A microbial-derived succinylated bile acid to safeguard liver health.

In this issue of Cell, Nie and co-authors report that the microbe-derived bile acid (BA) 3-succinylated cholic acid protects against the progression of metabolic dysfunction-associated liver disease. Intriguingly, its protective mechanism does not involve traditional BA signaling pathways but is instead linked to the proliferation of the commensal microbe Akkermansia muciniphila.

Binding of Akkermansia muciniphila to mucin is O-glycan specific.

The intestinal anaerobic bacterium Akkermansia muciniphila is specialized in the degradation of mucins, which are heavily O-glycosylated proteins that constitute the major components of the mucus lining the intestine. Despite that adhesion to mucins is considered critical for the persistence of A. muciniphila in the human intestinal tract, our knowledge of how this intestinal symbiont recognizes and binds to mucins is still limited. Here, we first show that the mucin-binding properties of A. muciniphila are independent of environmental oxygen concentrations and not abolished by pasteurization. We then dissected the mucin-binding properties of pasteurized A. muciniphila by use of a recently developed cell-based mucin array that enables display of the tandem repeats of human mucins with distinct O-glycan patterns and structures. We found that A. muciniphila recognizes the unsialylated LacNAc (Galß1-4GlcNAcß1-R) disaccharide selectively on core2 and core3 O-glycans. This disaccharide epitope is abundantly found on human colonic mucins capped by sialic acids, and we demonstrated that endogenous A. muciniphila neuraminidase activity can uncover the epitope and promote binding. In summary, our study provides insights into the mucin-binding properties important for colonization of a key mucin-foraging bacterium.

Heterologous expression of P9 from Akkermansia muciniphila increases the GLP-1 secretion of intestinal L cells.

Glucagon-like peptide-1(GLP-1) is an incretin hormone secreted primarily from the intestinal L-cells in response to meals. GLP-1 is a key regulator of energy metabolism and food intake. It has been proven that P9 protein from A. muciniphila could increase GLP-1 release and improve glucose homeostasis in HFD-induced mice. To obtain an engineered Lactococcus lactis which produced P9 protein, mature polypeptide chain of P9 was codon-optimized, fused with N-terminal signal peptide Usp45, and expressed in L. lactis NZ9000. Heterologous secretion of P9 by recombinant L. lactis NZP9 were successfully detected by SDS-PAGE and western blotting. Notably, the supernatant of L. lactis NZP9 stimulated GLP-1 production of NCI-H716 cells. The relative expression level of GLP-1 biosynthesis gene GCG and PCSK1 were upregulated by 1.63 and 1.53 folds, respectively. To our knowledge, this is the first report on the secretory expression of carboxyl-terminal processing protease P9 from A. muciniphila in L. lactis. Our results suggest that genetically engineered L. lactis which expressed P9 may have therapeutic potential for the treatment of diabetes, obesity and other metabolic disorders.

Gut symbionts alleviate MASH through a secondary bile acid biosynthetic pathway.

The gut microbiota has been found to play an important role in the progression of metabolic dysfunction-associated steatohepatitis (MASH), but the mechanisms have not been established. Here, by developing a click-chemistry-based enrichment strategy, we identified several microbial-derived bile acids, including the previously uncharacterized 3-succinylated cholic acid (3-sucCA), which is negatively correlated with liver damage in patients with liver-tissue-biopsy-proven metabolic dysfunction-associated fatty liver disease (MAFLD). By screening human bacterial isolates, we identified Bacteroides uniformis strains as effective producers of 3-sucCA both in vitro and in vivo. By activity-based protein purification and identification, we identified an enzyme annotated as ß-lactamase in B. uniformis responsible for 3-sucCA biosynthesis. Furthermore, we found that 3-sucCA is a lumen-restricted metabolite and alleviates MASH by promoting the growth of Akkermansia muciniphila. Together, our data offer new insights into the gut microbiota-liver axis that may be leveraged to augment the management of MASH.

Mechanism of 2'-fucosyllactose degradation by human-associated Akkermansia.

Among the first microorganisms to colonize the human gut of breastfed infants are bacteria capable of fermenting human milk oligosaccharides (HMOs). One of the most abundant HMOs, 2'-fucosyllactose (2'-FL), may specifically drive bacterial colonization of the intestine. Recently, differential growth has been observed across multiple species of Akkermansia on various HMOs including 2'-FL. In culture, we found growth of two species, A. muciniphila MucT and A. biwaensis CSUN-19,on HMOs corresponded to a decrease in the levels of 2'-FL and an increase in lactose, indicating that the first step in 2'-FL catabolism is the cleavage of fucose. Using phylogenetic analysis and transcriptional profiling, we found that the number and expression of fucosidase genes from two glycoside hydrolase (GH) families, GH29 and GH95, vary between these two species. During the mid-log phase of growth, the expression of several GH29 genes was increased by 2'-FL in both species, whereas the GH95 genes were induced only in A. muciniphila. We further show that one putative fucosidase and a ß-galactosidase from A. biwaensis are involved in the breakdown of 2'-FL. Our findings indicate that the plasticity of GHs of human-associated Akkermansia sp. enables access to additional growth substrates present in HMOs, including 2'-FL. Our work highlights the potential for Akkermansia to influence the development of the gut microbiota early in life and expands the known metabolic capabilities of this important human symbiont.IMPORTANCEAkkermansia are mucin-degrading specialists widely distributed in the human population. Akkermansia biwaensis has recently been observed to have enhanced growth relative to other human-associated Akkermansia on multiple human milk oligosaccharides (HMOs). However, the mechanisms for enhanced growth are not understood. Here, we characterized the phylogenetic diversity and function of select genes involved in the growth of A. biwaensis on 2'-fucosyllactose (2'-FL), a dominant HMO. Specifically, we demonstrate that two genes in a genomic locus, a putative ß-galactosidase and α-fucosidase, are likely responsible for the enhanced growth on 2'-FL. The functional characterization of A. biwaensis growth on 2'-FL delineates the significance of a single genomic locus that may facilitate enhanced colonization and functional activity of select Akkermansia early in life.

Interrelation between gut microbiota, SCFA, and fatty acid composition in pigs.

The gut microbiota is a key player in the host metabolism. Some bacteria are able to ferment non-digestible compounds and produce short-chain fatty acids that the host can later transform and accumulate in tissue. In this study, we aimed to better understand the relationships between the microorganisms and the short-chain fatty acid composition of the rectal content, including the possible linkage with the fatty acid composition in backfat and muscle of the pig. We studied a Duroc × Iberian crossbred population, and we found significant correlations between different bacterial and archaeal genera and the fatty acid profile. The abundance of n-butyric acid in the rectal content was positively associated with Prevotella spp. and negatively associated with Akkermansia spp., while conversely, the abundance of acetic acid was negatively and positively associated with the levels of Prevotella spp. and Akkermansia spp., respectively. The most abundant genus, Rikenellaceae RC9 gut group, had a positive correlation with palmitic acid in muscle and negative correlations with stearic acid in backfat and oleic acid in muscle. These results suggest the possible role of Prevotella spp. and Akkermansia spp. as biomarkers for acetic and n-butyric acids, and the relationship of Rikenellaceae RC9 gut group with the lipid metabolism, building up the potential, although indirect, role of the microbiota in the modification of the backfat and muscle fatty acid composition of the host.IMPORTANCEThe vital role of the gut microbiota on its host metabolism makes it essential to know how its modulation is mirrored on the fatty acid composition of the host. Our findings suggest Prevotella spp. and Akkermansia spp. as potential biomarkers for the levels of beneficial short-chain fatty acids and the possible influence of Rikenellaceae RC9 gut group in the backfat and muscle fatty acid composition of the pig.

Ficolin A knockout alleviates sepsis-induced severe lung injury in mice by restoring gut Akkermansia to inhibit S100A4/STAT3 pathway.

Acute lung injury (ALI) is a life-threatening disease with high morbidity and mortality. Our previous results demonstrated that Ficolin A (FcnA) protected against lipopolysaccharide (LPS)-induced mild ALI via activating complement, however the mechanism of severe lung damage caused by sepsis remains unclear. This study aimed to investigate whether FcnA modulated gut microbiota to affect the progression of sepsis-induced severe ALI. Fcna-/- and Fcnb-/- C57BL/6 mice were applied to establish the ALI model by injection of LPS intraperitoneally. Mice were treated with antibiotics, fecal microbiota transplantation (FMT), and intratracheal administration of recombinant protein S100A4. Changes in body weight of mice were recorded, and lung injury were assessed. Then lung tissue wet/dry weight was calculated. We found knockout of FcnA, but not FcnB, alleviated sepsis-induced severe ALI evidenced by increased body weight change, decreased wet/dry weight of lung tissue, reduced inflammatory infiltration, decreased lung damage score, decreased Muc-2, TNF-α, IL-1ß, IL-6, and Cr levels, and increased sIgA levels. Furthermore, knockout of FcnA restored gut microbiota homeostasis in mice. Correlation analysis showed that Akkermansia was significantly negatively associated with TNF-α, IL-1ß, and IL-6 levels in serum and bronchoalveolar lavage fluid (BALF). Moreover, knockout of FcnA regulated gut microbiota to protect ALI through S100A4. Finally, we found knockout of FcnA alleviated ALI by inhibiting S100A4 via gut Akkermansia in mice, which may provide further insights and new targets into treating sepsis-induced severe lung injury.

Akkermansia muciniphila plays critical roles in host health.

Akkermansia muciniphila, an intestinal microorganism, belongs to Verrucomicrobia, one of the most abundant microorganisms in the mammalian gut. It is a mucin-degrading bacterium that can colonise intestines of mammals such as humans and mice by utilising mucin as the only nitrogen and carbon source. When A. muciniphila colonises the intestine, its metabolites interact with the intestinal barrier, affecting host health by consolidating the intestinal barrier, regulating metabolic functions of the intestinal and circulatory systems, and regulating immune functions. This review summarised the mechanisms of A. muciniphila-host interactions that are relevant to host health. We focussed on characteristics of A. muciniphila in relation to its metabolites to provide a comprehensive understanding of A. muciniphila and its effects on host health and disease processes.

Outer membrane protein Amuc_1100 of Akkermansia muciniphila alleviates antibiotic-induced anxiety and depression-like behavior in mice.

Akkermansia muciniphila is present in the mucus layer of its host gut, and its outer membrane protein Amuc_1100 has a significant ameliorative effect on metabolic disorders and emotional memory aspects of enteritis, obesity, depression, and anxiety in the host. Antibiotics affect gut microbial composition, leading to imbalance and behavioral changes in the gut-brain axis, while probiotics have a protective effect against behavioral changes caused by gut flora disorders. In the present study, a depressed mouse model using a broad-spectrum cocktail mixture resulted in increased anxiety and depression-like behavior, decreased serum and hippocampal levels of 5-hydroxytryptamine (5-HT), and increased serum corticosterone (cort) levels. After application of A. muciniphila and Amuc_1100, anxiety and depression-like behavior in antibiotic-treated mice were significantly alleviated. In addition, the brain derived neurotrophic factor / Tropomyosin receptor kinase B (BDNF/TrkB) signaling pathway was altered, glial fibrillary acidic protein (GFAP) expression increased, and c-Fos protein expression decreased in the hippocampus of antibiotic-treated mice. After treatment with A. muciniphila and Amuc_1100, BDNF and TrkB levels were restored in the hippocampus and cortex. These results suggest that A. muciniphila and Amuc_1100 may alleviate antibiotic-induced anxiety and depression by affecting the BDNF/TrkB signaling pathway.

Effects of active, inactive, and derivatives of Akkermansia muciniphila on the expression of the endocannabinoid system and PPARs genes.

This study aimed to investigate the effects of active and heat-inactivated forms of Akkermansia muciniphila, bacterium-derived outer membrane vesicles (OMVs), and cell-free supernatant on the transcription of endocannabinoid system (ECS) members, including cannabinoid receptors 1 and 2 (CB1 and CB2), fatty acid amide hydrolase (FAAH), and peroxisome proliferator-activated receptors (PPARs) genes (i.e., α, ß/δ, and δ) in Caco-2 and HepG-2 cell lines. After the inoculation of A. muciniphila in brain heart infusion enriched medium, OMVs and cell-free supernatant were extracted. For the investigation of the effects of bacteria and its derivatives on the expression of ECS and PPARs genes, the aforementioned cells were treated by active and heat-inactivated bacteria, OMVs, and cell-free supernatant. Quantitative real-time polymerase chain reaction analysis revealed that both forms of the bacterium, bacterial-derived OMVs, and cell-free supernatant could affect the expression of CB1, CB2, FAAH, and PPARs genes (i.e., α, ß/δ, and δ) significantly (P < 0.05). Considering the engagement of the aforementioned genes in metabolic pathways, it might be suggested that both forms of the bacterium, OMVs, and cell-free supernatant might have the potential to serve as a probiotic, paraprobiotic, and postbiotic candidate to prevent obesity, metabolic disorders, and liver diseases.

Heat-Inactivated Akkermansia muciniphila Improves Gut Permeability but Does Not Prevent Development of Non-Alcoholic Steatohepatitis in Diet-Induced Obese Ldlr-/-.Leiden Mice.

The development of non-alcoholic steatohepatitis (NASH) has been associated with alterations in gut microbiota composition and reduced gut barrier function. Akkermansia muciniphila is a gut microbe that is thought to have health-promoting properties, including the ability to improve gut barrier function and host metabolism, both when administered live and after heat-inactivation. We questioned whether heat-inactivated A. muciniphila may reduce NASH development. Ldlr-/-.Leiden mice, a translational, diet-induced model for NASH, were fed a NASH-inducing high-fat diet (HFD) supplemented with heat-inactivated A. muciniphila. After 28 weeks, effects of the treatment on obesity and associated metabolic dysfunction in the gut (microbiota composition and permeability), adipose tissue, and liver were studied relative to an untreated HFD control. Treatment with heat-inactivated A. muciniphila did not affect body weight or adiposity and had no effect on plasma lipids, blood glucose, or plasma insulin. Heat-inactivated A. muciniphila had some minor effects on mucosal microbiota composition in ileum and colon and improved gut barrier function, as assessed by an in vivo functional gut permeability test. Epidydimal white adipose tissue (WAT) hypertrophy and inflammation were not affected, but heat-inactivated A. muciniphila did reduce hypertrophy in the mesenteric WAT which is in close proximity to the intestine. Heat-inactivated A. muciniphila did not affect the development of NASH or associated fibrosis in the liver and did not affect circulating bile acids or markers of liver fibrosis, but did reduce PRO-C4, a type IV collagen synthesis marker, which may be associated with gut integrity. In conclusion, despite beneficial effects in the gut and mesenteric adipose tissue, heat-inactivated A. muciniphila did not affect the development of NASH and fibrosis in a chronic disease setting that mimics clinically relevant disease stages.

Outlook on next-generation probiotics from the human gut.

Probiotics currently available on the market generally belong to a narrow range of microbial species. However, recent studies about the importance of the gut microbial commensals on human health highlighted that the gut microbiome is an unexplored reservoir of potentially beneficial microbes. For this reason, academic and industrial research is focused on identifying and testing novel microbial strains of gut origin for the development of next-generation probiotics. Although several of these are promising for the prevention and treatment of many chronic diseases, studies on human subjects are still scarce and approval from regulatory agencies is, therefore, rare. In addition, some issues need to be overcome before implementing their wide application on the market, such as the best methods for cultivation and storage of these oxygen-sensitive taxa. This review summarizes the most recent evidence related to NGPs and provides an outlook to the main issues that still limit their wide employment.

In vitro co-metabolism of epigallocatechin-3-gallate (EGCG) by the mucin-degrading bacterium Akkermansia muciniphila.

Akkermansia muciniphila is a Gram-negative bacterium that resides within the gut mucus layer, and plays an important role in promoting gut barrier integrity, modulating the immune response and inhibiting gut inflammation. Growth stimulation of A. muciniphila by polyphenols including epigallocatechin-3-gallate (EGCG) from difference sources is well-documented. However, no published in vitro culture data on utilization of polyphenols by A. muciniphila are available, and the mechanism of growth-stimulating prebiotic effect of polyphenols on it remains unclear. Here in vitro culture studies have been carried out on the metabolism of EGCG by A. muciniphila in the presence of either mucin or glucose. We found that A. muciniphila did not metabolize EGCG alone but could co-metabolize it together with both these substrates in the presence of mineral salts and amino acids for mucin and protein sources for glucose. Our metabolomic data show that A. muciniphila converts EGCG to gallic acid, epigallocatechin, and (-)-epicatechin through ester hydrolysis. The (-)-epicatechin formed is then further converted to hydroxyhydroquinone. Co-metabolism of A. muciniphila of EGCG together with either mucin or glucose promoted substantially its growth, which serves as a further demonstration of the growth-promoting effect of polyphenols on A. muciniphila and provides an important addition to the currently available proposed mechanisms of polyphenolic prebiotic effects on A. muciniphila.

Genomic convergence between Akkermansia muciniphila in different mammalian hosts.

BACKGROUND: Akkermansia muciniphila is a member of the human gut microbiota where it resides in the mucus layer and uses mucin as the sole carbon, nitrogen and energy source. A. muciniphila is the only representative of the Verrucomicrobia phylum in the human gut. However, A. muciniphila 16S rRNA gene sequences have also been found in the intestines of many vertebrates. RESULTS: We detected A. muciniphila-like bacteria in the intestines of animals belonging to 15 out of 16 mammalian orders. In addition, other species belonging to the Verrucomicrobia phylum were detected in fecal samples. We isolated 10 new A. muciniphila strains from the feces of chimpanzee, siamang, mouse, pig, reindeer, horse and elephant. The physiology and genome of these strains were highly similar in comparison to the type strain A. muciniphila MucT. Overall, the genomes of the new strains showed high average nucleotide identity (93.9 to 99.7%). In these genomes, we detected considerable conservation of at least 75 of the 78 mucin degradation genes that were previously detected in the genome of the type strain MucT. CONCLUSIONS: The low genomic divergence observed in the new strains may indicate that A. muciniphila favors mucosal colonization independent of the differences in hosts. In addition, the conserved mucus degradation capability points towards a similar beneficial role of the new strains in regulating host metabolic health.

Metformin alleviates choline diet-induced TMAO elevation in C57BL/6J mice by influencing gut-microbiota composition and functionality.

Trimethylamine-N-oxide (TMAO), a gut-microbiota-dependent metabolite generated from its dietary precursors such as choline, has been identified as an independent risk factor for atherosclerosis. Metformin is the most widely used drug for the treatment of type 2 diabetes (T2D), which has therapeutic effects on hyperglycemia accelerated atherosclerosis. A growing body of evidence suggest that metformin plays a therapeutic role by regulating the structure and metabolic function of gut microbiota. However, whether metformin has an impact on gut-microbiota-mediated TMAO production from choline remains obscure. In this study, the oral administration of metformin significantly reduced choline diet-increased serum TMAO in choline diet-fed C57BL/6J mice. The diversity analysis based on 16S rRNA gene sequencing of C57BL/6J mice fecal samples indicated that metformin markedly changed the gut-microbiota composition. Metformin was positively correlated with the enrichment of different intestinal bacteria such as Bifidobacterium and Akkermansia and a lower cutC (a choline utilization gene) abundance. Furthermore, the ex vivo and in vitro inhibitory effects of metformin on choline metabolism of TMA-producing bacteria were confirmed under anaerobic condition. The results suggested that metformin suppresses serum TMAO level by remodeling gut microbiota involved in TMA generation from choline.

Genomic diversity and ecology of human-associated Akkermansia species in the gut microbiome revealed by extensive metagenomic assembly.

BACKGROUND: Akkermansia muciniphila is a human gut microbe with a key role in the physiology of the intestinal mucus layer and reported associations with decreased body mass and increased gut barrier function and health. Despite its biomedical relevance, the genomic diversity of A. muciniphila remains understudied and that of closely related species, except for A. glycaniphila, unexplored. RESULTS: We present a large-scale population genomics analysis of the Akkermansia genus using 188 isolate genomes and 2226 genomes assembled from 18,600 metagenomes from humans and other animals. While we do not detect A. glycaniphila, the Akkermansia strains in the human gut can be grouped into five distinct candidate species, including A. muciniphila, that show remarkable whole-genome divergence despite surprisingly similar 16S rRNA gene sequences. These candidate species are likely human-specific, as they are detected in mice and non-human primates almost exclusively when kept in captivity. In humans, Akkermansia candidate species display ecological co-exclusion, diversified functional capabilities, and distinct patterns of associations with host body mass. Analysis of CRISPR-Cas loci reveals new variants and spacers targeting newly discovered putative bacteriophages. Remarkably, we observe an increased relative abundance of Akkermansia when cognate predicted bacteriophages are present, suggesting ecological interactions. A. muciniphila further exhibits subspecies-level genetic stratification with associated functional differences such as a putative exo/lipopolysaccharide operon. CONCLUSIONS: We uncover a large phylogenetic and functional diversity of the Akkermansia genus in humans. This variability should be considered in the ongoing experimental and metagenomic efforts to characterize the health-associated properties of A. muciniphila and related bacteria.

Decreased Abundance of Akkermansia muciniphila Leads to the Impairment of Insulin Secretion and Glucose Homeostasis in Lean Type 2 Diabetes.

Although obesity occurs in most of the patients with type 2 diabetes (T2D), a fraction of patients with T2D are underweight or have normal weight. Several studies have linked the gut microbiome to obesity and T2D, but the role of gut microbiota in lean individuals with T2D having unique clinical characteristics remains unclear. A metagenomic and targeted metabolomic analysis is conducted in 182 lean and abdominally obese individuals with and without newly diagnosed T2D. The abundance of Akkermansia muciniphila (A. muciniphila) significantly decreases in lean individuals with T2D than without T2D, but not in the comparison of obese individuals with and without T2D. Its abundance correlates inversely with serum 3ß-chenodeoxycholic acid (ßCDCA) levels and positively with insulin secretion and fibroblast growth factor 15/19 (FGF15/19) concentrations. The supplementation with A. muciniphila is sufficient to protect mice against high sucrose-induced impairment of glucose intolerance by decreasing ßCDCA and increasing insulin secretion and FGF15/19. Furthermore, ßCDCA inhibits insulin secretion and FGF15/19 expression. These findings suggest that decreased abundance of A. muciniphila is linked to the impairment of insulin secretion and glucose homeostasis in lean T2D, paving the way for new therapeutic options for the prevention or treatment of diabetes.

Gut Akkermansia muciniphila ameliorates metabolic dysfunction-associated fatty liver disease by regulating the metabolism of L-aspartate via gut-liver axis.

The gut bacterium Akkermansia muciniphila has been increasingly recognized for its therapeutic potential in treating metabolic disorders, including obesity, diabetes, and metabolicdysfunction-associated fatty liver disease (MAFLD). However, its underlying mechanism involved in its well-known metabolic actions needs further evaluation. The present study explored the therapeutic effect and mechanism of A. muciniphila in intervening MAFLD by using a high-fat and high-cholesterol (HFC) diet induced obese mice model. Mice treated with A. muciniphila efficiently reversed MAFLD in the liver, such as hepatic steatosis, inflammatory, and liver injury. These therapeutic effects persisted after long-term drug withdrawal and were slightly weakened in the antibiotics-treated obese mice. A. muciniphila treatment efficiently increased mitochondrial oxidation and bile acid metabolism in the gut-liver axis, ameliorated oxidative stress-induced cell apoptosis in gut, leading to the reshaping of the gut microbiota composition. These metabolic improvements occurred with increased L-aspartate levels in the liver that transported from the gut. The administration of L-aspartate in vitro or in mice displayed the similar beneficial metabolic effects mentioned above and efficiently ameliorated MAFLD. Together, these data indicate that the anti-MAFLD activity of A. muciniphila correlated with lipid oxidation and improved gut-liver interactions through regulating the metabolism of L-aspartate. A. muciniphila could be a potential agent for clinical intervention in MAFLD.