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. 2017 Feb 8;21(2):208-219.
doi: 10.1016/j.chom.2017.01.005.

Microbial Respiration and Formate Oxidation as Metabolic Signatures of Inflammation-Associated Dysbiosis

Elizabeth R Hughes  1 , Maria G Winter  1 , Breck A Duerkop  2 , Luisella Spiga  1 , Tatiane Furtado de Carvalho  3 , Wenhan Zhu  1 , Caroline C Gillis  1 , Lisa Büttner  1 , Madeline P Smoot  1 , Cassie L Behrendt  2 , Sara Cherry  4 , Renato L Santos  3 , Lora V Hooper  5 , Sebastian E Winter  6
Affiliations

Microbial Respiration and Formate Oxidation as Metabolic Signatures of Inflammation-Associated Dysbiosis

Elizabeth R Hughes et al. Cell Host Microbe. .

Abstract

Intestinal inflammation is frequently associated with an alteration of the gut microbiota, termed dysbiosis, which is characterized by a reduced abundance of obligate anaerobic bacteria and an expansion of facultative Proteobacteria such as commensal E. coli. The mechanisms enabling the outgrowth of Proteobacteria during inflammation are incompletely understood. Metagenomic sequencing revealed bacterial formate oxidation and aerobic respiration to be overrepresented metabolic pathways in a chemically induced murine model of colitis. Dysbiosis was accompanied by increased formate levels in the gut lumen. Formate was of microbial origin since no formate was detected in germ-free mice. Complementary studies using commensal E. coli strains as model organisms indicated that formate dehydrogenase and terminal oxidase genes provided a fitness advantage in murine models of colitis. In vivo, formate served as electron donor in conjunction with oxygen as the terminal electron acceptor. This work identifies bacterial formate oxidation and oxygen respiration as metabolic signatures for inflammation-associated dysbiosis.

Keywords: bacterial respiration; dysbiosis; formate metabolism; gut microbiota; intestinal inflammation; metagenomics.

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Figures

Figure 1
Figure 1. Intestinal inflammation induces changes in the cecal microbiome
Groups of specific pathogen free (SPF) mice (N = 6 per group) were treated with 3 % dextran sulfate sodium (DSS) in the drinking water for 9 days or mock-treated. DNA, extracted from cecal content, was subjected to shotgun metagenomic sequencing. (A) Phylogenetic composition of the gut microbiota at the phylum level. (B) Percent of total reads corresponding to the taxonomic groups Clostridia (class), Bacteroidia (class), and Enterobacteriaceae (family). Each dot represents one animal. Lines represent the geometric mean ± standard deviation. ns, not statistically significant; **, P < 0.01 (C) Enrichment of pathways related to molybdopterin-cofactor biosynthesis and the electron transport chain in DSS-treated (red) compared to mock-treated mice (green). The size of the circle chart is proportional to the combined number of reads for all animals. Each slice represents one animal with the arc length being a measure of the number of reads from each mouse (see also Figure S2).
Figure 2
Figure 2. Molybdopterin cofactor contributes to fitness of Enterobacteriaceae in the DSS-induced colitis model
(A-C) Groups of SPF C57BL/6 mice were treated with 3 % DSS or water (mock). After four days, animals were intragastrically inoculated with the E. coli Nissle 1917 (EcN) wild-type strain (WT) or an isogenic ΔmoaA mutant. Samples were analyzed five days after inoculation (see also Figure S3A – E). (A) Representative images of hematoxylin and eosin-stained cecal sections. Scale bar, 200 µm. (B and C) Abundances of the experimentally introduced E. coli strains in the cecum (B) and colon (C) content. (D) Groups of SPF C57BL/6 mice, treated with 3 % DSS for 4 days, were intragastrically inoculated with an equal mixture of the indicated wild-type (WT) Enterobacteriaceae strains and isogenic moaA mutants and DSS treatment was continued. Five days after inoculation, the abundance of each strain was determined and the ratio (competitive index) of the two strains calculated. Bars represent geometric means ± standard error. The number of mice per group (N) is indicated in each panel. *, P < 0.05; ***, P < 0.001.
Figure 3
Figure 3. Contribution of anaerobic respiratory pathways to fitness of E. coli during DSS-induced inflammation
(A) 3 % DSS-or mock-treated SPF C57BL/6 mice were intragastrically inoculated with an equal mixture of the indicated strains. Five days after inoculation, abundance of each strain was determined and the ratio (competitive index) of the two strains calculated (see also Figure S4A). Bars represent geometric means ± standard error. The number of mice per group (N) is indicated above each bar. (B) Paired end reads from the metagenomic sequencing experiment in Figure 1 were mapped to a non-redundant dataset of representative fdo (triangles) and fdn (squares) operons. Each symbol represents one animal (N = 6 per treatment group). The lines show the geometric mean ± standard error. *, P < 0.05, **, P < 0.01; ***, P < 0.001.
Figure 4
Figure 4. Formate dehydrogenases enhance growth of E. coli in the inflamed gut
(A-B) SPF C57BL/6 mice, treated with 3 % DSS for 4 days, were intragastrically inoculated with an equal mixture of the indicated E. coli wild-type (WT) strains and isogenic mutants. (A) Competitive index in the colon content five days after inoculation (see also Figure S5C). (B) mRNA levels of Tnfa in the colon tissue were determined by RT-qPCR. (C) Competitive index in the colon content. SPF mice with different models of murine colitis were intragastrically inoculated with a mouse commensal E. coli strain (SL1). Mice received an equal mixture of WT and the isogenic ΔfdnG ΔfdoG mutant. Wild-type C57BL/6 mice were mock-or DSS-treated (2 %) and colonized for 9 days. Il10 knockout (KO) C57BL/6 mice were treated with piroxicam 2 days prior to colonization and colonized for 7 days. Rag1 KO mice on a C57BL/6 background received a T cell transfer, and after developing signs of inflammation were colonized for 9 days (see also Figure S6A and B). Il10 KO BALB/c mice were treated with piroxicam 2 days prior to colonization and were colonized for 14 days (gray bar). Bars represent geometric means ± standard error. The number of mice per group (N) is indicated in each panel. *, P < 0.05, **, P < 0.01; ***, P < 0.001; ns, not statistically significant.
Figure 5
Figure 5. Formate oxidation contributes to the expansion of the E. coli population during colitis
(A) SPF C57BL/6 mice were pre-colonized with an equal mixture of the E. coli Nissle 1917 (EcN) wild-type strain (WT) and the isogenic ΔfdnG fdoG mutant. Inflammation was induced by 3 % DSS treatment for 9 days and the competitive index in the cecum and colon determined. (B – E) Groups of SPF animals were pre-colonized with either the EcN WT or the ΔfdnG fdoG mutant and inflammation induced for 9 days by administration of 3 % DSS. (B) Representative images of hematoxylin and eosin-stained sections of the colon. Scale bar, 200 µm. (C) Combined histopathology score of lesions in the colon. Lines represent the mean ± standard deviation. (D) mRNA levels of pro-inflammatory markers (Tnfa, black bars; Nos2, white bars; Cxcl2, gray bars) as determined by RT-qPCR. (E) Abundance of the experimentally introduced E. coli strains in the colon content (see also Figure S7B). Bars represent geometric means ± standard error. The number of mice per group (N) is indicated in each panel. *, P < 0.05, **, P < 0.01; ***, P < 0.001; ns, not statistically significant.
Figure 6
Figure 6. Formate in the intestinal lumen is microbiota-derived
(A and B) Germ-free Swiss Webster mice were pre-colonized for three days with E. coli alone, E. coli and B. thetaiotaomicron or E. coli and a fecal transplant from a healthy, conventionally raised mouse. The E. coli inoculum consisted of an equal mixture of the E. coli Nissle 1917 (EcN) wild-type strain (WT) and the ΔfdnG fdoG mutant. Inflammation was induced by 3 % DSS. Conventional SPF Swiss Webster mice received 3 % DSS and an equal mixture of EcN WT and the .fdnG fdoG mutant. Samples were analyzed 9 days after the start of DSS treatment (see also Figure S7B). (A) mRNA levels of the pro-inflammatory markers (Tnfa, black bars; Nos2, white bars; Cxcl2; gray bars) as determined by RT-qPCR. (B) Competitive index of the EcN WT and the ΔfdnG fdoG mutant. (C and D) Conventionally raised SPF and germ-free C57BL/6 mice were treated with 2 % DSS or mock-treated for 7 days. (C) mRNA levels of the pro-inflammatory markers (Tnfa, black bars; Nos2, white bars; Cxcl2; gray bars) as determined by RT-qPCR. (D) Extracellular formate levels in the lumen of the colon as determined by GC/MS. Bars represent geometric means ± standard error. The number of mice per group N) is indicated above each bar. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant.
Figure 7
Figure 7. Utilization of oxygen by E. coli in the DSS colitis model
(A) Groups of SPF C57BL/6 mice, treated with 3 % DSS for 4 days, were inoculated with the indicated strains. The competitive index of the EcN strains was determined in the colon content after 5 days (see also Figure S7F). (B) Sequencing reads from the metagenomics data set were mapped to representative cydAB (black circles and lines) and cyoABCDE (white squares and gray lines) operons. Each symbol represents one animal. Lines show the mean ± standard deviation. (C and D) Groups of SPF C57BL/6 mice were treated for 4 days with 3 % DSS. Animals were intragastrically inoculated with an equal mixture of the EcN wild-type (WT; black bars) and the ΔcydA mutant (white bars) or the EcN WT and the ΔcyoABCD (Δcyo; gray bar) mutant. The population of each strain after 5 days in the colon (C) and cecum (D) is shown. Bars represent geometric means ± standard error. The number of mice used (N) is indicated in each panel. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not statistically significant.
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