Figure 4. L-serine catabolism regulates intraspecific competition of E. coli in the inflamed gut.

(a) E. coli LF82 WT, LF82 ΔtdcΔsda (ΔTS) mutant, C. rodentium DBS100 WT, and C. rodentium ΔtdcΔsda (ΔTS) mutant strains (1 x 10³ CFU) were inoculated into sterilized cecal content isolated from healthy and colitic (Salmonella-induced colitis) and cultured for 8 hrs at 37°C with 20% O₂ and 5% CO₂. After 8hrs, culture media were plated onto LB agar and bacterial CFUs were measured. Fold increase of bacterial strains in the inflammation (+) luminal content to the inflammation (−) control condition is shown. Dots indicate individual replicates with mean ± s.e.m (N=4, biologically independent samples). N.S.; not significant, *; P < 0.05 by Mann–Whitney U test (two-sided). (b) Each indicated E. coli strain (MG1655, HS, dn15.6244.1 (dn), Nissle 1917 (EcN), SK460, and CUMT8) was cultured in cecal content isolated from control and colitic mice as described in (a). Fold increase of bacterial strains in the inflammation (+) luminal content to the inflammation (−) control condition is shown. Dots indicate individual replicates with mean ± s.e.m (N=4-7, biologically independent samples). N.S.; not significant, ***; P < 0.001 by Mann–Whitney U test (two-sided). (c) Competitive growth assay of LF82 (Amp^R) (LF) and MG1655 (Str^R) (MG) in a minimal medium supplemented with L-asparagine (Asn) or L-serine (Ser). Bacterial CFUs were quantified by culture on LB plates supplemented with ampicillin or streptomycin. Dots indicate individual replicates with mean ± s.e.m (N=10, biologically independent samples). N.S.; not significant, **; P < 0.01, ****; P < 0.0001 by 1-Way ANOVA followed by Bonferroni post-hoc test. (d) Germ-free (GF) C57BL/6 mice were mono-colonized either with LF82 or MG1655, or co-colonized both strains (1 x 10⁹/mouse each). On day 10, mice were treated with 1.5% DSS for 5 days. Bacterial CFUs in each mouse was quantified by culturing fecal matter at indicated time points. Data are given as geometric mean ± s.d. (N=3-4, biologically independent animals). Results are representative of 3 independent experiments. N.S.; not significant, *; P < 0.05 by Mann–Whitney U test (day 10 (non-inflamed) vs day 15 (inflamed)). (e) GF C57BL/6 mice were co-colonized with LF82 WT or LF82 ΔtdcΔsda double mutant (ΔTS) and a commensal E. coli strain (MG1655 or HS) (1 x 10⁹ CFU each/mouse) for 10 days. On day 10, colitis was induced by 1.5% DSS (for 5 days). Colonization of LF82 WT (LF), LF82ΔTS (ΔTS), and MG1655 (MG) (f) or HS (h) was assessed at indicated time points. Data are presented as a geometric mean ± s.d. The competitive index of LF82 WT/MG1655 (WT) and LF82ΔTS/MG1655 (ΔTS) (g) and LF82 WT/HS (WT) and LF82ΔTS/HS (ΔTS) (i) in the non-inflamed (Non-inf) gut (day 10) and the inflamed gut (day 15) are shown. Dots indicate individual mice with as geometric mean ± s.d (N=5-8, biologically independent animals). (g, i) N.S.; not significant, ***; P < 0.001, ****; P < 0.0001 by 1-Way ANOVA followed by Bonferroni post-hoc test.