Volume 21, Issue 2, Pages 195-207 (February 2017) The Bactericidal Lectin RegIIIβ Prolongs Gut Colonization and Enteropathy in the Streptomycin Mouse Model for Salmonella Diarrhea  Tsuyoshi Miki, Ryosuke Goto, Mayuka Fujimoto, Nobuhiko Okada, Wolf-Dietrich Hardt  Cell Host & Microbe  Volume 21, Issue 2, Pages 195-207 (February 2017) DOI: 10.1016/j.chom.2016.12.008 Copyright © 2017 Elsevier Inc. Terms and Conditions

Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Impaired Colonization of S. Tmatt in RegIIIβ−/− Mice (A–F) Streptomycin-treated C57BL/6 mice (WT) or RegIIIβ−/− mice (n = 10) were infected with S. Tmatt (ΔspiB::kan) by gavage. At 2, 4, or 10 days postinfection (dpi), mice were sacrificed and S. Tmatt loads in the feces (A), cecal content (B), mesenteric lymph node (C), and spleen (D) were determined. (E) Cecal pathology was scored in H&E-stained cecal tissue section. (F) Lipocalin-2 was monitored by ELISA. Bar indicates median. ns, not significant (p ≥ 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; Mann-Whitney U test. See also Figure S2. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 Colonic Microbiota Analysis in C57BL/6 Wild-Type Mice and RegIIIβ−/− Mice (A) Colonic microbiota of mice before streptomycin treatment (baseline, n = 3) or after streptomycin treatment (Sm-treated, n = 3) or infected with S. Tmatt (2 dpi and 10 dpi, n = 5) was analyzed by 16S rRNA pyrosequencing. Relative abundances of colonic microbial communities in the mice are denoted. (B) Principal coordinate analysis (PCoA) of 32 samples from 16S rRNA pyrosequencing. (C and D) Quantification of colonic microbiota in mice infected with S. Tmatt at 2 dpi (C) and 10 dpi (D) by qPCR of 16S rRNA gene copy number. n = 5. Eubac, Eubacteria; Bact, Bacteroides spp.; MIB, mouse intestinal Bacteroides; Erec, Eubacterium rectale/Clostridium coccoides; Lact, Lactobacillus sp.; SFB, segmented filamentous bacteria. All bars represent mean ± SD. ns, not significant (p ≥ 0.05); ∗p< 0.05; ∗∗p < 0.01; Mann-Whitney U test. (E) The microbiota from RegIIIβ−/− mice was exposed to RegIIIβ or polymyxin B for 30 min at 37°C. Killing effect was evaluated by comparing the sample with buffer control, meaning that less than 1 indicates bacteria were killed. n = 3. The bar shows mean ± SD. ns, not significant (p ≥ 0.05); ∗p < 0.05; one sample t test. (F) Recombinant RegIIIβ (200 μg) or buffer control was infused into isolated colonic segment from RegIIIβ−/− mice. After 30 minutes, luminal contents from each segment were harvested and then plated on the Bacteroides agar plate. n = 3. The bars represent mean ± SD. ns, not significant (p ≥ 0.05); ∗∗∗p < 0.001; Student’s t test. See also Figure S3. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 Administration of Bacteroides Reduces Levels of S. Tmatt Colonization in the Inflamed Gut (A) Experimental strategy. sm, streptomycin. (B) S. Tmatt loads in the feces were determined. Bar indicates median. n = at least 11. (C) Cecal pathology was scored in H&E-stained cecal tissue section. Bar indicates median. (D) Lipocalin-2 ELISA. Bar indicates median. (E) Quantification of colonic Eubacteria and Bacteroides spp. in mice infected with S. Tmatt and supplemented with Bacteroides spp. by qPCR of 16S rRNA gene copy number. The bar shows mean ± SD. ns, not significant (p ≥ 0.05); ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; Mann-Whitney U test. See also Figures S4 and S5. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Metabolomics of the Colonic Content in C57BL/6 Wild-Type Mice and RegIIIβ−/− Mice (A–D) Streptomycin-treated WT or RegIIIβ−/− mice (n = 6) were infected with S. Tmatt (ΔspiB::kan) by gavage. At 8 dpi, mice were sacrificed and colonic contents were subjected to metabolomic analysis. (A) A principal component analysis (PCA) plot of the processed data from the colonic metabolome. (B) A heatmap of the relative abundance of all identified metabolites, showing expression pattern of them. Red color indicates upregulated (high concentrations), whereas green color indicates downregulated (low concentrations). (C) A plot showing over-represented pathways resulted from analysis using Metaboanalyst in RegIIIβ−/− mice. Y axis indicates the log (p value; Welch’s t test), and x axis shows the pathway impact value calculated from pathway topology analysis. Size of the plot represents the number of metabolites included in the identified pathway. (D) Concentrations of vitamin B6 in colonic content of WT and RegIIIβ−/− mice were measured at 8 days post infection with S. Tm. n = 7. Bar indicates median. ns, not significant (p ≥ 0.05); ∗∗∗p < 0.001; Mann-Whitney U test. See also Figure S6. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Commensal Bacteria Produce Vitamin B6 in the Gut (A) Concentrations of vitamin B6 in colonic content of WT mice treated with antibiotics (Abs) mixture (AVNM; ampicillin, vancomycin, neomycin, and metronidazole) (+) or without (−). n = 7. (B) Experimental strategy. B. acidifaciens was inoculated by gavage into mice treated with AVNM. (C) Concentrations of vitamin B6 in colonic content of WT mice treated with AVNM and subsequently inoculated with B. acidifaciens (+) or not (−). n = 7. Bar indicates median. ∗p < 0.01; ∗∗∗p < 0.001; Mann-Whitney U test. See also Figure S6. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Administration of Vitamin B6 Promotes the S. Tm Clearance and Recovery from the Gut Inflammation (A) Experimental strategy. sm, streptomycin. (B) S. Tmatt loads in the feces were determined. Bar indicates median. n = 10. (C) Concentrations of vitamin B6 in colonic content. Bar indicates median. (D) Cecal pathology was scored in H&E-stained cecal tissue section. Bar indicates median. ns, not significant (p ≥ 0.05); ∗p < 0.05; ∗∗p < 0.01; Mann-Whitney U test. See also Figure S6. Cell Host & Microbe 2017 21, 195-207DOI: (10.1016/j.chom.2016.12.008) Copyright © 2017 Elsevier Inc. Terms and Conditions