D-Tryptophan suppresses enteric pathogen and pathobionts and prevents colitis by modulating microbial tryptophan metabolism

Abstract

_D-Amino acids (_D-AAs) have various functions in mammals and microbes. _D-AAs are produced by gut microbiota and can act as potent bactericidal molecules. Thus, _D-AAs regulate the ecological niche of the intestine; however, the actual impacts of _D-AAs in the gut remain unknown. In this study, we show that _D-Tryptophan (_D-Trp) inhibits the growth of enteric pathogen and colitogenic pathobionts. The growth of Citrobacter rodentium in vitro is strongly inhibited by _D-Trp treatment. Moreover, _D-Trp protects mice from lethal C. rodentium infection via reduction of the pathogen. Additionally, _D-Trp prevents the development of experimental colitis by the depletion of specific microbes in the intestine. _D-Trp increases the intracellular level of indole acrylic acid (IA), a key molecule that determines the susceptibility of enteric microbes to _D-Trp. Treatment with IA improves the survival of mice infected with C. rodentium. Hence, _D-Trp could act as a gut environmental modulator that regulates intestinal homeostasis.

Keywords: cell biology; cellular physiology; microbiology; microbiome.

Graphical abstract

Figure 1

Effects of _D-Tryptophan on the growth of enteric pathogen (A and B) Optical density (OD) measured at 600 nm after culturing Citrobacter rodentium in LB broth supplemented with or without 50 mM of each _D-amino acid (A) (n = 4 samples per group) or with or without various concentrations of _D-Methionine (_D-Met) or _D-Tryptophan (_D-Trp) (B) (n = 4 samples per group) for 24 h. (C-H) Mice were fed chow diets supplemented with _D-Methionine (_D-Met) or _D-Tryptophan (_D-Trp) at concentrations of 0% (Control; Cont), 0.2%, 1%, or 5% and then infected orally with 2 × 10⁹ colony-forming units (CFU) of C. rodentium (n = 5-6 mice per group). The mice were fed with their respective diets beginning 10 days before infection and continuing through the end of the experiment. (C and F) Survival rates for 21 days after infection with C. rodentium. (D, E, G, H) _D-Trp levels in (D and G) feces and (E and H) plasma derived from mice fed supplemented chow diets for 10 days before the infection. (I-M) Mice were fed with the respective diets (Cont, unsupplemented diet; _L-Trp, 5% _L-Tryptophan supplemented diet; _D-Trp, 5% _D-Tryptophan supplemented diet) and then infected orally with 2 × 10⁹ CFU of C. rodentium (n = 7-8 mice per group). The mice were fed their respective diets beginning 2 weeks before infection and continuing through the end of the experiment. (I) Fecal C. rodentium load on days 3, 6, and 9 post-infection. (J) Colon length and (K) colon weight per cm on day 10 post-infection. (L) Fecal Lipocalin-2 (LCN2) concentration on day 9 post-infection. (M) C. rodentium load in the liver and the spleen at day 10 post-infection. Each dot represents one sample or mouse. Horizontal bars indicate mean values. Statistical significance was assessed using the Log-rank test in panels C and F, one-way ANOVA with Dunnett’s multiple comparison test in panels D, E, G, and H, and Tukey’s multiple comparison test in panels I, J, K, L, and M. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. All the experiments were conducted at least three independent times.

Figure 2

Protective effect of _D-Tryptophan against experimental colitis (A-H) Acute colitis was induced in SPF wild-type mice via treatment with 2% dextran sulfate sodium (DSS) for 5 days, followed by providing plain water for 9 days. The mice were treated orally every day with 1 mL of 0.5% carboxymethyl cellulose (CMC) or 5% _D-Tryptophan suspended in 0.5% CMC beginning 1 week before DSS treatment and continuing through the end of the experiment (n = 12 mice per group). The mice were euthanized, and the colon was harvested 9 days following DSS treatment (B-H). (A) Changes in body weight over 14 days. (B) Colon weight per cm. (C) Representative hematoxylin and eosin-stained colonic sections (scale bar: 100 μm). (D) Histological score. (E-H) Representative flow cytometry plots and numbers of (E) CD45⁺CD11b⁺Ly6G⁺ cells (Neutrophils), (F) CD45⁺CD11b⁺F4/80⁺Siglec-F^-Ly6C⁺ cells (Inflammatory monocytes), (G) CD45⁺CD4⁺TCRβ⁺ cells (T helper cells), and (H) CD45⁺B220⁺TCRβ⁻ cells (B cells) in the colonic lamina propria. Each dot represents one mouse or the mean ± SEM. Horizontal bars indicate mean values. Statistical significance was assessed using unpaired Student’s t test in panels E, F, G, and H, Welch’s t-test in panels B and D, and two-way ANOVA with Šidák corrections for multiple comparisons in panel A. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; N.S., not significant. All the experiments were conducted at least three independent times.

Figure 3

Effect of _D-Tryptophan on the composition of gut microbiota (A-D) Specific pathogen-free (SPF) wild-type mice were fed with respective diets (Cont, unsupplemented diet; _D-Trp, 5% _D-Tryptophan supplemented diet) for 2 weeks, after which fecal samples were obtained to analyze the composition of the gut microbiota (A-C, n = 6; D, n = 12 mice per group). (A) Principal coordinate analysis (PCA) plot generated using weighted UniFrac metric. (B) Relative abundance of operational taxonomic units (OTUs) in fecal samples. Various colors correspond to each indicated bacterial family. (C) Histogram of the linear discriminant analysis (LDA) scores computed for differentially abundant bacterial taxa in fecal samples. (D) The total number of bacteria per milligram of the fecal samples. (E) Optical density (OD) of individual bacterial species cultured in a medium supplemented with or without 20 mM_D-Tryptophan (_D-Trp) measured over time at 600 nm (n = 8 samples per group). (F) OD of Citrobacter rodentium cultured in a medium supplemented with or without 20 mM _D-Tryptophan (_D-Trp) or _L-Tryptophan (_L-Trp) measured over time at 600 nm (n = 8 samples per group). Each dot represents one sample or mouse, or the mean ± SD. Horizontal bars indicate mean values. Statistical significance was assessed using unpaired Student’s t-test in panel D, and two-way ANOVA with Šidák corrections for multiple comparisons in panels E and F. The asterisks in E and F indicate significant differences after 24 h. ∗∗∗p < 0.001; N.S., not significant. All the experiments were conducted at least three independent times.

Figure 4

Production of indoleacrylic acid (IA) by _D-Tryptophan in bacteria and its protective effect against enteric pathogens (A-D) Metabolomic analysis of intracellular contents from the Citrobacter rodentium cultured in the presence or absence of 50 mM _L-Tryptophan (_L-Trp) or _D-Tryptophan (_D-Trp) for 24 h (n = 4 samples per group). (A) Pathway of tryptophan metabolism. Colored dots represent significantly increased metabolite levels compared to None. Red: _D-Trp, Blue: _L-Trp. (B) Volcano plot showing the significance and magnitude of differences in the relative abundance of intracellular metabolites grown in the presence of 50 mM _L-Trp or _D-Trp. (C) The relative abundance of intracellular tryptophan and tryptophan derivatives. (D) The relative abundance of intracellular indole-3-acrylic acid (IA) in Escherichia coli, Lactobacillus johnsonii, and Limosilactobacillus reuteri grown in the presence or absence of 50 mM _L-Trp or _D-Trp. IS; internal standard. (E) Optical density (OD) of E. coli, L. reuteri, L. johnsonii, and C. rodentium grown in a medium supplemented with or without 2 mM IA measured over time at 600 nm (n = 8 samples per group). (F-H) Mice were fed with respective diets (Cont, unsupplemented diet; IA, 2.5% IA supplemented diet) and then infected orally with 2 × 10⁹ colony-forming units (CFU) of C. rodentium (n = 10 mice per group). (F) Changes in body weight, (G) survival rate, and (H) fecal C. rodentium load on days 3, 6, and 9 post-infection. Each dot represents one sample or mouse, or the mean ± SD (E) or SEM (F). Horizontal bars indicate mean values. Statistical significance was assessed using Tukey’s multiple comparison test in panels C and D, two-way ANOVA with Šidák corrections for multiple comparisons in panels E and F, Log-rank test in panels G, and unpaired Student’s t-test in panel H, Asterisks in (E) indicate significant differences observed after 24 h. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; N.S., not significant. All the experiments were conducted at least three independent times.