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.
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Data availability
Analysed data, including INSeq, RNA-seq and mass spectrometry outputs, as well as primer and adaptor sequences are available in Supplementary Data 1. Sequencing data used for this study can be found in the National Center for Biotechnology Information BioProject database with the accession code PRJNA955715. Unprocessed data from mass spectrometry and additional supporting data are available upon reasonable request from the corresponding authors. Source data are provided with this paper.
Code availability
Code for analysing the INSeq data is available at https://github.com/pmalkus/Akk_INseq_paper.
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Acknowledgements
We are thankful to O. Kuddar and E. Rivas for support with the assembly of arrayed Tn mutant libraries; J. Granek for the base trimming code; A. Sharma for preparing sequencing libraries; and members of the R.H.V. laboratory for critical reading of the manuscript. We thank L. Augenlicht at the Albert Einstein College of Medicine for providing the Muc2−/− mice. This work was supported by National Institutes of Health awards AI142376 and DK110496 (to R.H.V.), American Heart Association award 18POST34070017 (to L.E.D.) and a fellowship from the Natural Sciences and Engineering Research Council of Canada (PDF4878642016 to L.E.D.).
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R.H.V., L.E.D. and P.N.M. designed the research. L.E.D., P.N.M., M.V., L.D. and E.A. performed the experiments and analysed the data. L.E.D. prepared the figures. P.M.N. wrote the INSeq analysis code. Z.C.H. and J.L. contributed to running the SCFA analysis. M.V. performed the microfluidic droplet experiments. L.D. performed the live imaging experiments. L.E.D. and R.H.V. wrote the manuscript. R.H.V. supervised the project. All authors reviewed and edited the manuscript.
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Extended data
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 labelled with FM4-64. Experiments were repeated 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 3 h, 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 3 h 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 16 S 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-labelled 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) Droplet-seq 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 (72 h). 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/Verrucomicrobia.
(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 RNA-seq reads generated from wild type A. muciniphila grown in mucin medium mapped to genes in the mul1 and mul2 loci. (b) Growth curves for wild type A. muciniphila and mutants in mul1B and mul2B 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 (colours) 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 16 S 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). (b) Principal Coordinates Analysis (PCoA) performed on weighted UniFrac distances. Statistical significance was determined by Permutational Multivariate Analysis of Variance (PERMANOVA). (c) Relative abundances of potential mucin-degrading taxa at the family level. The centre line is the mean, and the whiskers show the minimum and maximum. (d) 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). (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 Muris47. Violin plots show the expression of Ldlr and Hmgcr in mouse colonic epithelial and goblet cells.
Supplementary information
Supplementary Video 1
View through an orthogonal section of a 3D reconstructed STED image of a mucinosome inside A. muciniphila. Fluorescein-labelled mucin is shown in magenta and the A. muciniphila cell surface (anti-Akk) is cyan.
Supplementary Video 2
Live imaging of A. muciniphila grown with fluorescein-labelled mucin under an agarose pad, with and without the addition of 50 μM CCCP. Images were captured every 30 s for 20 min. The arrows indicate cells with active mucinosome formation (untreated) and locations where mucin accumulates at the cell surface but fails to form mucinosomes in the presence of CCCP.
Supplementary Data 1
Data tables containing INSeq, RNA-seq and mass spectrometry outputs and primer and adaptor sequences.
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Davey, L.E., Malkus, P.N., Villa, M. et al. A genetic system for Akkermansia muciniphila reveals a role for mucin foraging in gut colonization and host sterol biosynthesis gene expression. Nat Microbiol 8, 1450–1467 (2023). https://doi.org/10.1038/s41564-023-01407-w
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DOI: https://doi.org/10.1038/s41564-023-01407-w
