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Science 339 (6120): 708-711

Copyright © 2013 by the American Association for the Advancement of Science

Host-Derived Nitrate Boosts Growth of E. coli in the Inflamed Gut

Sebastian E. Winter1, Maria G. Winter1, Mariana N. Xavier1, Parameth Thiennimitr1,2, Victor Poon1, A. Marijke Keestra1, Richard C. Laughlin3, Gabriel Gomez3, Jing Wu3, Sara D. Lawhon3, Ina E. Popova4, Sanjai J. Parikh4, L. Garry Adams3, Renée M. Tsolis1, Valley J. Stewart5, and Andreas J. Bäumler1,*

1 Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, One Shields Avenue, Davis, CA, USA.
2 Department of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
3 Department of Veterinary Pathobiology, College of Veterinary Medicine and Biomedical Sciences, Texas A&M University, College Station, TX, USA.
4 Department of Land, Air and Water Resources, University of California, Davis, One Shields Avenue, Davis, CA, USA.
5 Department of Microbiology, College of Biological Sciences, University of California, Davis, One Shields Avenue, Davis, CA, USA.


Figure 1
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Fig. 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 was determined 5 days after inoculation. A plasmid (pMOA1) carrying the cloned moaA gene was used to complement the moaA mutant (moaA). The number of animals (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+AG (n = 4). Bars represent geometric means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not statistically significant (Student's t test).

 

Figure 2
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Fig. 2. Wild-type E. coli (WT) outcompetes a nitrate respiration–deficient mutant during colitis. Mock-treated (Mock), DSS-treated (DSS), or DSS+AG-treated 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. 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 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) and (B)] or E. coli LF82 [(C) and (D)]. (B) and (D) Bars represent geometric means ± SE. **P < 0.01, ***P < 0.001 (Student's t test). n is given in (A) and (C).

 

Figure 3
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Fig. 3. Nitrate respiration enhances luminal growth of EcN during inflammation. (A to 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 hematoxylin and eosin (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) CIs recovered from the luminal fluid (open bars) or mucus scrapings (solid bars). (E to H) DSS-treated mice were inoculated either with EcN (WT) or with the narG napA narZ mutant. Inflammation in the colon (E to G) and bacterial numbers recovered from colon contents (E) were determined 5 days after inoculation. (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 analyzed by quantitative real-time polymerase chain reaction (fold increases over mock-treated mice). (H) Bacterial numbers [colony-forming units (CFU)] recovered from colon contents. In (C), (D), (G), and (H), bars represent geometric means ± SE. *P < 0.05, **P < 0.01; ns, not statistically significant (Student's t test). n is given in (B) and (F).

 


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