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Microcins mediate competition among Enterobacteriaceae in the inflamed gut

Abstract

The Enterobacteriaceae are a family of Gram-negative bacteria that include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine’s microbiota1, some of these organisms can bloom in the inflamed gut2,3,4,5; expansion of enterobacteria is a hallmark of microbial imbalance known as dysbiosis6. Microcins are small secreted proteins that possess antimicrobial activity in vitro7,8, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit the expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limits the growth of competitors in the inflamed intestine, including commensal E. coli, adherent–invasive E. coli and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild-type, microcin-producing EcN to mice previously infected with S. enterica substantially reduced intestinal colonization by the pathogen. Our work provides the first evidence that microcins mediate inter- and intraspecies competition among the Enterobacteriaceae in the inflamed gut. Moreover, we show that microcins can act as narrow-spectrum therapeutics to inhibit enteric pathogens and reduce enterobacterial blooms.

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Figure 1: Microcins do not promote bacterial competition in the absence of intestinal inflammation.
Figure 2: Microcins enable E. coli Nissle to limit the expansion of a commensal E. coli in the inflamed gut.
Figure 3: E. coli Nissle utilizes microcins to outcompete an enteric pathogen when the strains are co-administered.
Figure 4: Microcins therapeutically reduce colonization of an enteric pathogen in the inflamed gut.

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Acknowledgements

We would like to acknowledge E. Nolan for critically reading the manuscript, J. Behnsen for setting up the Raffatellu laboratory germ-free mouse facility, and M. Valeri, A. Perez-Lopez, V. Diaz-Ochoa and E. Hoover for contributing to the maintenance of the germ-free facility. We would also like to thank W. Zhu and S. Winter for providing us with the protocol for cleaning up DNA stool samples from DSS, and M. Rolston at the UC Davis Host-Microbe Systems Biology Core for processing samples for Illumina MiSeq Analysis. We would like to acknowledge the students of the 2015 Summer course ‘Frontiers in Host–Microbe Interactions’, Marine Biology Laboratory, who helped with the generation of one dataset for the manuscript. Work in the laboratory of M.R. is supported by Public Health Service Grants AI083663, AI126277, AI101784, AI114625, AI105374, and DK058057. M.R. holds an Investigator in the Pathogenesis of Infectious Disease Award from the Burroughs Wellcome Fund.

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Authors and Affiliations

Authors

Contributions

M.S.-C. performed most bacterial growth assays and all animal experiments, and analysed the results. S.-P.N. analysed the microbiota data. R.A.E. scored histological sections. H.L., D.H., C.T.V., and A.A.T. assisted with mutant construction, bacterial growth assays, and animal experiments. M.S.-C. and M.R. were responsible for the overall study design. M.S.-C., S.-P.N. and M.R. wrote the manuscript.

Corresponding author

Correspondence to Manuela Raffatellu.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Wild-type EcN and microcin mutant (EcN mchDEF) gut colonization in competition with commensal E. coli in the absence of intestinal inflammation.

a, Illustration of the microcin gene cluster in EcN. b–g, Intragastric inoculation of SPF mice with cEc alone or in competition with wild-type EcN or EcN mchDEF at a 1:1 ratio. b, Ratio (EcN over cEc) in faecal content at days 1–5 after inoculation (n = 5 per group). c, d, CFU mg−1 of (c) cEc or (d) wild-type EcN or EcN mchDEF in faecal content at days 1–5 after inoculation, when cEc was administered alone or in competition as indicated. e, f, Caecal histopathology scores (e) or gene expression (f) (data are expressed as fold change over mock-treated mice) at day 5 after inoculation for panels b–d (n = 5 per group). g, Haematoxylin and eosin-stained sections from representative animals at day 5 after inoculation. Scale bar, 100 μm. Each individual symbol represents one mouse (ce). Bars represent the geometric mean ± s.e.m. (b, f), geometric mean (c, d), or mean (e). n.s., not significant.

Extended Data Figure 2 Colonization and histopathology of wild-type EcN and microcin mutants (mchDEF and mcmA mchB) in competition with commensal E. coli, in mice with DSS-mediated colitis.

a–i, Experimental design as in Fig. 2a with SPF mice. a, Ratio (EcN over cEc) in caecal content at day 5 after intragastric inoculation (n = 6 per group). b, c, CFU mg−1 of (b) cEc or (c) wild-type EcN or EcN mchDEF in faecal content at day 5 after inoculation when in competition as indicated (n = 6 per group). d, e, Caecal (d) gene expression (n = 5 per group; data are presented as fold change over mock-treated mice) or (e) histopathology scores at day 5 after inoculation from mice shown in panels b, c and Fig. 2b–d (DSS only, n = 3; all others, n = 5). Scale bar, 100 μm. f, Detailed histopathology scoring of mice in e. g, Haematoxylin and eosin-stained sections from representative animals at day 5 following inoculation. h, i, CFU mg−1 of (h) cEc or (i) wild-type EcN or EcN mcmA mchB in sample content at days 1–5 following intragastric inoculation when cEc was administered alone or in competition as indicated (n = 5 per group). Each individual symbol represents one mouse (b, c, e, h, i). Bars represent the geometric mean ± s.e.m. (a, d), geometric mean (b, c, h, i) or mean (e). *P < 0.05, **P < 0.01; n.s., not significant.

Extended Data Figure 3 In vitro growth curves of EcN and microcin mutants when grown alone or in competition with EcN mutants or commensal E. coli.

a–i, Strains were grown overnight in nutrient broth supplemented with 0.2 mM 2,2′-dipyridyl. Growth assays were performed in iron-limiting conditions (DMEM/F12 supplemented with 10% FBS) or in medium supplemented with 1 μM iron citrate. Time points at 0, 5, 8 and 11 h after inoculation were collected. a, b, cEc CFU ml−1 when grown alone or in competition with wild-type or mutant EcN in iron-limiting conditions (a) or in medium supplemented with 1 μM iron citrate (b). c, d, CFU ml−1 of wild-type or mutant EcN when grown alone or in competition with cEc in iron-limiting conditions (c) or in medium supplemented with 1 μM iron citrate (d). eg, Under iron-limiting conditions, CFU ml−1 of wild-type or mutant EcN when grown alone (e), of wild-type EcN (f) or EcN mchDEF or EcN mcmA mchB (g) when grown competitively as indicated. h, i, CFU ml−1 of the indicated EcN mutants (immunity gene(s) and/or mchDEF) grown in competition with wild-type EcN in (h) iron-limiting conditions or in (i) medium supplemented with 1 μM iron citrate. Symbols represent the geometric mean of three independent experiments ± s.e.m. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 4 Gut colonization of wild-type EcN and microcin mutants in the DSS–colitis model.

a, Experimental design for bd with SPF mice. b, CFU mg−1 of wild-type EcN or EcN mchDEF in faecal content at days 1–5 following intragastric inoculation (n = 5 per group). c, d, Caecal gene expression (c; n = 5 per group; data are expressed as fold change over mock-treated mice) or haematoxylin and eosin-stained sections (d; representative) from panel b mice at day 5 following inoculation. Scale bar, 100 μm. e, Triple co-administration design for fh with SPF mice. f, g, CFU mg−1 of cEc (f) or indicated EcN strain (g) in faecal content at days 1–5 following intragastric inoculation with cEc, wild-type EcN and the indicated EcN mutant (n = 5 per group). h, Competitive index (CI; EcN wild-type over mutant) for EcN data presented in panel g (n = 5 per group). b, f, g, Each individual symbol represents one mouse. Bars represent the geometric mean (b, f, g) or the geometric mean ± s.e.m (c, h). *P < 0.05, **P < 0.01; n.s., not significant.

Extended Data Figure 5 Impact of microcins on the microbiota and impact of a high iron diet on microcin-mediated competition.

a, High-iron diet design for be with SPF mice (n = 5 per group) b, Ratio (EcN over cEc) in faecal content at days 1–5 after intragastric inoculation. c, Caecal gene expression at day 5 following inoculation (n = 3–4 per group). d, e, CFU mg−1 of cEc (d) or wild-type EcN or EcN mchDEF (e) in indicated samples at days 1–5 after inoculation when cEc was administered alone or in competition as indicated (n = 5 per group). f, g, See Fig. 2a for experimental design with SPF mice. 16S ribosomal rRNA gene sequence analysis (V4 region) of faecal DNA obtained from mice prior to DSS administration (day −4), after DSS administration (Day 0), and day 5 after intragastric inoculation of cEc with either wild-type EcN or EcN mchDEF at a 1:1 ratio. f, Eubacterial alpha diversity (inverse Simpson index); Shannon index yielded similar results. g, Principal coordinates (PCo) analysis plot (PCo1 versus PCo2) of eubacterial beta diversity (weighted UniFrac); symbols as in panel f. Each individual symbol represents one mouse (d, e, f, g). Bars represent the geometric mean ± s.e.m. (b, c), geometric mean (d, e) or mean ± s.d. (f). n.s., not significant. Black symbols represent comparisons within same day; red symbols represent comparisons between days.

Extended Data Figure 6 In vitro activity of EcN microcins against S. Typhimurium and AIEC.

ai, Strains were grown overnight in nutrient broth supplemented with 0.αM 2,2′-dipyridyl. Growth assays were performed in iron-limiting conditions (DMEM/F12 supplemented with 10% FBS) or in medium supplemented with 1 μM iron citrate. Time points at 0, 5, 8 and 11 h after inoculation were collected. a, b, STm CFU ml−1 when grown alone or in competition with wild-type or mutant EcN in iron-limiting conditions (a) or in medium supplemented with 1 μM iron citrate (b). c, STm CFU ml−1 in iron-limiting conditions when in competition with wild-type EcN or an EcN mchDEF strain harbouring either pWSK29::mchDEF or the empty-vector control. d, e, CFU ml−1 of wild-type or mutant EcN when grown alone or in competition with STm in (d) iron-limiting conditions or in (e) medium supplemented with 1 μM iron citrate. f, g, AIEC CFU ml−1 when grown alone or in competition with wild-type or mutant EcN in iron-limiting conditions (f) or in medium supplemented with 1 μM iron citrate (g). h, i, CFU ml−1 of wild-type or mutant EcN when grown alone or in competition with AIEC in iron-limiting conditions (h) or in medium supplemented with 1 μM iron citrate (i). Symbols represent the geometric mean (three independent experiments) ± s.e.m. *P < 0.05, **P <0.01, ***P < 0.001, ****P < 0.0001.

Extended Data Figure 7 Co-administration of S. Typhimurium with wild-type EcN or EcN mchDEF.

a–e, See Fig. 3d for co-administration design with SPF mice. a, b, Intragastric inoculation with wild-type STm. Caecal (a) gene expression (STm only, n = 7; all others, n = 9); data are expressed as fold change over mock-treated mice or (b) histopathology (STm only and STm + wild-type EcN, n = 5; STm + EcN mchDEF, n = 4) from mice shown in Fig. 3f, g at Day 7 following infection. c-e, Intragastric inoculation with STm invA spiB. c, d, CFU mg−1 of (c) STm invA spiB (n = 4) or (d) wild-type EcN or EcN mchDEF in faecal content at designated time points after infection when STm invA spiB was administered alone or in competition as indicated (n = 5 per group). e, Ratio of wild-type EcN or EcN mchDEF over STm invA spiB in faecal content at designated time points after infection (n = 5 per group). Each individual symbol represents one mouse (bd). Bars represent the geometric mean ± s.e.m. (a, e), mean (b), or geometric mean (c, d). n.s., not significant.

Extended Data Figure 8 Gut colonization of AIEC in DSS-treated mice when competing with wild-type EcN or EcN mchDEF.

a, Experimental design for bf with SPF mice. b, Ratio of wild-type EcN or EcN mchDEF over AIEC in faecal content at days 1–5 following intragastric inoculation (n = 9 per group). c, d, CFU mg−1 of (c) AIEC or (d) wild-type EcN or EcN mchDEF in faecal content at days 1–5 following inoculation when AIEC was administered alone or in competition as indicated (n = 9 per group). e, Caecal histopathology scores at day 5 after inoculation for panels c, d (n = 5 per group). f, Detailed histopathology scoring of panel e mice. Each individual symbol represents one mouse (c–e). Bars represent the geometric mean ± s.e.m. (b), geometric mean (c, d) or mean (e). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; n.s., not significant.

Extended Data Figure 9 Therapeutic administration of wild-type EcN, EcN mchDEF or mock during S. Typhimurium infection.

aj, See Fig. 4a for therapeutic design with SPF mice. af, Intragastric inoculation with wild-type STm. a, Ratio of wild-type EcN or EcN mchDEF over STm in faecal content on days 4–7 after infection with STm (n = 8 per group). b, c, e, STm CFU mg−1 at designated time points after infection in faecal content of mice therapeutically treated with mock (b; n = 7), wild-type EcN (c; n = 8) or EcN mchDEF (e; n = 8). d, f, CFU mg−1 of wild-type EcN (d) or EcN mchDEF (f) in faecal content at designated time points after STm infection. g, h, Intragastric inoculation with STm pMcmI (n = 10 per group). CFU mg−1 of STm pMcmI (g) or wild-type EcN (h) in faecal content at designated time points following STm pMcmI infection. Grey box represents average STm CFU mg−1 in mock-treated mice (panel b). i, Caecal histopathology scores for panels bf (STm alone, n = 6; all others, n = 7). j, Detailed histopathology scoring of mice in i. Each individual symbol represents one mouse (bh). Bars represent the geometric mean ± s.e.m. (a) or mean (i). *P < 0.05, **P < 0.01, ***P < 0.001; n.s., not significant.

Extended Data Figure 10 In vitro growth curves of microcin M (mcmA) and microcin H47 (mchB) EcN mutants in competition with commensal E. coli, S. Typhimurium, or AIEC.

ah, Strains were grown overnight in Nutrient Broth supplemented with 0.2 mM 2,2′-dipyridyl. Growth assays were performed in iron-limiting conditions (DMEM/F12 supplemented with 10% FBS) and time points at 0, 5, 8 and 11 h following inoculation were collected. ac, CFU ml−1 of commensal E. coli (a), STm (b) or AIEC (c) when grown alone or in competition with the indicated EcN strain. d, CFU ml−1 of complemented and uncomplemented EcN microcin immunity gene mutants when in competition with EcN wild-type. e, CFU ml−1 of wild-type STm or STm harbouring pMchI or pMcmI when in competition with EcN wild-type. fh, CFU ml−1 of STm fepA (f), STm iroN (g) and STm fepA iroN (h) in competition with either wild-type EcN or EcN mcmA mchB. Symbols represent the geometric mean for three independent experiments ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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Sassone-Corsi, M., Nuccio, SP., Liu, H. et al. Microcins mediate competition among Enterobacteriaceae in the inflamed gut. Nature 540, 280–283 (2016). https://doi.org/10.1038/nature20557

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