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. 2015 Sep 22:6:8292.
doi: 10.1038/ncomms9292.

The outer mucus layer hosts a distinct intestinal microbial niche

Hai Li  1 , Julien P Limenitakis  1 , Tobias Fuhrer  2 , Markus B Geuking  1 , Melissa A Lawson  1 , Madeleine Wyss  1 , Sandrine Brugiroux  3 , Irene Keller  1 , Jamie A Macpherson  1 , Sandra Rupp  1 , Bettina Stolp  4 , Jens V Stein  4 , Bärbel Stecher  3 , Uwe Sauer  2 , Kathy D McCoy  1 , Andrew J Macpherson  1
Affiliations

The outer mucus layer hosts a distinct intestinal microbial niche

Hai Li et al. Nat Commun. .

Abstract

The overall composition of the mammalian intestinal microbiota varies between individuals: within each individual there are differences along the length of the intestinal tract related to host nutrition, intestinal motility and secretions. Mucus is a highly regenerative protective lubricant glycoprotein sheet secreted by host intestinal goblet cells; the inner mucus layer is nearly sterile. Here we show that the outer mucus of the large intestine forms a unique microbial niche with distinct communities, including bacteria without specialized mucolytic capability. Bacterial species present in the mucus show differential proliferation and resource utilization compared with the same species in the intestinal lumen, with high recovery of bioavailable iron and consumption of epithelial-derived carbon sources according to their genome-encoded metabolic repertoire. Functional competition for existence in this intimate layer is likely to be a major determinant of microbiota composition and microbial molecular exchange with the host.

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Figures

Figure 1
Figure 1. Microbial communities in the mucus layer and content vary in the intestine.
(a) The microbial composition within the mucus layer (Muc) and luminal content (Con) of different segments along the caecum and colon of SPF mice was determined by 16S amplicon analysis. Representative bar graphs from one mouse out of four to six mice per group are shown. (b) Principal coordinates analysis on weighted UniFrac distances was performed on all operational taxonomic units. P-values to determine the statistical significance of clustering were calculated using the Adonis method. Analysis was performed using QIIME 1.8.0.
Figure 2
Figure 2. B. thetaiotaomicron in colonic mucus and contents has different transcriptional patterns.
(a) A heat map of gene expression values shows differentially expressed genes identified by RNA-seq in bacteria isolated from colonic mucus and contents of B. thetaiotaomicron monocolonized C57BL/6 mice. Data shown are three experimental repeats from the two compartments. (b) The gene expression levels of B. thetaiotaomicron in colonic mucus were compared with colonic contents and the log2 transformed fold change was plotted against log10 transformed P-value adjusted (padj). Red-coloured dots indicate genes with log2 FoldChange ≥2 and padj ≤0.05. (c) A network analysis was performed on genes highlighted in red in b according to translated protein–protein interactions using STRING database. The formed gene clusters were differentially coloured and functional definitions were provided to according clusters. Original transcriptomic data with annotations are given in Supplementary Data 1.
Figure 3
Figure 3. E. coli in colonic mucus and contents has different transcriptional patterns.
(a) A heat map of gene expression values shows differentially expressed genes identified by RNA-seq in bacteria isolated from colonic mucus and contents of E. coli monocolonized C57BL/6 mice. Data include three experimental repeats. (b) A volcano plot was constructed as in Fig. 2b visualizing E. coli transcriptional patterns. (c) Network analysis on E. coli was performed as in Fig. 2c. Original transcriptomic data with annotations are given in Supplementary Data 2.
Figure 4
Figure 4. Iron recovery predominates behaviours of E. coli in colonic mucus.
Genes (a) belonging to Fur regulon or (b) repressed by RyhB or (c) responding to oxygen levels (ArcA/FNR) of E. coli as reported previously were listed out. The ratio of expression levels of the whole panel of genes in colonic mucus relative to colonic contents is shown. Several genes/clusters were highlighted in indicated colours referring to same genes shown in Supplementary Fig. 9. The annotations of all displayed dots were listed in Supplementary Data 4. The red dotted line indicates a ratio of unity between colonic mucus and contents.
Figure 5
Figure 5. Ex vivo and in vivo metabolites consumption assay and bacterial replication dynamics.
(a) E. coli was cultured on colonic mucus and luminal contents harvested from germ-free mice (n=4). The consumed and secreted metabolites were identified at sequential time points comparing metabolic patterns before and after E. coli growth by non-targeted mass spectrometry analysis. R2 values representing the goodness of exponential fit of nonlinear regression indicate the continuity of metabolite variation trend among the time course. Metabolites with annotations labelled in green indicate possible alternative molecular species desigations (listed in full in Supplementary Data 5). (b) E. coli and (c) B. thetaiotaomicron replication rates in colonic mucus and contents from monocolonized mice were addressed by 32P-radioactivity decay after metabolic labelling of the test bacteria. Data are pooled from three independent experiments. (d) The average intensity of classified phospholipids detected directly ex vivo from colonic mucus from E. coli monocolonized mice (n=4), B. thetaiotaomicron monocolonized mice (n=4) were compared with germ-free mice (n=4). Comparison of detected common amino acids served as a control group. The red dotted line indicates ratios of unity between monocolonized and germ-free status. (e,f) Metabolites classified as oligosaccharides from (e) colonic contents or (f) colonic mucus were compared directly ex vivo as described for d. NS, not significant.
Figure 6
Figure 6. RNAseq and STRING analysis on E. coli and B. thetaiotaomicron from bicolonized mice mucus.
Network analysis on transcriptional patterns of E. coli in colonic mucus (a) and B. thetaiotaomicron in colonic mucus (b) from E. coli and B. thetaiotaomicron bicolonized mice were performed as in Fig. 2c. Original transcriptomic data with annotations are given in Supplementary Data 10 and 11.
Figure 7
Figure 7. RNAseq and STRING analysis on E. coli and B. thetaiotaomicron from bicolonized mice contents.
Network analysis on transcriptional patterns of E. coli in colonic contents (a) and B. thetaiotaomicron in colonic contents (b) from E. coli and B. thetaiotaomicron bicolonized mice were performed as in Fig. 2c. Original transcriptomic data with annotations are given in Supplementary Data 10 and 11.
Figure 8
Figure 8. Gene cluster distribution allows discrimination between mucus and content compartments.
A heat map of the correlation of each cluster identified in the STRING analysis and the number of genes each cluster is shown. The unsupervised profiles for each experimental condition clustered E. coli and B. thetaiotaomicron separately, whereas regrouping profiles for monocolonization and bicolonization for each compartment and microbe.
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