A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression

Abstract

Akkermansia muciniphila, a mucophilic member of the gut microbiota, protects its host against metabolic disorders. Because it is genetically intractable, the mechanisms underlying mucin metabolism, gut colonization and its impact on host physiology are not well understood. Here we developed and applied transposon mutagenesis to identify genes important for intestinal colonization and for the use of mucin. An analysis of transposon mutants indicated that de novo biosynthesis of amino acids was required for A. muciniphila growth on mucin medium and that many glycoside hydrolases are redundant. We observed that mucin degradation products accumulate in internal compartments within bacteria in a process that requires genes encoding pili and a periplasmic protein complex, which we term mucin utilization locus (MUL) genes. We determined that MUL genes were required for intestinal colonization in mice but only when competing with other microbes. In germ-free mice, MUL genes were required for A. muciniphila to repress genes important for cholesterol biosynthesis in the colon. Our genetic system for A. muciniphila provides an important tool with which to uncover molecular links between the metabolism of mucins, regulation of lipid homeostasis and potential probiotic activities.

Extended Data Fig. 1. Akkermansia sp. are mucin specialists and the acquisition of mucin by A. muciniphila is selective and energy dependent

(a) Growth curves, as assessed by optical density (OD₆₀₀) of a range of Gram-positive and Gram-negative mucin-degrading intestinal microbes, including A. muciniphila and A. glycaniphila, in the indicated medium. (b) A. muciniphila and Bacteroides thetaiotaomicron grown with fluorescein-mucin. The cells were grown with fluorescein mucin in a modified version of synthetic media with 0.25% mucin as the sole carbon source. Membranes were labeled with FM4–64. Experiments were three twice. (c-d) Mucin uptake is a specific and active process. A. muciniphila grown in the presence of either fluorescein-mucin or fluorescein-dextran (green) for 3 h and stained with anti-Akkermansia anti-sera (anti-Akk). All microscopy was performed at least three times (c). Flow cytometric analysis of cells grown in the presence of fluorescein-mucin for 3h, with or without pre-treatment with CCCP. Cells for flow cytometry were gated for the anti-Akkermansia positive population and the numbers under each curve represent the mean fluorescent intensity of fluorescein-mucin (d). A. muciniphila grown with fluorescein-mucin for 3h without CCCP or with CCCP treatment (e). Flow cytometry analyses of A. muciniphila grown with the cell permanent esterase carboxyfluorescein diacetate (CFDA) in the presence and absence of CCCP, and after heat inactivation (f). Scale bar = 1 μm. Error bars represent the standard error of the mean.

Extended Data Fig. 2. Transposon mutagenesis in A. muciniphila

(a) Map of the A. muciniphila optimized INSeq plasmid. (b) Overview of the A. muciniphila conjugation protocol. (c) PCR analysis confirming transposition. DNA from representative Tn mutants was amplified with primers for A. muciniphila specific 16S rRNA, the bla gene located on the delivery plasmid backbone, and the cat gene located with the transposon. This analysis was performed for every transposition experiment. (d) Southern blot analysis of Tn mutant DNA digested with HindIII and probed with DIG-labeled probes that recognize the cat gene in the Tn insert. Data is representative of two experiments with similar results. (e-f) A Cartesian mapping strategy to identify Tn insertions. (e) Trade-off between genome coverage and clonal redundancy, using simulated subsets of the arrayed collection optimized for low redundancy. A series of 96-well plates drawn from the arrayed collection that minimizes clonal redundancy was identified by simulation. The tradeoff between increasing genome coverage (orange) and increasing clonal redundancy (blue) as the number of plates included from the optimized series grows (X-axis). (f) Estimating location mapping accuracy for different sizes of the optimized library. The distribution of clonal replicates for increasing sizes of the optimized library was drawn from the simulation. The estimated number of clones present in one, two, three, and four replicates are shown as a function of increasing collection size. For orthogonal pooling and Cartesian location mapping the search space for an individual clone scales as the number of replicates to the power of the number of pooling dimensions. A clone present in only one well has a unique plate-well address (1^3), while a clone with present three times in the collection would be mapped to 27 potential Plate-Row-Col locations (3^3).

Extended Data Fig. 3. INSeq analysis of relative nutritional requirements for A. muciniphila to grow in mucin medium and the role of putative glycan hydrolases

Plot of INSeq data from Tn mutant pools grown for eight generations in mucin medium where each dot represents all inserts in a specific gene. Genes that belong to KEGG amino acid biosynthesis pathways are highlighted for cultures grown in (a) mucin medium and (b) mucin medium supplemented with Phytone. Predicted glycosyl hydrolases for A. muciniphila BAA-835 were identified using the CAZy database and highlighted on the INSeq plot for cultures grown in (c) mucin and in (d) mucin medium with Phytone. Statistical analysis on INSeq data was performed with a Mann-Whitney Utest. (e) Dropletseq analysis of A. muciniphila grown in mucin medium microdroplets. Tn mutants (Arrayed Pool) were injected into a microfluidic device at a low density to generate on average less than one bacterium per droplet. The graph displays the INSeq analysis and Log₂ fold change for cultures grown in mucin in batch culture (8 generations) versus single cell growth in droplets (72h). Selected genes that were depleted in one condition relative to the other are highlighted on the plot. GH, glycosyl hydrolase.

Extended Data Fig. 4. A significant proportion of A. muciniphila genes required for growth in mucin medium are specific to Akkermansia/Verru comicrobia

(a) Number of genes required for optimal A. muciniphila growth in mucin medium that lack functional annotations. Genes corresponding to Tn mutants with a Log₂ > 2 fold decrease in abundance in mucin medium were used as the query for a BLAST search to identify potential homologs. The plot represents the number of genes encoding hypothetical proteins that were unique to Akkermansia spp. (Akk), homologs in other members of the PVC super phylum (PVC), homologs in other bacteria (other), and genes annotated as conserved hypothetical proteins (conserved). (b) Distribution of genes with Pfam designations belonging to pili or type II secretion families (Pili/T2SS), or TPR families in the INSeq analysis of genes required for growth in mucin medium in vitro, (c) in the cecum of germ-free mice, and (d) in the cecum of conventional mice.

Extended Data Fig. 5. Evidence for the presence of a stable Mul1A-Mul1B protein complex

Transcriptional analysis of Mul1 operons. (a) View of RNAseq reads generated from wild type A. muciniphila grown in mucin medium mapped to genes in the mul1A and mul1B loci. (b) Growth curves for wild type A. muciniphila and mutants in mul1A and mul1B grown in triplicate in synthetic medium or with mucin as the sole carbon and nitrogen source and corresponding microscopy with FL-mucin (green). Cells are stained with anti-Akkermansia antisera (white). The scale bar is 1 μm. (c) Coomassie blue stained SDS-PAGE gel showing eluted proteins following immunoprecipitation with anti-Mul1 antibodies. Immunoprecipitations were performed with cell lysates from wild type A. muciniphila and in mul1A mutants. (d) Depiction of Conserved Domains (colors) in Muc5AC and locations of peptides identified as co-precipitating with Mul1A (vertical bars). The experiment was performed in triplicate.

Extended Data Fig. 6. Mucin utilization is required for A. muciniphila to compete in CONV mice and in Muc2^−/− mice

A breeding colony of Akkermansia-free mice (Akk-free) was generated to facilitate mouse colonization without antibiotic pre-treatment. (a-c) Comparison of the microbiota of Akkermansia colonized (Akk-colonized) and Akk-free mice by 16S rRNA gene sequencing. (a) Relative abundances of fecal bacteria at the genus level in Akk-colonized and Akk-free mice. Each sample was obtained from separately housed mice (n = 3 per group). The center line is the mean and the whiskers show the minimum and maximum. (b) Principal Coordinates Analysis (PCoA) performed on weighted UniFrac distances. Statistical significance was determined by Permutational Multivariate Analysis of Variance (PERMANOVA). (c) Linear discriminant analysis Effect Size (LEfSe) analysis of Akk-colonized and Akk-free mice. The Kruskal-Wallis test was used to detect features with a significant differential abundance (p < 0.05). (d) Relative abundances of potential mucin-degrading taxa at the family level. (e) CONV mice were pre-treated with antibiotics and gavaged with a 1:1 mix of WT and mutant A. muciniphila prepared with a fecal slurry from Akk-free mice to partially reconstitute the microbiota (n = 6 per group). Bacterial loads in fecal pellets were quantified by qPCR, each point represents one cage. (f) Colonization of mucin deficient Muc2^−/− mice with A. muciniphila. Each point represents the average A. muciniphila per gram of feces (WT, n = 4; mul1A::Tn, n = 4; mul2A::Tn, n = 6). (g) Competition between wild type A. muciniphila and the mul1A::Tn mutant in Muc2^−/− mice. Mice were gavaged with a 1:1 mix of wild type and mutant and abundance was monitored over time using strain specific primers. Each point represents the average amount of A. muciniphila (n = 4), error bars represent the standard error.

Extended Data Fig. 7. The impact of mucin utilization by A. muciniphila in colonization along the GI tract, SCFA production and transcriptional responses

(a) Abundance of A. muciniphila wild type and mul1A mutants along the GI tract of female GF mice (n =3). Intestinal contents were scraped from sections along the GI tract and A. muciniphila levels were quantified by qPCR. Data are presented as mean values +/− SEM. The analysis was carried out with the same female mice that were used for RNAseq. (b) Expression of cholesterol biosynthesis genes in male and female mice, and control mice gavaged with sterile PBS. (c) Normalized expression of genes that are pivotal to cholesterol biosynthesis (Hmgcr) and uptake (Ldlr) in relation to cecal acetate and propionate levels. (d) Representative single cell RNAseq expression data from the Tabula Muris. Violin plots show the expression of Ldlr and Hmgcr in mouse colonic epithelial and goblet cells.

Figure 1.. A. muciniphila accumulates mucin glycans and requires amino acid biosynthesis for replication in mucin as a sole carbon and nitrogen source.

(a-c) Mucin accumulates within A. muciniphila intracellular compartments. (a) STED imaging of A. muciniphila showing mucin glycans or mucin degradation products within intracellular compartments (“mucinosomes”). Bacteria grown with fluorescein (FL) labeled mucin (purple) were stained with anti-Akkermansia antisera (anti-Akk, cyan). The lower panel shows orthogonal views of A. muciniphila (white lines denote the orthogonal plane) and the right panel shows a 3D reconstruction of a single cell. Images are representative of three independent experiments. (b) Flow cytometry of A. muciniphila incubated with FL-mucin over a 24 h. (c) Live cell imaging over 3 min shows accumulation of FL-mucin (green) at the A. muciniphila cell pole (Extended Video S2). (d-f) Amino acid biosynthesis is required for optimal growth of A. muciniphila on mucin. A complex Tn mutant pool was grown in synthetic medium (Input) or mucin medium for eight generations (Output). Each dot represents the pooled Tn insertions for each gene as determined by INSeq. Unadjusted P values were determined by Mann-Whitney U-test. (d). A comparison of the transcriptional response to growth on mucin (RNAseq) to the fitness of the corresponding mutants (IN-Seq) (e). Each dot represents one gene, and only genes that were detected in both the INSeq and RNAseq datasets are shown. RNAseq represents the Log₂ fold change in expression in mucin vs synthetic media, while INSeq represents the Log₂ fold change in the abundance between input Tn mutant pools versus the output. Dashed lines represent no change. (f) Overrepresentation analysis to identify differentially abundant KEGG pathways (Log₂ fold change > 1.5, p < 0.05) detected by INSeq and RNAseq following growth in mucin. P values were calculated by hypergeometric distribution. (Scale bar =1μm)

Figure 2.. The metabolic requirements for A. muciniphila to colonize the GI tract increase as the host microbiota becomes more complex.

(a) Chromosomal location of genes with Tn insertions that produced a significant change in abundance after growth in mucin medium (Mucin), or in the cecum of the following mouse models: germ-free (GF), Altered Schaedler Flora mice (ASF), conventional (CONV), and Muc2-deficient (Muc2^−/−). Lines represent the location of Tn inserts that result in growth defects (Log₂ fold change > 5, p < 0.05, unadjusted Mann-Whitney U-Test). Dots along the outer ring indicate locations of all Tn insertions and numbers represent chromosomal location (bp). (b) KEGG pathways overrepresented (q < 0.05) among mutants that displayed significant fitness defects. (c) Global analysis of the abundance of mutants with fitness defects in mucin medium as determined by INSeq and analyzed with Omics Dashboard. The larger nodes represent the mean Log₂ fold change for components of a metabolic pathway, and the smaller nodes represent individual genes within each pathway. The boxed figure shows the mean Log₂ fold change for individual amino acid biosynthesis pathways. (d) Venn diagram showing overlapping genes required for growth under various conditions. For each condition, genes with a Log₂ fold change in abundance >1 were compared. The number of shared genes and the percentage of the total mutant pool are shown for each condition. (e) Omics Dashboard analysis of genes encoding putative cell surface components. Genes with annotations corresponding to predicted functions in capsule and exopolysaccharide biosynthesis were manually curated (Exopolysacchar). Additional functions related to plasma membrane biogenesis (Plasma Mem), cell wall biogenesis (Cell Wall Gen), and transport of amino acids, carbohydrate, and ions across the cell wall (Transport) were predicted in BioCyc. (f) Detailed view of exopolysaccharide biosynthesis or capsule genes and the relative fitness of respective Tn mutants in each environment. (g) INSeq analysis of genes in the assimilatory sulfate reduction (ASR) pathway. Heatmaps represent the Log₂ fold change of input versus output for mutants in the pathway.

Figure 3.. MUL loci encode for a mucin transport complex in A. muciniphila.

(a) Map of the Mucin Utilization Loci, mul1 (Amuc_0543 – Amuc_0550) and mul2 (Amuc_1098 – Amuc_1102). Blue arrows represent genes with Tn inserts and their Log₂ fold change in abundance after culturing in mucin. Genes without Tn inserts are represented as grey arrows. Bars represent the mean normalized reads for each Tn insert in the input pool and the output after growth in mucin. (b-g) Characterization of A. muciniphila mul mutants defective for mucin utilization display different patterns of association with FL-mucin. Growth curves of mutants grown in synthetic or mucin medium (b-e), where mucin is the sole carbon and nitrogen source, and (f) flow cytometric analysis of fluorescein-mucin acquisition by mul mutants. Mean fluorescein intensity was quantified for bacteria detected with anti-Akkermansia antibodies. (g) Mutants lacking mul1A and mul2A, but not a galactose epimerase (Amuc_0029), failed to accumulate mucin or mucin degradation products in intracellular compartments. Cultures were grown in synthetic medium supplemented with FL-mucin prior to staining with anti-Akkermansia antibodies (anti-Akk). The insets show the corresponding image with enhanced brightness to visualize FL-mucin in mul1A and mul2A mutants. Images are representative of three independent experiments. The scale bar is 1 μm. (h) Multiple proteins associate with the TPR domain protein Mul1A. Mass spectrometric (LC-MS/MS) analysis of proteins that co-immunoprecipitated with Mul1A. Numbers refer to the gene ID (amuc). Node size reflects the Log₂ fold change in normalized spectral counts over immunoprecipitations performed with mul1A mutants and edge thickness is scaled to the -Log₁₀(P value). Nodes are color coded based on Pfam. Statistical analysis of peptides used two-tailed heteroscedastic t-test on Log2-transformed data. (i) Proposed model of the MUL transporter that imports mucin glycans or mucin degradation products. Mul1A and Mul1B form a complex with accessory proteins that include sulfatases, GHs, and potential inner and outer membrane transporters (sodium solute symporter (SSS) and TonB dependent transporter (TBDT)).

Figure. 4.. Mucin utilization enables A. muciniphila to compete against members of the microbiota and leads to repression of genes in cholesterol biosynthesis.

(a-c) Mucin utilization gives A. muciniphila a competitive advantage. In GF mice, growth on mucin is not required for mono-colonization (a), but mul mutants are outcompeted by wild type A. muciniphila (WT) (b). Similarly, mul mutants were outcompeted in mice with a conventional microbiota that lacks A. muciniphila (Akk-free) (c). Each point represents the average A. muciniphila per gram of feces per cage (GF, n = 5; Akk-free, n = 6). (d) SCFA content of cecal contents from GF mice colonized with wild type A. muciniphila (WT, n=15), mul1A Tn mutants (n=13), or vehicle controls (PBS, n=12). Acetate and propionate were analyzed by One Way ANOVA (F statistic). The ratio of propionate to acetate was analyzed by a two-tailed student’s t test. Data are presented as mean values +/− SD. (e-h) Comparison of the transcriptional profiles of colonic tissue from GF mice colonized with mul1A mutants or wild type A. muciniphila. (e) RNAseq of colon tissue. Each point represents a gene (grey), and colors indicate a Log₂ fold change > 1 (green, dashed line), a Benjamini-Hochberg adjusted P value < 0.05 (blue, dashed line), or both (red). Significance was determined using the Wald test. (f) MetaScape enrichment plot and (g) network visualization showing pathways enriched in mice colonized with the mul1A mutants. Node colors correspond to cluster annotation and edge thickness denotes relatedness of the pathways. BH adjusted P values were determined in MetaScape with a hypergeometric test. (h) Differential expression of genes along the mevalonate and cholesterol biosynthesis super pathways visualized with BioCyc. Enzyme names are color coded to indicate relative expression levels between mice colonized with mul1A mutants versus wild type A. muciniphila. (i) Expression of selected genes in response to mul1A mutants (Wald test with BH adjusted P values). (LoD = limit of detection)