and the intestinal microbiota are emerging as key players in health and disease. Shifts in intestinal microbiota composition affect a variety of systems; however, evidence of their direct impact on cognitive functions is still lacking. We tested whether faecal microbiota transplant (FMT) from aged donor mice into young adult recipients altered the hippocampus, an area of the central nervous system (CNS) known to be affected by the ageing process and related functions. RESULTS: Young adult mice were transplanted with the microbiota from either aged or age-matched donor mice. Following transplantation, characterization of the microbiotas and metabolomics profiles along with a battery of cognitive and behavioural tests were performed. Label-free quantitative proteomics was employed to monitor protein expression in the hippocampus of the recipients. We report that FMT from aged donors led to impaired spatial learning and memory in young adult recipients, whereas anxiety, explorative behaviour and locomotor activity remained unaffected. This was paralleled by altered expression of proteins involved in synaptic plasticity and neurotransmission in the hippocampus. Also, a strong reduction of bacteria associated with short-chain fatty acids (SCFAs) production (Lachnospiraceae, Faecalibaculum, and Ruminococcaceae) and disorders of the CNS (Prevotellaceae and Ruminococcaceae) was observed. Finally, the detrimental effect of FMT from aged donors on the CNS was confirmed by the observation that microglia cells of the hippocampus fimbria, acquired an ageing-like phenotype; on the contrary, gut permeability and levels of systemic and local (hippocampus) cytokines were not affected. CONCLUSION: These results demonstrate that age-associated shifts of the microbiota have an impact on protein expression and key functions of the CNS. Furthermore, these results highlight the paramount importance of the gut-brain axis in ageing and provide a strong rationale to devise therapies aiming to restore a young-like microbiota to improve cognitive functions and the declining quality of life in the elderly. Video Abstract.

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis.

BACKGROUND & AIMS: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota. METHODS: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy. RESULTS: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of Faecalibacterium prausnitzii, a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels. CONCLUSION: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation. LAY SUMMARY: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis.

The early life microbiota protects neonatal mice from pathological small intestinal epithelial cell shedding.

The early life gut microbiota plays a crucial role in regulating and maintaining the intestinal barrier, with disturbances in these communities linked to dysregulated renewal and replenishment of intestinal epithelial cells. Here we sought to determine pathological cell shedding outcomes throughout the postnatal developmental period, and which host and microbial factors mediate these responses. Surprisingly, neonatal mice (Day 14 and 21) were highly refractory to induction of cell shedding after intraperitoneal administration of liposaccharide (LPS), with Day 29 mice showing strong pathological responses, more similar to those observed in adult mice. These differential responses were not linked to defects in the cellular mechanisms and pathways known to regulate cell shedding responses. When we profiled microbiota and metabolites, we observed significant alterations. Neonatal mice had high relative abundances of Streptococcus, Escherichia, and Enterococcus and increased primary bile acids. In contrast, older mice were dominated by Candidatus Arthromitus, Alistipes, and Lachnoclostridium, and had increased concentrations of SCFAs and methyamines. Antibiotic treatment of neonates restored LPS-induced small intestinal cell shedding, whereas adult fecal microbiota transplant alone had no effect. Our findings further support the importance of the early life window for microbiota-epithelial interactions in the presence of inflammatory stimuli and highlights areas for further investigation.

Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism.

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiIMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer's disease pathophysiology.

Apolipoprotein E (APOE) genotype is the strongest prevalent genetic risk factor for Alzheimer's disease (AD). Numerous studies have provided insights into the pathologic mechanisms. However, a comprehensive understanding of the impact of APOE genotype on microflora speciation and metabolism is completely lacking. In this study, we investigated the association between APOE genotype and the gut microbiome composition in human and APOE-targeted replacement (TR) transgenic mice. Fecal microbiota amplicon sequencing from matched individuals with different APOE genotypes revealed no significant differences in overall microbiota diversity in group-aggregated human APOE genotypes. However, several bacterial taxa showed significantly different relative abundance between APOE genotypes. Notably, we detected an association of Prevotellaceae and Ruminococcaceae and several butyrate-producing genera abundances with APOE genotypes. These findings were confirmed by comparing the gut microbiota of APOE-TR mice. Furthermore, metabolomic analysis of murine fecal water detected significant differences in microbe-associated amino acids and short-chain fatty acids between APOE genotypes. Together, these findings indicate that APOE genotype is associated with specific gut microbiome profiles in both humans and APOE-TR mice. This suggests that the gut microbiome is worth further investigation as a potential target to mitigate the deleterious impact of the APOE4 allele on cognitive decline and the prevention of AD.-Tran, T. T. T., Corsini, S., Kellingray, L., Hegarty, C., Le Gall, G., Narbad, A., Müller, M., Tejera, N., O'Toole, P. W., Minihane, A.-M., Vauzour, D. APOE genotype influences the gut microbiome structure and function in humans and mice: relevance for Alzheimer's disease pathophysiology.