Abstract
Bacterial toxins are well-studied virulence factors; however, recent studies have revealed their importance in bacterial niche adaptation. Enterotoxigenic Bacteroides fragilis (ETBF) expresses B. fragilis toxin (BFT) that we hypothesized may contribute to both colonic epithelial injury and niche acquisition. We developed a vertical transmission model for ETBF in mice that showed that BFT enabled ETBF to access a lamina propria (LP) niche during colonic microbiome development that was inaccessible to non-toxigenic B. fragilis. LP entry by ETBF required BFT metalloprotease activity, and showed temporal restriction to the pre-weaning period, dependent on goblet-cell-associated passages. In situ single-cell analysis showed bft expression at the apical epithelial surface and within the LP. BFT expression increased goblet cell number and goblet-cell-associated passage formation. These findings define a paradigm by which bacterial toxin expression specifies developmental niche acquisition, suggesting that a selective advantage conferred by a toxin may impact long-term host health.
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Data availability
All data supporting the findings of this study are available within the paper and its Supplementary Information. Raw images supporting each relevant figure are available at https://www.ebi.ac.uk/bioimage-archive/ via BioStudies accession number S-BIAD922. Source data are provided with this paper.
Code availability
No specific code was used in the analysis of data in this article.
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Acknowledgements
This work was supported by a Pilot and Feasibility Award from the Digestive Diseases Research Core Center at the University of Chicago (NIDDK P30DK42086), National Institutes of Health (NIH) Award DK085025 to J.L.S., and NIH Awards AI138565 and AI157196 to J.B.W. B.W.C. was supported by the National Institute of Allergy and Infectious Diseases of the NIH (F30AI126791). B.W.C. and A.L.H. were trainees of the NIH Medical Scientist Training Program at the University of Chicago (GM007281). Math1fl/fl Vil-Cre-ERT2 mice were a gift from R. D. Newberry.
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C.A.H., B.W.C., E.V. and J.B.W. designed, performed and analysed the experiments. C.A.H., A.L.H., E.S.S. and J.L.S. generated the fluorescent bacterial strains. C.A.H., B.W.C., E.V. and J.B.W. wrote the article.
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Nature Microbiology thanks Kathryn Knoop, Sarkis Mazmanian and Benjamin Ross for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Vertical transmission model of ETBF acquisition.
(a) Timeline of vertical transmission experiments. (b) Dams (open circles) were mated, decolonized with clindamycin in drinking water beginning on E14, inoculated with 109 CFU ETBF on E15, maintained on clindamycin through E17, and assayed for faecal colonization weekly. Offspring (closed circles) were weaned at age 3 weeks (P21) and feces were collected for CFU analysis at the indicated time points. Average CFU ± standard deviation of n = 14 litters is shown, representing two independent replicates. (c) Weight of P21 mice colonized with NTBF, ETBF, or Δbft. (d) Stool CFU from mice in (c). For (c) and (d), each data point represents a litter average.
Extended Data Fig. 2 ETBF can access the lamina propria in adult mice.
Ceca, proximal colons, and distal colons were extracted at eight weeks from adult mice that were gavaged at six weeks with (a) ETBF, (b) Δbft, (c) NTBF, or (d) ETBF in the absence of antibiotic treatment. (e) Quantification of LP burden of each strain as in a-d. All strains constitutively express sfGFP. Tissue was sectioned at 30 µm, stained with phalloidin (grey) and DAPI (blue), and visualized by confocal microscopy. Scale bars = 20 µm. For each strain, images are representative of six adults.
Extended Data Fig. 3 The ccf locus facilitates crypt infiltration but is not required for BFT-mediated niche acquisition.
At P23, tissues were harvested from pups born to dams colonized with (a) ETBF or ETBF Δccf, (b) ETBF Δbft or ETBF ΔbftΔccf, (c) NTBF or NTBF Δccf, or (d) NTBF Δccf::bft. All strains constitutively express sfGFP. Sections were stained with DAPI (blue) and phalloidin (gray) and visualized by confocal microscopy. (e, f) To normalize crypt infiltration across strains, an infiltration ratio was calculated per strain by dividing the distance bacteria invaded by the total length of the crypt. (g) To illustrate total abundance in tissue versus crypt, the total number of bacteria present in crypts was divided by the total number of bacteria in adjacent lamina propria. (h) Analysis of the ratio of LP burden to total tissue burden (TTB = crypt + LP burden) of the noted strains. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 by one-way ANOVA. Scale bars = 10 μm. Data are representative of 4 biological replicates, n = 6-8 mice per strain. Displayed images are representative of a minimum of 6 independent images.
Extended Data Fig. 4 Early intervention with NTBF displaces ETBF in proximal colon.
Pups born to ETBF-colonized dams were inoculated with NTBF WT or ΔtssC on (a, b) P12, (c, d) P16, or (e, f) P20 and tissues were harvested from P23 mice to visualize ETBF (green) and NTBF or its ΔtssC variant (red) in the proximal colon (left and center panel) and enumerate CFU (right panel). * p < 0.05, *** p < 0.001, and **** p < 0.0001 in pairwise comparisons (two-tailed t-test). Scale bar = 20 µm. Data represents n = 11-14 mice in total per group (b), n = 18-23 mice in total per group (d), and n = 13 mice in total per group (f).
Extended Data Fig. 5 Early intervention with NTBF displaces ETBF in distal colon.
Pups born to ETBF-colonized dams were inoculated with NTBF WT or ΔtssC on (a, b) P12, (c, d) P16, or (e, f) P20 and tissues were harvested P23 mice to visualize ETBF (green) and NTBF or its ΔtssC variant (red) in the distal colon (left and center panel) and enumerate CFU (right panel). * p < 0.05, ** p < 0.01, and **** p < 0.0001 in pairwise comparisons (two-tailed t-test). Scale bar = 20 µm. Data represents n = 11-14 mice in total per group (b), n = 18-23 mice in total per group (d), and n = 13 mice in total per group (f).
Extended Data Fig. 6 BFT expression does not alter interbacterial competition in the pre-weaning period.
Pups born to (a) ETBF (ATCC 43859) or (b) ETBF Δbft-colonized dams were inoculated with NTBF (TM4000/638 R) on P16 and tissues were harvested from P23 mice to enumerate CFU (right panel). No statistically significant differences were found in pairwise comparisons (two-tailed t-test). Data represents n = 7–10 mice in total per group.
Extended Data Fig. 7 Recombinant BFT is sufficient to elicit an increase in colonic GCs.
Five-week-old mice born to dams colonized with ETBF Δbft were subjected to either apical (a, c) or basolateral (b, d) exposure of active BFT (BFTWT) or inactive toxin (BFTE349A) before tissue harvest 24-48 hours later. Sections were stained with AB-PAS and visualized with brightfield microscopy for enumeration of GCs and measurement of crypt length. ** p < 0.01, *** p < 0.01, and **** p < 0.0001 in pairwise comparisons (two-tailed t-test). Data represents 2-4 biological replicates with n = 5–7 mice in total per group.
Extended Data Fig. 8 Genetic ablation of GCs results in neonatal lethality in pups colonized with ETBF.
(a) Survival data for Math1fl/flVil-Cre-ERT2 mice either homozygous null (−/−) or heterozygous (+/−) for Vil-Cre-ERT2, born to dams colonized with ETBF. Math1 deletion was induced by daily tamoxifen injections starting at P5 and ending at P10. Ear tissue was collected from dead neonates for genotyping. Otherwise, mice were sacrificed at P23 for genotyping and to assess faecal ETBF CFUs (b). Math1fl/fl Vil-Cre-ERT2+/− = 14 mice; Math1fl/fl Vil-Cre-ERT2−/− = 6 mice. Gehan-Breslow-Wilcoxon test showed statistically significant differences between survival curves. p-value = 0.0176. Data representative of two independent experiments.
Extended Data Fig. 9 Lamina propria niche acquisition and toxin expression is dependent on antibiotic treatment in adult mice.
RNA in situ hybridization analysis with probes for gfp (green) and bft (red) was performed on 14 µm fixed-frozen sections of ceca that were extracted at eight weeks from adults gavaged at six weeks with (a) ETBF, (b) ETBF in the absence of antibiotic treatment, (c) Δbft, or (d) NTBF. Sections were stained with phalloidin (gray) and DAPI (blue) and visualized with Airyscan. Scale bar = 3 µm. For each strain, images are representative of six adults over two independent replicates (24 mice total).
Extended Data Fig. 10 Toxin exerts distinct effects based upon anatomical location.
Five-week-old mice born to dams colonized with ETBF Δbft were subjected to either apical (a–c) or basolateral (d–f) exposure of active (BFTWT) or inactive toxin (BFTE349A) before tissue harvest 24-48 hours later. All strains constitutively express sfGFP. Sections were stained with DAPI (blue) and phalloidin (gray) and visualized by confocal microscopy. (b, e) Tissue infiltration was assessed as bacilli per cubic micron of lamina propria (L.P.), and (e, f) crypt lengths were measured across all four groups. For a-c, n = 5 mice per strain, and for d-f, n = 8 mice per strain.** p < 0.01 and **** p < 0.0001 in pairwise comparisons (two-tailed t-test).
Supplementary information
Supplementary Information
Supplementary Figs. 1–3 and Table 1.
Supplementary Video 1
ETBF is visualized within the LP.
Supplementary Video 2
ETBF Δbft is not able to access the LP.
Supplementary Video 3
ETBF can enter the LP in antibiotic-treated adult mice.
Supplementary Video 4
ETBF Δbft is restricted from the LP in antibiotic-treated adult mice.
Supplementary Video 5
ETBF and the bft transcript are visualized in the LP.
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Hill, C.A., Casterline, B.W., Valguarnera, E. et al. Bacteroides fragilis toxin expression enables lamina propria niche acquisition in the developing mouse gut. Nat Microbiol 9, 85–94 (2024). https://doi.org/10.1038/s41564-023-01559-9
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DOI: https://doi.org/10.1038/s41564-023-01559-9
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