Probing Peptidoglycan Synthesis in the Gut Commensal Akkermansia Muciniphila with Bioorthogonal Chemical Reporters.

Our gut microbiota directly influences human physiology in health and disease. The myriad of surface glycoconjugates in both the bacterial cell envelope and our gut cells dominate the microbiota-host interface and play a critical role in host response and microbiota homeostasis. Among these, peptidoglycan is the basic glycan polymer offering the cell rigidity and a basis on which many other glycoconjugates are anchored. To directly study peptidoglycan in gut commensals and obtain the molecular insight required to understand their functional activities we need effective techniques like chemical probes to label peptidoglycan in live bacteria. Here we report a chemically guided approach to study peptidoglycan in a key mucin-degrading gut microbiota member of the Verrucomicrobia phylum, Akkermansia muciniphila. Two novel non-toxic tetrazine click-compatible peptidoglycan probes with either a cyclopropene or isonitrile handle allowed for the detection and imaging of peptidoglycan synthesis in this intestinal species.

Exploring the effect of a microencapsulated citrus essential oil on in vitro fermentation kinetics of pig gut microbiota.

Essential oils (EOs) have emerged as a potential alternative to antibiotics in pig breeding due to their antimicrobial properties. Citrus EOs, a common by-product of the orange juice industry, can be an interesting alternative from a financial perspective due to their huge offer in the global market. Thus, the effect of a citrus EO, and specifically different formulations of Brazilian Orange Terpenes (BOT), on pig gut microbiota was evaluated by means of an in vitro fermentation model simulating different sections of the pig gut (stomach, ileum, and colon). Treatments consisted in: BOT in its unprotected form (BOT, 1.85 and 3.70 mg/mL), microencapsulated BOT (MBOT, 3.50 and 7.00 mg/mL), colistin (2 µg/mL), and a control. BOT and MBOT altered in a similar way the total bacterial 16S rRNA gene copies in the stomach only from 18 h of incubation onwards, and no metabolite production in terms of short-chain fatty acids (SCFAs) was detected. In ileal and colonic fermentations, BOT and MBOT affected ileal and colonic microbiota in terms of total bacterial 16S rRNA gene copies, reduced phylogenetic diversity, and altered composition (p < 0.05) as evidenced by the significant reduction of certain bacterial taxa. However, more pronounced effects were found for MBOT, indicating its higher antimicrobial effects compared to the unprotected BOT, and suggesting that the antibacterial efficiency of the unprotected BOT was probably enhanced by microencapsulation. Furthermore, MBOT stimulated lactate production in ileal fermentations and greatly stimulated overall SCFA production in colonic fermentations. This indicates that besides the shifts in ileal and colonic microbiota by the delivered EO (BOT), the wall material of microcapsules (chitosan/modified starch) might have worked as an additional carbon source with prebiotic functioning, stimulating growth and metabolic activity (SCFAs) of colonic bacteria.

Inter-species Metabolic Interactions in an In-vitro Minimal Human Gut Microbiome of Core Bacteria.

Knowledge of the functional roles and interspecies interactions are crucial for improving our understanding of the human intestinal microbiome in health and disease. However, the complexity of the human intestinal microbiome and technical challenges in investigating it pose major challenges. In this proof-of-concept study, we rationally designed, assembled and experimentally tested a synthetic Diet-based Minimal Microbiome (Db-MM) consisting of ten core intestinal bacterial species that together are capable of efficiently converting dietary fibres into short chain fatty acids (SCFAs). Despite their genomic potential for metabolic competition, all ten bacteria coexisted during growth on a mixture of dietary fibres, including pectin, inulin, xylan, cellobiose and starch. By integrated analyses of metabolite production, community composition and metatranscriptomics-based gene expression data, we identified interspecies metabolic interactions leading to production of key SCFAs such as butyrate and propionate. While public goods, such as sugars liberated from colonic fibres, are harvested by non-degraders, some species thrive by cross-feeding on energetically challenging substrates, including the butyrogenic conversion of acetate and lactate. Using a reductionist approach in an in-vitro system combined with functional measurements, our study provides key insights into the complex interspecies metabolic interactions between core intestinal bacterial species.

Duodenal Anaerobutyricum soehngenii infusion stimulates GLP-1 production, ameliorates glycaemic control and beneficially shapes the duodenal transcriptome in metabolic syndrome subjects: a randomised double-blind placebo-controlled cross-over study.

OBJECTIVE: Although gut dysbiosis is increasingly recognised as a pathophysiological component of metabolic syndrome (MetS), the role and mode of action of specific gut microbes in metabolic health remain elusive. Previously, we identified the commensal butyrogenic Anaerobutyricum soehngenii to be associated with improved insulin sensitivity in subjects with MetS. In this proof-of-concept study, we investigated the potential therapeutic effects of A. soehngenii L2-7 on systemic metabolic responses and duodenal transcriptome profiles in individuals with MetS. DESIGN: In this randomised double-blind placebo-controlled cross-over study, 12 male subjects with MetS received duodenal infusions of A. soehngenii/ placebo and underwent duodenal biopsies, mixed meal tests (6 hours postinfusion) and 24-hour continuous glucose monitoring. RESULTS: A. soehngenii treatment provoked a markedly increased postprandial excursion of the insulinotropic hormone glucagon-like peptide 1 (GLP-1) and an elevation of plasma secondary bile acids, which were positively associated with GLP-1 levels. Moreover, A. soehngenii treatment robustly shaped the duodenal expression of 73 genes, with the highest fold induction in the expression of regenerating islet-protein 1B (REG1B)-encoding gene. Strikingly, duodenal REG1B expression positively correlated with GLP-1 levels and negatively correlated with peripheral glucose variability, which was significantly diminished in the 24 hours following A. soehngenii intake. Mechanistically, Reg1B expression is induced upon sensing butyrate or bacterial peptidoglycan. Importantly, A. soehngenii duodenal administration was safe and well tolerated. CONCLUSIONS: A single dose of A. soehngenii improves peripheral glycaemic control within 24 hours; it specifically stimulates intestinal GLP-1 production and REG1B expression. Further studies are needed to delineate the specific pathways involved in REG1B induction and function in insulin sensitivity. TRIAL REGISTRATION NUMBER: NTR-NL6630.

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.

A Continuous Battle for Host-Derived Glycans Between a Mucus Specialist and a Glycan Generalist in vitro and in vivo.

The human gastrointestinal tract is colonized by a diverse microbial community, which plays a crucial role in human health. In the gut, a protective mucus layer that consists of glycan structures separates the bacteria from the host epithelial cells. These host-derived glycans are utilized by bacteria that have adapted to this specific compound in the gastrointestinal tract. Our study investigated the close interaction between two distinct gut microbiota members known to use mucus glycans, the generalist Bacteroides thetaiotaomicron and the specialist Akkermansia muciniphila in vitro and in vivo. The in vitro study, in which mucin was the only nutrient source, indicated that B. thetaiotaomicron significantly upregulated genes coding for Glycoside Hydrolases (GHs) and mucin degradation activity when cultured in the presence of A. muciniphila. Furthermore, B. thetaiotaomicron significantly upregulated the expression of a gene encoding for membrane attack complex/perforin (MACPF) domain in co-culture. The transcriptome analysis also indicated that A. muciniphila was less affected by the environmental changes and was able to sustain its abundance in the presence of B. thetaiotaomicron while increasing the expression of LPS core biosynthesis activity encoding genes (O-antigen ligase, Lipid A and Glycosyl transferases) as well as ABC transporters. Using germ-free mice colonized with B. thetaiotaomicron and/or A. muciniphila, we observed a more general glycan degrading profile in B. thetaiotaomicron while the expression profile of A. muciniphila was not significantly affected when colonizing together, indicating that two different nutritional niches were established in mice gut. Thus, our results indicate that a mucin degrading generalist adapts to its changing environment, depending on available carbohydrates while a mucin degrading specialist adapts by coping with competing microorganism through upregulation of defense related genes.

Production of inactivated gram-positive and gram-negative species with preserved cellular morphology and integrity.

There are many approaches available to produce inactive bacteria by termination of growth, each with a different efficacy, impact on cell integrity, and potential for application in standardized inactivation protocols. The aim of this study was to compare these approaches and develop a standardized protocol for generation of inactivated Gram-positive and Gram-negative bacteria, yielding cells that are metabolically dead with retained cellular integrity i.e., preserving the surface and limited leakage of intracellular proteins and DNA. These inactivated bacteria are required for various applications, for instance, when investigating receptor-triggered signaling or bacterial contact-dependent analysis of cell lines requiring long incubation times. We inactivated eight different bacterial strains of different species by treatment with beta-propiolactone, ethanol, formalin, sodium hydroxide, and pasteurization. Inactivation efficacy was determined by culturing, and cell wall integrity assessed by quantifying released DNA, bacterial membrane and intracellular DNA staining, and visualization by scanning electron microscopy. Based on these results, we discuss the bacterial inactivation methods, and their advantages and disadvantages to study host-microbe interactions with inactivated bacteria.

Selection and characterization of a SpaCBA pilus-secreting food-grade derivative of Lacticaseibacillus rhamnosus GG.

Many studies have established the functional properties of Lacticaseibacillus rhamnosus GG, previously known as Lactobacillus rhamnosus GG, marketed worldwide as a probiotic. The extraordinary capacity of L. rhamnosus GG to bind to human mucus and influence the immune system especially stand out. Earlier, we have shown the key role of its SpaCBA sortase-dependent pili encoded by the spaCBA-srtC1 gene cluster herein. These heterotrimeric pili consist of a shaft pilin SpaA, a basal pilin SpaB, and tip pilin SpaC that contains a mucus-binding domain. Here, we set out to characterize a food-grade non-GMO mutant of L. rhamnosus GG, strain PA11, which secretes its pilins, rather than coupling them to the cell surface, due to a defect in the housekeeping sortase A. The sortase-negative strain PA11 was extensively characterized using functional genomics and biochemical approaches and found to secrete the SpaCBA pili into the supernatant. Given the functional importance and uniqueness of the mucus-binding pili of L. rhamnosus GG, strain PA11 offers novel opportunities towards the characterization and further therapeutic application of SpaCBA pili and their low-cost, large-scale production. KEY POINTS: •Creation of pilus-secreting mutant (PA11) of the key probiotic LGG. •Strain PA11 is defective in a functional housekeeping sortase SrtA. •Strain PA11 opens novel biotherapeutic application avenues. Graphical abstract.

Bacteroides thetaiotaomicron Fosters the Growth of Butyrate-Producing Anaerostipes caccae in the Presence of Lactose and Total Human Milk Carbohydrates.

The development of infant gut microbiota is strongly influenced by nutrition. Human milk oligosaccharides (HMOSs) in breast milk selectively promote the growth of glycan-degrading microbes, which lays the basis of the microbial network. In this study, we investigated the trophic interaction between Bacteroides thetaiotaomicron and the butyrate-producing Anaerostipes caccae in the presence of early-life carbohydrates. Anaerobic bioreactors were set up to study the monocultures of B. thetaiotaomicron and the co-cultures of B. thetaiotaomicron with A. caccae in minimal media supplemented with lactose or a total human milk carbohydrate fraction. Bacterial growth (qPCR), metabolites (HPLC), and HMOS utilization (LC-ESI-MS2) were monitored. B. thetaiotaomicron displayed potent glycan catabolic capability with differential preference in degrading specific low molecular weight HMOSs, including the neutral trioses (2'-FL and 3-FL), neutral tetraoses (DFL, LNT, LNnT), neutral pentaoses (LNFP I, II, III, V), and acidic trioses (3'-SL and 6'-SL). In contrast, A. caccae was not able to utilize lactose and HMOSs. However, the signature metabolite of A. caccae, butyrate, was detected in co-culture with B. thetaiotaomicron. As such, A. caccae cross-fed on B. thetaiotaomicron-derived monosaccharides, acetate, and d-lactate for growth and concomitant butyrate production. This study provides a proof of concept that B. thetaiotaomicron could drive the butyrogenic metabolic network in the infant gut.

Growth rate alterations of human colorectal cancer cells by 157 gut bacteria.

Several bacteria in the human gut microbiome have been associated with colorectal cancer (CRC) by high-throughput screens. In some cases, molecular mechanisms have been elucidated that drive tumorigenesis, including bacterial membrane proteins or secreted molecules that interact with the human cancer cells. For most gut bacteria, however, it remains unknown if they enhance or inhibit cancer cell growth. Here, we screened bacteria-free supernatants (secretomes) and inactivated cells of over 150 cultured bacterial strains for their effects on cell growth. We observed family-level and strain-level effects that often differed between bacterial cells and secretomes, suggesting that different molecular mechanisms are at play. Secretomes of Bacteroidaceae, Enterobacteriaceae, and Erysipelotrichaceae bacteria enhanced cell growth, while most Fusobacteriaceae cells and secretomes inhibited growth, contrasting prior findings. In some bacteria, the presence of specific functional genes was associated with cell growth rates, including the virulence genes TcdA, TcdB in Clostridiales and FadA in Fusobacteriaceae, which both inhibited growth. Bacteroidaceae cells that enhanced growth were enriched for genes of the cobalamin synthesis pathway, while Fusobacteriaceae cells that inhibit growth were enriched for genes of the ethanolamine utilization pathway. Together, our results reveal how different gut bacteria have wide-ranging effects on cell growth, contribute a better understanding of the effects of the gut microbiome on host cells, and provide a valuable resource for identifying candidate target genes for potential microbiome-based diagnostics and treatment strategies.

Akkermansia muciniphila uses human milk oligosaccharides to thrive in the early life conditions in vitro.

Akkermansia muciniphila is a well-studied anaerobic bacterium specialized in mucus degradation and associated with human health. Because of the structural resemblance of mucus glycans and free human milk oligosaccharides (HMOs), we studied the ability of A. muciniphila to utilize human milk oligosaccharides. We found that A. muciniphila was able to grow on human milk and degrade HMOs. Analyses of the proteome of A. muciniphila indicated that key-glycan degrading enzymes were expressed when the bacterium was grown on human milk. Our results display the functionality of the key-glycan degrading enzymes (α-L-fucosidases, ß-galactosidases, exo-α-sialidases and ß-acetylhexosaminidases) to degrade the HMO-structures 2'-FL, LNT, lactose, and LNT2. The hydrolysation of the host-derived glycan structures allows A. muciniphila to promote syntrophy with other beneficial bacteria, contributing in that way to a microbial ecological network in the gut. Thus, the capacity of A. muciniphila to utilize human milk will enable its survival in the early life intestine and colonization of the mucosal layer in early life, warranting later life mucosal and metabolic health.

Treatment with Anaerobutyricum soehngenii: a pilot study of safety and dose-response effects on glucose metabolism in human subjects with metabolic syndrome.

Dysbiosis of the intestinal microbiota has been implicated in insulin resistance, although evidence regarding causality in humans is scarce. We performed a phase I/II dose-finding and safety study on the effect of oral intake of the anaerobic butyrogenic strain Anaerobutyricum soehngenii on glucose metabolism in 24 subjects with metabolic syndrome. We found that treatment with A. soehngenii was safe and observed a significant correlation between the measured fecal abundance of administered A. soehngenii and improvement in peripheral insulin sensitivity after 4 weeks of treatment. This was accompanied by an altered microbiota composition and a change in bile acid metabolism. Finally, we show that metabolic response upon administration of A. soehngenii (defined as improved insulin sensitivity 4 weeks after A. soehngenii intake) is dependent on microbiota composition at baseline. These data in humans are promising, but additional studies are needed to reproduce our findings and to investigate long-term effects, as well as other modes of delivery.

Development of omics-based protocols for the microbiological characterization of multi-strain formulations marketed as probiotics: the case of VSL#3.

The growing commercial interest in multi-strain formulations marketed as probiotics has not been accompanied by an equal increase in the evaluation of quality levels of these biotechnological products. The multi-strain product VSL#3 was used as a model to setup a microbiological characterization that could be extended to other formulations with high complexity. Shotgun metagenomics by deep Illumina sequencing was applied to DNA isolated from the commercial VSL#3 product to confirm strains identity safety and composition. Single-cell analysis was used to evaluate the cell viability, and ß-galactosidase and urease activity have been used as marker to monitor the reproducibility of the production process. Similarly, these lots were characterized in detail by a metaproteomics approach for which a robust protein extraction protocol was combined with advanced mass spectrometry. The results identified over 1600 protein groups belonging to all strains present in the VSL#3 formulation. Of interest, only 3.2 % proteins showed significant differences mainly related to small variations in strain abundance. The protocols developed in this study addressed several quality criteria that are relevant for marketed multi-strain products and these represent the first efforts to define the quality of complex probiotic formulations such as VSL#3.

Akkermansia muciniphila ameliorates the age-related decline in colonic mucus thickness and attenuates immune activation in accelerated aging Ercc1 -/Δ7 mice.

BACKGROUND: The use of Akkermansia muciniphila as potential therapeutic intervention is receiving increasing attention. Health benefits attributed to this bacterium include an improvement of metabolic disorders and exerting anti-inflammatory effects. The abundance of A. muciniphila is associated with a healthy gut in early mid- and later life. However, the effects of A. muciniphila on a decline in intestinal health during the aging process are not investigated yet. We supplemented accelerated aging Ercc1 -/Δ7 mice with A. muciniphila for 10 weeks and investigated histological, transcriptional and immunological aspects of intestinal health. RESULTS: The thickness of the colonic mucus layer increased about 3-fold after long-term A. muciniphila supplementation and was even significantly thicker compared to mice supplemented with Lactobacillus plantarum WCFS1. Colonic gene expression profiles pointed towards a decreased expression of genes and pathways related to inflammation and immune function, and suggested a decreased presence of B cells in colon. Total B cell frequencies in spleen and mesenteric lymph nodes were not altered after A. muciniphila supplementation. Mature and immature B cell frequencies in bone marrow were increased, whereas B cell precursors were unaffected. These findings implicate that B cell migration rather than production was affected by A. muciniphila supplementation. Gene expression profiles in ileum pointed toward a decrease in metabolic- and immune-related processes and antimicrobial peptide production after A. muciniphila supplementation. Besides, A. muciniphila decreased the frequency of activated CD80+CD273- B cells in Peyer's patches. Additionally, the increased numbers of peritoneal resident macrophages and a decrease in Ly6Cint monocyte frequencies in spleen and mesenteric lymph nodes add evidence for the potentially anti-inflammatory properties of A. muciniphila. CONCLUSIONS: Altogether, we show that supplementation with A. muciniphila prevented the age-related decline in thickness of the colonic mucus layer and attenuated inflammation and immune-related processes at old age. This study implies that A. muciniphila supplementation can contribute to a promotion of healthy aging.

Antibiotics-induced monodominance of a novel gut bacterial order.

OBJECTIVE: The composition of the healthy human adult gut microbiome is relatively stable over prolonged periods, and representatives of the most highly abundant and prevalent species have been cultured and described. However, microbial abundances can change on perturbations, such as antibiotics intake, enabling the identification and characterisation of otherwise low abundant species. DESIGN: Analysing gut microbial time-series data, we used shotgun metagenomics to create strain level taxonomic and functional profiles. Community dynamics were modelled postintervention with a focus on conditionally rare taxa and previously unknown bacteria. RESULTS: In response to a commonly prescribed cephalosporin (ceftriaxone), we observe a strong compositional shift in one subject, in which a previously unknown species, UBorkfalki ceftriaxensis, was identified, blooming to 92% relative abundance. The genome assembly reveals that this species (1) belongs to a so far undescribed order of Firmicutes, (2) is ubiquitously present at low abundances in at least one third of adults, (3) is opportunistically growing, being ecologically similar to typical probiotic species and (4) is stably associated to healthy hosts as determined by single nucleotide variation analysis. It was the first coloniser after the antibiotic intervention that led to a long-lasting microbial community shift and likely permanent loss of nine commensals. CONCLUSION: The bloom of UB. ceftriaxensis and a subsequent one of Parabacteroides distasonis demonstrate the existence of monodominance community states in the gut. Our study points to an undiscovered wealth of low abundant but common taxa in the human gut and calls for more highly resolved longitudinal studies, in particular on ecosystem perturbations.

Trichococcus shcherbakoviae sp. nov., isolated from a laboratory-scale anaerobic EGSB bioreactor operated at low temperature.

A new species of the genus Trichococcus, strain Art1T, was isolated from a psychrotolerant syntrophic propionate-oxidizing consortium, obtained before from a low-temperature EGSB reactor fed with a mixture of VFAs (acetate, propionate and butyrate). The 16S rRNA gene sequence of strain Art1T was highly similar to those of other Trichococcus species (99.7-99.9 %) but digital DNA-DNA hybridization values were lower than those recommended for the delineation of a novel species, indicating that strain Art1T is a novel species of the genus Trichococcus. Cells of strain Art1T are non-motile cocci with a diameter of 0.5-2.0 µm and were observed singularly, in pairs, short chains and irregular conglomerates. Cells of Art1T stained Gram-positive and produced extracellular polymeric substances . Growth was optimal at pH 6-7.5 and cells could grow in a temperature range of from -2 to 30 °C (optimum 25-30 °C). Strain Art1T can degrade several carbohydrates, and the main products from glucose fermentation are lactate, acetate, formate and ethanol. The genomic DNA G+C content of strain Art1T is 46.7 %. The major components of the cellular fatty acids are C16 : 1 ω9c, C16 : 0 and C18 : 1 ω9c. Based on genomic and physiological characteristics of strain Art1T, a new species of the genus Trichococcus, Trichococcusshcherbakoviae, is proposed. The type strain of Trichococcusshcherbakoviae is Art1T (=DSM 107162T = VKM B-3260T).

Reclassification of Eubacterium hallii as Anaerobutyricum hallii gen. nov., comb. nov., and description of Anaerobutyricum soehngenii sp. nov., a butyrate and propionate-producing bacterium from infant faeces.

A bacterial strain designated L2-7T, phylogenetically related to Eubacterium hallii DSM 3353T, was previously isolated from infant faeces. The complete genome of strain L2-7T contains eight copies of the 16S rRNA gene with only 98.0-98.5 % similarity to the 16S rRNA gene of the previously described type strain E. hallii. The next closest validly described species is Anaerostipes hadrus DSM 3319T (90.7 % 16S rRNA gene similarity). A polyphasic taxonomic approach showed strain L2-7T to be a novel species, related to type strain E. hallii DSM 3353T. The experimentally observed DNA-DNA hybridization value between strain L2-7T and E. hallii DSM 3353T was 26.25 %, close to that calculated from the genomes (34.3 %). The G+C content of the chromosomal DNA of strain L2-7T was 38.6 mol%. The major fatty acids were C16 : 0, C16 : 1cis9 and a component with summed feature 10 (C18 : 1c11/t9/t6c). Strain L2-7T had higher amounts of C16 : 0 (30.6 %) compared to E. hallii DSM 3353T (19.5 %) and its membrane contained phosphatidylglycerol and phosphatidylethanolamine, which were not detected in E. hallii DSM 3353T. Furthermore, 16S rRNA gene phylogenetic analysis advocates that E. hallii DSM 3353T is misclassified, and its reclassification as a member of the family Lachnospiraceae is necessary. Using a polyphasic approach, we propose that E. hallii (=DSM 3353T=ATCC 27751T) be reclassified as the type strain of a novel genus Anaerobutyricum sp. nov., comb. nov. and we propose that strain L2-7T should be classified as a novel species, Anaerobutyricum soehngenii sp. nov. The type strain is L2-7T (=DSM 17630T=KCTC 15707T).

Deciphering the trophic interaction between Akkermansia muciniphila and the butyrogenic gut commensal Anaerostipes caccae using a metatranscriptomic approach.

Host glycans are paramount in regulating the symbiotic relationship between humans and their gut bacteria. The constant flux of host-secreted mucin at the mucosal layer creates a steady niche for bacterial colonization. Mucin degradation by keystone species subsequently shapes the microbial community. This study investigated the transcriptional response during mucin-driven trophic interaction between the specialised mucin-degrader Akkermansia muciniphila and a butyrogenic gut commensal Anaerostipes caccae. A. muciniphila monocultures and co-cultures with non-mucolytic A. caccae from the Lachnospiraceae family were grown anaerobically in minimal media supplemented with mucin. We analysed for growth, metabolites (HPLC analysis), microbial composition (quantitative reverse transcription PCR), and transcriptional response (RNA-seq). Mucin degradation by A. muciniphila supported the growth of A. caccae and concomitant butyrate production predominantly via the acetyl-CoA pathway. Differential expression analysis (DESeq 2) showed the presence of A. caccae induced changes in the A. muciniphila transcriptional response with increased expression of mucin degradation genes and reduced expression of ribosomal genes. Two putative operons that encode for uncharacterised proteins and an efflux system, and several two-component systems were also differentially regulated. This indicated A. muciniphila changed its transcriptional regulation in response to A. caccae. This study provides insight to understand the mucin-driven microbial ecology using metatranscriptomics. Our findings show that the expression of mucolytic enzymes by A. muciniphila increases upon the presence of a community member. This could indicate its role as a keystone species that supports the microbial community in the mucosal environment by increasing the availability of mucin sugars.

Model-driven design of a minimal medium for Akkermansia muciniphila confirms mucus adaptation.

The abundance of the human intestinal symbiont Akkermansia muciniphila has found to be inversely correlated with several diseases, including metabolic syndrome and obesity. A. muciniphila is known to use mucin as sole carbon and nitrogen source. To study the physiology and the potential for therapeutic applications of this bacterium, we designed a defined minimal medium. The composition of the medium was based on the genome-scale metabolic model of A. muciniphila and the composition of mucin. Our results indicate that A. muciniphila does not code for GlmS, the enzyme that mediates the conversion of fructose-6-phosphate (Fru6P) to glucosamine-6-phosphate (GlcN6P), which is essential in peptidoglycan formation. The only annotated enzyme that could mediate this conversion is Amuc-NagB on locus Amuc_1822. We found that Amuc-NagB was unable to form GlcN6P from Fru6P at physiological conditions, while it efficiently catalyzed the reverse reaction. To overcome this inability, N-acetylglucosamine needs to be present in the medium for A. muciniphila growth. With these findings, the genome-scale metabolic model was updated and used to accurately predict growth of A. muciniphila on synthetic media. The finding that A. muciniphila has a necessity for GlcNAc, which is present in mucin further prompts the adaptation to its mucosal niche.