Strain competition restricts colonization of an enteric pathogen and prevents colitis

Abstract

The microbiota is a major source of protection against intestinal pathogens; however, the specific bacteria and underlying mechanisms involved are not well understood. As a model of this interaction, we sought to determine whether colonization of the murine host with symbiotic non-toxigenic Bacteroides fragilis could limit acquisition of pathogenic enterotoxigenic B. fragilis We observed strain-specific competition with toxigenic B. fragilis, dependent upon type VI secretion, identifying an effector-immunity pair that confers pathogen exclusion. Resistance against host acquisition of a second non-toxigenic strain was also uncovered, revealing a broader function of type VI secretion systems in determining microbiota composition. The competitive exclusion of enterotoxigenic B. fragilis by a non-toxigenic strain limited toxin exposure and protected the host against intestinal inflammatory disease. Our studies demonstrate a novel role of type VI secretion systems in colonization resistance against a pathogen. This understanding of bacterial competition may be utilized to define a molecularly targeted probiotic strategy.

Keywords: colonization resistance; enterotoxigenic Bacteroides fragilis; in vivo strain competition; probiotics; type VI secretion.

Figure 1. NTBF strain dominance of ETBF through T6S

1. A–C

   SPF C57BL/6J mice were co‐colonized with E1 and N1 wild type (WT, A, n = 5 mice), N1 T6SS mutant (ΔtssC, B, n = 4), or N1 complemented (ΔtssC pTssC, C, n = 5). Fecal CFU was quantified for E1 (open squares) and N1 (closed squares) weekly.

2. D

   Four weeks post‐colonization, E1 fecal recovery was compared between the N1 WT, ΔtssC, and ΔtssC pTssC groups.

Data information: Results are representative of three independent experiments. Data are presented as mean ± SEM (A–C) or mean ± SD (D). n.s., not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by unpaired, parametric, two‐tailed Student's t‐test at each time point (applying Bonferroni correction), comparing the co‐colonizing strains (A–C) or one‐way ANOVA, Tukey's multiple comparisons test (D).

Figure EV1. The N1 T6SS is required for strain dominance of E1 both in vivo and in vitro

1. A, B

   SPF mice were co‐colonization with E1 WT and N1 WT (n = 5), T6SS mutant (ΔtssC, n = 4), or complemented (ΔtssC pTssC, n = 5). Fecal CFU was monitored for 4 weeks post‐colonization. N1 clone fecal CFU was compared between groups at the 4‐week time point (A) and the competitive index of E1 over N1 was determined for each mouse (B).

2. C, D

   Mice (n = 4) were mono‐colonized with N1 WT, T6SS mutant (ΔtssC), or complemented (ΔtssC pTssC), and fecal CFU was determined for 4 weeks (C). Comparison of fecal CFU was made between groups after 4 weeks (D).

3. E–G

   SPF mice were co‐colonized with E1 and N1 WT (n = 5), T6SS mutant (ΔtssC, n = 4), or complemented (ΔtssC pTssC, n = 5). Fecal CFU at 1 day post co‐colonization was determined for N1 (E) and E1 (F) and competitive index of E1 over N1 was calculated for each mouse (G).

4. H

   SPF mice (n = 4) were mono‐colonized with E1 and fecal CFU determined over time.

5. I

   In vitro competitions were performed between E1 WT and N1 WT, N1 ΔtssC, or N1 Δbte2, or between E1 Bti2a and N1 WT (n = 3 competitions). Recovered CFU of E1 was quantified after each competition, and statistical difference from E1 WT recovered after competition with N1 WT was determined.

Data information: Results are representative of three independent experiments. Data are presented as mean ± SD (A, B, D–G, I) or mean ± SEM (C and H). A dashed line denotes equal recovery of competitors. n.s., not significant; **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by one‐way ANOVA, Tukey's multiple comparisons test (A, B, D–G, I).

Figure 2. An effector–immunity pair is required for E1 colonization resistance

1. A

   Nucleotide alignment of the T6SS locus from N1 and E1. Percent identity is indicated as height, green representing high homology with red highlighting non‐conserved regions.

2. B–E

   Co‐colonization of N1 WT (B and D, n = 4 mice) or N1 Δbte2 (C, n = 4) with E1 WT (B and C) or E1 overexpressing Bti2a (E1 pBti2a, D). Fecal CFU was monitored over time (B–D) and E1 CFU compared to N1 WT‐E1 WT group at 4 weeks post co‐colonization (E).

Data information: Results are representative of two independent experiments. Data are presented as mean ± SEM (B–D) or mean ± SD (E). **P < 0.01, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by unpaired, parametric, two‐tailed Student's t‐test at each time point (applying Bonferroni correction) comparing the co‐colonizing strains (B–D) or one‐way ANOVA, Tukey's multiple comparisons test (E).

Figure 3. B. fragilis provides strain‐specific colonization resistance

1. A, B

   Initial colonization of gnotobiotic (A) or SPF (B) mice (n = 4 mice per group) with N1 followed by secondary challenge with N1 (closed squares) or E1 (open squares). Fecal CFU was determined for the primary and secondary colonization strains through 4 weeks post‐secondary challenges.

2. C

   All primary colonization and secondary challenge pairs were tested with 3 NTBF and 2 ETBF strains. Stable colonization of the secondary challenge strain significantly above the limit of detection is denoted as a “+” while non‐significance is denoted as a “−” (n = 4 mice per group). The diagonal gray bar indicates self‐secondary challenge, the horizontal red bars show strains that provide broad colonization resistance against non‐self strains, and the vertical dashed box indicates a strain that has an enhanced secondary colonization phenotype.

Data information: Results illustrate a single experiment (A) or are representative of at least two independent experiments (B and C). Data are presented as mean ± SEM. Arrows denote day of primary colonization and secondary challenge. A dashed line denotes limit of detection. **P < 0.01, ****P < 0.0001. Statistical significance was determined by unpaired, parametric, two‐tailed Student's t‐test at each time point (applying Bonferroni correction) comparing the secondary challenge strains.

Figure EV2. Successful B. fragilis secondary challenge is strain‐dependent

1. A–J

   Mice (n = 4 per group) were initially colonized with N1 (A and B), N2 (C and D), N3 (E and F), E1 (G and H), or E2 (I and J) followed by secondary challenge by all strains or a mock inoculum at 1 week post‐primary colonization. Fecal CFU of primary and secondary strains were monitored for 4 weeks post‐secondary challenge (A, C, E, G, and I). At the last time point, the secondary challenge strain was tested for statistical significance above the mock‐inoculated group (B, D, F, H, and J).

Data information: Results are representative of at least two independent experiments. Data are presented as mean ± SEM (A, C, E, G, and I) or mean ± SD (B, D, F, H, J). Limit of detection is denoted by a dashed line. ***P < 0.001, ****P < 0.0001. Statistical significance was determined by one‐way ANOVA, Tukey's multiple comparisons test (B, D, F, H, and J).

Figure 4. T6S is required for strain‐specific colonization resistance

1. A–F

   Primary colonization of SPF mice with N2 WT, T6SS mutant (ΔtssC), and complemented (ΔtssC pTssC) followed by secondary challenge with N1 WT (A and B, n = 5 mice), N2 WT (C and D, n = 5), or E1 WT (E and F, n = 4) was performed. Fecal CFU for primary and secondary strains was determined for 4 weeks post‐secondary challenge (A, C, E). Selected time points were tested for statistical difference of secondary challenge between groups. This includes 4 weeks post‐secondary challenge (B) and 3 days post‐challenge (D and F).

Data information: Results are representative of three independent experiments. Data are presented as mean ± SEM (A, C, and E) or mean ± SD (B, D, and F). Arrows denote day of primary colonization and secondary challenge. A dashed line denotes limit of detection. n.s., not significant; ****P < 0.0001. Statistical significance was determined by unpaired, parametric, two‐tailed Student's t‐test at each time point (applying Bonferroni correction, D and F) or one‐way ANOVA, Tukey's multiple comparisons test (B).

Figure EV3. T6SS‐dependent colonization resistance does not extend to related Bacteroides species in vivo

1. A

   B. thetaiotaomicron recovered after in vitro competition (n = 3 competitions) with N2 WT, N2 ΔtssC, or N2 ΔtssC pTssC.

2. B–D

   SPF mice (n = 4) were sequentially colonized with N2 WT, N2 ΔtssC, or N2 ΔtssC pTssC strains, followed by secondary challenge of B. thetaiotaomicron 1 week after primary colonization. Four weeks post‐secondary challenge, fecal CFU was determined for B. thetaiotaomicron (B). Fecal CFU for primary (C) and secondary strains (D) were determined for 4 weeks post‐secondary challenge.

3. E

   B. vulgatus recovered after in vitro competition with N2 WT, N2 ΔtssC, or N2 ΔtssC pTssC.

4. F–H

   SPF mice (n = 4) were sequentially colonized with N2 WT, N2 ΔtssC, or N2 ΔtssC pTssC followed by secondary challenge with B. vulgatus 1 week after primary colonization. Four weeks post‐secondary challenge, fecal CFU was determined for B. vulgatus (F). Fecal CFU for primary (G) and secondary strains (H) were determined for 4 weeks post‐secondary challenge.

Data information: Results are representative of three independent experiments. Data are presented as mean ± SEM (C, D, G, and H) or mean ± SD (A, B, E, and F). Limit of detection is denoted by a dashed line. n.s., not significant; **P < 0.01, ***P < 0.001. Statistical significance was determined by one‐way ANOVA, Tukey's multiple comparisons test (A, B, E, and F).

Figure 5. The N1 T6SS protects against ETBF‐induced disease

1. A

   Mice were co‐colonized with E1 and either N1 WT (n = 4) or N1 ΔtssC (n = 3). Five days post‐inoculation, fecal RNA was extracted and tested for BFT expression via qRT–PCR.

2. B

   Four weeks after co‐colonization with E1 and either N1 WT or N1 ΔtssC (n = 4 mice per group), the sera were collected, tested via ELISA for anti‐BFT IgG, and endpoint titer calculated.

3. C–F

   Mice pre‐treated with DSS were inoculated with no organisms (sham), E1 only, or E1 competed with N1 WT or N1 ΔtssC. Five days post‐inoculation, the ceca were weighed (C) and fixed for histopathological examination after sham (D), E1 only (E) and E1‐N1 WT (F) colonizations. Scale bars denote 100 μm (main image) and 200 μm (inset).

Data information: Experiments are a pooling of two independent repeats (A and B) or are representative of three independent trials (C–F). Data are presented as mean ± SD (A, B and C). *P < 0.05, ***P < 0.001, ****P < 0.0001. Statistical significance was determined by unpaired, parametric, two‐tailed Student's t‐test (A and B) or one‐way ANOVA, Tukey's multiple comparisons test (C).

Figure EV4. N1 protects from ETBF‐mediated intestinal damage via competition with E1 in DSS‐treated mice

1. A–D

   Mice pre‐treated with DSS were inoculated with no organisms (sham), E1 only, E1 with N1 WT, or E1 with N1 ΔtssC. Gross examination of ceca (A, n = 5 per group) and histopathological examination of colonic tissue (B–D, n = 5 per group) were performed after intestinal dissection. Scale bars (B–D) denote 100 μm (main image) and 200 μm (inset). Results are representative of three independent experiments.