Host-derived nitrate boosts growth of E. coli in the inflamed gut

Abstract

Changes in the microbial community structure are observed in individuals with intestinal inflammatory disorders. These changes are often characterized by a depletion of obligate anaerobic bacteria, whereas the relative abundance of facultative anaerobic Enterobacteriaceae increases. The mechanisms by which the host response shapes the microbial community structure, however, remain unknown. We show that nitrate generated as a by-product of the inflammatory response conferred a growth advantage to the commensal bacterium Escherichia coli in the large intestine of mice. Mice deficient in inducible nitric oxide synthase did not support the growth of E. coli by nitrate respiration, suggesting that the nitrate generated during inflammation was host-derived. Thus, the inflammatory host response selectively enhances the growth of commensal Enterobacteriaceae by generating electron acceptors for anaerobic respiration.

Figure 1. Anaerobic respiration enhances luminal growth of E. coli during DSS-induced colitis

(A) Competitive index (CI) of the EcN wild type (WT) and the moaA mutant after anaerobic growth in mucin broth supplemented with 40 mM of the indicated electron acceptors (N = 3). (B) Mock-treated mice (Mock), DSS-treated mice (DSS) or mice treated with DSS and AG (DSS+AG) were inoculated with the indicated mixtures of E. coli strains and the CI in colon contents determined 5 days after inoculation. A plasmid (pMOA1) carrying the cloned moaA gene was used to complement the moaA mutant (moaA). N is given in Fig. S3C. (C) Concentration of nitrate (NO₃^-) determined in the cecal mucus layer of mock-treated mice (N = 4), DSS-treated mice (DSS, N = 3) or mice treated with DSS and AG (DSS+AG, N = 4). Bars represent geometric means ± standard error. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant (Student’s t-test).

Figure 2. Wild-type E. coli outcompetes a nitrate respiration-deficient mutant during colitis

Mock-treated (Mock), DSS-treated (DSS) or DSS+AG-treated (DSS+AG) wild-type mice, Nos2-deficient mice (Nos2) or mice harboring T cells deficient for the production of IL-10 (Cd4 Il10 mice) were inoculated with the indicated mixtures of E. coli strains. WT, E. coli wild type; narG narZ napA, E. coli nitrate respiration-deficient mutant. The narG narZ napA mutant was complemented by introducing a functional chromosomal napA allele and a plasmid (pNARG1) carrying the cloned narG gene (narG narZ napA[rest] [pNARG1]). Pathological changes in the colon (A and C) and the competitive index (CI) recovered from colon contents (B and D) were determined 5 days after inoculation. (A and C) Combined histopathology score in the colon. Each dot represents data from an individual animal. Experiments were performed with EcN (A-B) or E. coli LF82 (C-D). (B and D) Bars represent geometric means ± standard error. **, P < 0.01; ***, P < 0.001 (Student’s t-test). N is given in panels A and C.

Figure 3. Nitrate respiration enhances luminal growth of EcN during inflammation

(A-D) Bovine ligated ileal loops treated with thapsigargin or mock-treated (vehicle) were inoculated with a mixture of EcN (WT) and a nitrate respiration-deficient mutant (narG napA narZ). Samples were collected 8 hours after inoculation. (A) Representative H&E stained ileal sections. Scale bar, 200 μm. (B) Combined histopathology score in the ileum. Each dot represents data from an individual animal. (C) Fluid accumulation in ligated ileal loops. (D) Competitive indices (CI) recovered from the luminal fluid (open bars) or mucus scrapings (closed bars). (E-H) DSS-treated mice were inoculated either with EcN (WT) or with the narG napA narZ mutant. Inflammation in the colon (E, F and G) and bacterial numbers recovered from colon contents (E) were determined 5 days after inouclation. (E) Representative H&E stained colonic sections. Scale bar, 100 μm. (F) Combined histopathology score in the colon. (G) Expression of Nos2, Kc and Tnfa in colonic RNA samples using qRT-PCR (fold-increases over mock-treated, mock-treated mice). (H) Bacterial numbers (CFU) recovered from colon contents. (C, D, G, H) Bars represent geometric means ± standard error. *, P < 0.05; **, P < 0.01; ns, not statistically significant (Student’s t-test). N is given in panels B and F.