Host-compound foraging by intestinal microbiota revealed by single-cell stable isotope probing

Abstract

The animal and human intestinal mucosa secretes an assortment of compounds to establish a physical barrier between the host tissue and intestinal contents, a separation that is vital for health. Some pathogenic microorganisms as well as members of the commensal intestinal microbiota have been shown to be able to break down these secreted compounds. Our understanding of host-compound degradation by the commensal microbiota has been limited to knowledge about simplified model systems because of the difficulty in studying the complex intestinal ecosystem in vivo. In this study, we introduce an approach that overcomes previous technical limitations and allows us to observe which microbial cells in the intestine use host-derived compounds. We added stable isotope-labeled threonine i.v. to mice and combined fluorescence in situ hybridization with high-resolution secondary ion mass spectrometry imaging to characterize utilization of host proteins by individual bacterial cells. We show that two bacterial species, Bacteroides acidifaciens and Akkermansia muciniphila, are important host-protein foragers in vivo. Using gnotobiotic mice we show that microbiota composition determines the magnitude and pattern of foraging by these organisms, demonstrating that a complex microbiota is necessary in order for this niche to be fully exploited. These results underscore the importance of in vivo studies of intestinal microbiota, and the approach presented in this study will be a powerful tool to address many other key questions in animal and human microbiome research.

Fig. 1.

Imaging ¹⁵N enrichment (δ¹⁵N) in the cecum tissue and intestinal lumen 8 h after i.v. injection of ¹³C,¹⁵N threonine. A mosaic of 16 individual high-resolution NanoSIMS images is shown. ¹²C¹⁴N⁻ secondary ion intensity distribution images of the same area are shown to illustrate the structure of the tissue and lumen biomass. ¹⁵N hotspots are indicated by white arrows. The patchy distribution of ¹⁵N hotspots in the tissue is likely attributable to the heterogeneity in mucus release activity both within and between mucus cells (24, 25).

Fig. 2.

Representative NanoSIMS (at% ¹³C and ¹⁵N) and FISH images 8 h after i.v. injection of ¹³C,¹⁵N threonine. (A) B. acidifaciens (blue) and Ruminococcaceae OTU_5807 (red) cells (bar: 5 μm; prepared without embedding; other Bacteria shown in green), (B) Akkermansia spp. (white) cells (bar: 2 μm; prepared without embedding), and (C) Lactobacillaceae/Enterococcaceae spp. (red) cells (bar: 5 μm; prepared without embedding; other Bacteria shown in green). (D) Semithin sections of lumen contents with Lachnospiraceae OTU_11021 (yellow/white, overlap of green and red signals), all other Bacteria (blue), and autofluorescent dietary fibers (green) (bar: 10 μm). Exemplary cells with or without significant enrichment (white or green arrows) are indicated. No enrichment in carbon is visible in D because of dilution by unlabeled carbon in the resin.

Fig. 3.

Single-cell stable isotope labeling of selected bacterial groups in the mouse intestine 8 h after i.v. injection of ¹³C,¹⁵N threonine. At% ¹³C and ¹⁵N was calculated for each cell. FISH probes targeted Akkermansia spp. (Akk), Mucispirillum spp. (Mcs), B acidifaciens (Bac), Ruminococcaceae OTU_5807 (Rum), and Lactobacillaceae/Enterococcaceae spp. (Lab) (probe details in Table S1). Eub338-targeted unlabeled cells (control) from the gut were used as controls. Each point represents a single cell, and box plots summarize the quartiles of the target population. Red points are significantly enriched cells (>95% confidence intervals).

Fig. 4.

Mice that host a four-member intestinal microbiota (4S microbiota) consisting of Lactobacillus acidophilus [Altered Schaedler Flora (ASF) 360], Lactobacillus murinus (ASF 361), Mucispirillum schaedleri (ASF 457), and a Parabacteroides sp. (ASF 519) were colonized with either A. muciniphila (4S-Akk) or B. acidifaciens (4S-Bac) or both together (4S-Akk/Bac). Isotope ratios (δ) are given in units of permil (‰) and are relative to Vienna PeeDee Belemnite (V-PDB) for δ¹³C and to atmospheric air (atm. air) for δ¹⁵N. Colonization with A. muciniphila or B. acidifaciens or both strains together had no significant effect in enrichment of ¹³C or ¹⁵N in the lumen. (A) EA–IRMS data of carbon and nitrogen in lumen contents 8 h after i.v. injection of ¹³C¹⁵N threonine in mice that had been colonized for 10 d. (B) Amount of total ¹³C in lumen acetate, propionate, and butyrate, measured with LC–IRMS. Values from NC microbiota mice used in (Fig. 1 B and C) are shown in A and B for comparison. (C) Per-cell at% ¹³C and ¹⁵N of selected bacterial groups. Measurements of ASF 360/361, 457, and 519 cells from 4S, 4S-Akk, and 4S-Bac mice were pooled because there were no significant differences between mouse types and Eub338-targeted cells that were not labeled (control) are shown for comparison. Each point represents the at% of a single cell, and box plots summarize the quartiles of the target population. Red points are significantly enriched cells (>95% confidence interval).