Infection trains the host for microbiota-enhanced resistance to pathogens

Abstract

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

Keywords: Citrobacter rodentium; Enterococcus faecalis; Klebsiella pneumoniae; aerobic respiration; bile acid; bismuth subsalicylate; colonization resistance; gut microbiome; hydrogen sulfide; taurine.

Figure 1.. Infection-trained microbiota enhance colonization resistance

(A) Kpn colony forming units (CFU) in the gut luminal contents and mucosal scrapings of SPF mice at 1 day (21–24 hours) post-Kpn infection (1 experiment with 2 paired cages, n = 10). (B) Kpn CFU in the feces of control (untreated) and streptomycin-treated mice. Points show median ± 95% confidence intervals (1 experiment, n = 4–5). (C) Scheme for wildR and post-∆yopM mice. For infection with Kpn, naïve SPF mice served as the control. (D) Kpn CFU in the feces of control and F10 wildR mice at 1 day post-Kpn infection (2 pooled experiments, n = 14, fold decrease = 14). An outlier was identified and removed using Grubbs’ method with α = 0.0001. (E) Kpn CFU, at 1 day post-Kpn infection, in the feces of control and post-∆yopM mice at 4 weeks post-∆yopM infection (2 experiments, n = 10–14, fold decrease = 6) and >15 weeks post-∆yopM infection (4 experiments, n = 19–20, fold decrease = 13). (F) Kpn CFU in the feces of ex-GF mice, conventionalized with the microbiota of control or post-∆yopM mice, at 1 day post-Kpn infection (2 pooled experiments, n = 13–15, fold decrease = 7). (G-H) Principal coordinates analysis (PCoA) plots of unweighted UniFrac distances between the 16S profiles of (G) control and F2 wildR mice (n = 11–12) and (H) control and post-∆yopM mice at >15 weeks post-∆yopM infection (n = 9–10). Percentages represent the variance explained by each PC. (I) Relative abundance of Proteobacteria in the gut microbiota of post-Yptb ∆yopM (4 experiments, n = 18–20, fold increase = 69), post-Yptb WT (1 experiment, n = 3–9, fold increase = 11), F2 wildR (3 experiments, n = 11–12, fold increase = 164), and respective control mice. (J) Fold change in the median abundance of Proteobacteria classes in the gut microbiota of post-Yptb vs. control mice (circles, Yptb ∆yopM; triangles, Yptb WT; n = 23–27) and F2 wildR vs. control mice (n = 11–12). Post-Yptb bars show mean ± SEM of 5 paired cages. The control for post-Yptb (∆yopM and WT) and F10 wildR mice was naïve SPF mice. The control for F2 wildR mice was F2 labR mice. In box and whisker plots, lines connect the medians of paired cages. Dotted lines indicate the detection limit. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney test (A-F, I; in J, with Benjamini-Hochberg correction) or PERMANOVA (G-H). See also Figures S1–S2 and Table S1.

Figure 2.. Infection-trained microbiota are enriched for taurine-utilizing taxa

(A) Fold changes in the median abundance of functions (EC numbers with log₂ fold change > 0.5, P < 0.05) in the shotgun metagenome of post-∆yopM mice at >15 weeks post-∆yopM infection (compared to naïve SPF mice) vs. F2 wildR mice (compared to F2 labR mice). Functions differentially abundant in only post-∆yopM mice are black diamonds; only wildR, gray circles; enriched in both post-∆yopM and wildR, blue; differentially abundant in both post-∆yopM and wildR, red. (B) Number of functions co-enriched in the post-∆yopM and F2 wildR metagenomes. (C) Fold change in the median abundance of functions enriched in the metagenome of post-∆yopM vs. naïve SPF mice (n = 11) and F2 wildR vs. F2 labR mice (n = 10). Post-∆yopM bars show mean ± SEM of 3 paired cages. (D) Pathways enriched in the post-∆yopM metagenome. Pie charts represent the relative contribution of taxa to a function. A, mqnA (same for E, X, B, and C); MQ, menaquinone; other, aggregate of rare taxa. (E) Model for Deltaproteobacteria energy generation. MQ, oxidized menaquinone; MQH₂, reduced menaquinone. (F) Abundance of taurine in the cecal contents of control and post-∆yopM mice at >15 weeks post-∆yopM infection (3 pooled experiments, n = 13–14, fold increase = 4.7). a.u., arbitrary units. (G) The microbiota converts primary (1°) bile acids to taurine and secondary (2°) bile acids. (H) Abundance of total taurine-conjugated bile acids in the cecal contents of control and post-∆yopM mice at >15 weeks post-∆yopM infection (3 pooled experiments, n = 12–14, fold increase = 1.6). (I) Growth yield (measured by absorbance at 600 nm) of B. wadsworthia in rich media + vehicle, taurine, or sulfate. Bars show mean ± SEM (1 experiment, n = 4). (J) Kpn CFU in the feces of ex-GF mice, conventionalized with SPF microbiota ± pre-engraftment with B. wadsworthia (Bw), at 1 day post-Kpn infection (2 pooled experiments, n = 10, fold decrease = 5.3). The dotted line indicates the detection limit. (K) Model: Elevated taurine post-infection enriches for gut taxa that utilize taurine. In box and whisker plots, lines connect the medians of paired cages. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.01 by Mann-Whitney (A, C, F, H-J) or hypergeometric (B) test. See also Figure S3 and Tables S2–S3.

Figure 3.. Taurine-trained microbiota enhances colonization resistance

(A) Scheme for taurine treatment. Mice were placed onto drinking water (vehicle) ± 200 mM taurine for 2–3 weeks prior to Kpn infection. (B) Abundance of taurine and total bile acids in the feces of vehicle and taurine-treated mice (1 experiment with 2 paired cages, n = 10, taurine fold increase = 2.1). (C) Kpn CFU in the feces of vehicle and taurine-treated mice at 1 day post-Kpn infection (8 pooled experiments, n = 58–59). Lines connect the means of paired cages (fold decrease = 4.5; for medians of paired cages, fold decrease = 2.1). (D) Kpn CFU in the feces of ex-GF mice, conventionalized with the microbiota of vehicle or taurine-treated mice, at 1 day post-Kpn infection (2 pooled experiments, n = 12, fold decrease = 11). (E) C. rodentium CFU in the feces of vehicle and taurine-treated mice at 5 days post-C. rodentium infection (5 pooled experiments, n = 39–40, fold decrease = 241). (F) Ten most differentially abundant species that were significant in at least 1 of 3 paired groups of vehicle and taurine-treated mice (experiment A, B, and/or C). Species are named according to their lowest taxonomic classification. Order, o; family, f; genus, g; Lachno., Lachnospiraceae; Rumino., Ruminococcaceae. (G) Median abundance of functions (EC numbers) in the shotgun metagenome of taurine vs. vehicle-treated mice. Significant functions (log₂ fold change > 0.5, P < 0.05) are red (2 pooled experiments, n = 9). dsr, dissimilatory sulfite reductase (fold increase = 8.3); ppm, parts per million (read counts assigned to a function per million total read counts). (H) Pie chart representing the relative contribution of species to dsr. Species are named according to their lowest taxonomic classification (order, o; genus, g). Figure 1. Infection-trained microbiota enhance colonization resistance (I) Hydrogen sulfide released by the gut microbiota of vehicle and taurine-treated mice during ex vivo culture ± taurine (1 experiment with 2 paired cages, n = 9–10, fold increase = 1.6). Data are representative of 2 experiments. (J) Model: Taurine enriches for gut taxa that generate sulfide. In box and whisker plots, lines connect the medians of paired cages, unless otherwise indicated. Dotted lines in C, D, and E indicate the detection limit. n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney test (B-G, I). See also Figure S4 and Tables S1–S2.

Figure 4.. Taurine-derived sulfide inhibits pathogen respiration

(A) Model for taurine-enhanced colonization resistance. (B) Kpn 1,2-propanediol utilization (pdu) gene organization. Numbers above genes in gray indicate fold decrease in control mice relative to post-∆yopM mice (2 pooled experiments, n = 6–7 samples/group). (C) Fold increase in Kpn CFU after growth on 1,2-propanediol (5 mM) as the sole carbon source ± an electron acceptor (50 mM). tetra., tetrathionate; DMSO, dimethyl sulfoxide; TMAO, trimethylamine N-oxide (2 pooled experiments, n = 6). (D) The competitive index (CI, output ratio divided by input ratio of WT:mutant) of the Kpn WT and cyxB^- mutant after growth on 1,2-propanediol ± oxygen (2 pooled experiments, n = 6). (E) Fold increase in Kpn CFU after growth on 1,2-propanediol ± sodium hydrosulfide (NaHS), taurocholate (5 mM), or taurine (5 mM) (2 pooled experiments, n = 4). (F) The CI of Kpn WT:cyxB^- and WT:pduQ^- in the feces of vehicle and taurine-treated mice at 1 day post-Kpn infection (2 pooled experiments, n = 14–15, fold decrease = 2). The dotted line indicates equal fitness between WT and mutant. Lines connect the medians of paired cages. (G) Model: Sulfide generated by the gut microbiota from taurine inhibits pathogen respiration. Bars show mean ± SEM. n.s., not significant; †, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney (C-F) or Wald (B) test. See also Figure S5 and Table S5.

Figure 5.. Sulfide sequestration unleashes pathogen respiration

(A) Scheme for bismuth treatment. Mice were placed onto drinking water (vehicle) ± 20 mM bismuth subsalicylate for 12 hours prior to Kpn infection. (B) Hydrogen sulfide released by the gut microbiota of vehicle and bismuth-treated mice during ex vivo culture (1 experiment with 2 paired cages, n = 8, fold decrease = 1.3). (C) Model for the impact of sulfide on gut ecology. (D) PCoA plot of unweighted UniFrac distances between the 16S profiles of mice at 0, 20, and 36 hours post-bismuth treatment (1 experiment with 2 paired cages, n = 5–7). Percentages represent the variance explained by each PC. (E-F) Relative abundance of (E) taxonomic classes and Kpn and (F) E. faecalis (Bacilli) and E. coli (Gammaproteobacteria) in the gut microbiota of mice at 0 and 36 hours post-bismuth treatment (1 experiment with 2 paired cages, n = 5–7). (G) Kpn CFU in the feces of vehicle and bismuth-treated mice at 24 hours post-Kpn infection. Data are representative of 3 experiments. The dotted line indicates the detection limit. (H) Model: Infectious exposures increase the availability of taurine, which the gut microbiota converts to sulfide, serving to inhibit pathogen respiration and regulate the microbiota. In box and whisker plots, lines connect the medians of paired cages. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by Mann-Whitney test (B, F, G) or PERMANOVA (D). See also Table S5.