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Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Veillonella parvula in the intestine

Nature Microbiology volume 7, pages 1673–1685 (2022)Cite this article

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Abstract

Colonization of the intestine by oral microbes has been linked to multiple diseases such as inflammatory bowel disease and colon cancer, yet mechanisms allowing expansion in this niche remain largely unknown. Veillonella parvula, an asaccharolytic, anaerobic, oral microbe that derives energy from organic acids, increases in abundance in the intestine of patients with inflammatory bowel disease. Here we show that nitrate, a signature metabolite of inflammation, allows V. parvula to transition from fermentation to anaerobic respiration. Nitrate respiration, through the narGHJI operon, boosted Veillonella growth on organic acids and also modulated its metabolic repertoire, allowing it to use amino acids and peptides as carbon sources. This metabolic shift was accompanied by changes in carbon metabolism and ATP production pathways. Nitrate respiration was fundamental for ectopic colonization in a mouse model of colitis, because a V. parvula narG deletion mutant colonized significantly less than a wild-type strain during inflammation. These results suggest that V. parvula harness conditions present during inflammation to colonize in the intestine.

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Fig. 1: The abundance of nitrate-respiring Veillonella spp. is increased during IBD.
Fig. 2: Nitrate respiration expands the metabolic repertoire of V. parvula.
Fig. 3: Metabolic changes associated with nitrate respiration.
Fig. 4: Nitrate respiration-mediated changes in membrane potential are harnessed for substrate transport.
Fig. 5: The conditional dispensability of ATP synthase unveils mechanistic aspects associated with fermentation and nitrate respiration.
Fig. 6: Nitrate reductase activity is required for efficient colonization in a mouse model of inflammation.

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Data availability

RNA-seq data are available on the Sequence Read Archive (SRA) under BioProject accession no. PRJNA861991. Human microbiome data were accessed through data deposited on the SRA under BioProject accession no. PRJNA436359. Source data are provided with this paper.

Code availability

No customized code was generated or used in the present study.

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Acknowledgements

We thank J. L. Merritt at Oregon Health & Science University for the SKV38 strain of V. parvula and the pBSLJ1 plasmid, as well as J. Vargas Asencio at MIT for helpful discussions and T. Reimels for editorial assistance. RNA-seq libraries were constructed and sequenced at the Broad Institute of MIT and Harvard by the Microbial ’Omics Core and Genomics Platform, respectively. The Microbial ’Omics Core also provided preliminary analysis for all RNA-seq data. We thank the gnotobiotic animal facility at the Broad Institute, specifically C. Umana and A. Discua, for helping with experiments and T. Caron for providing support for running the facility. This work was supported by grants from the National Institutes of Health (grant no. P30 DK043351), Center for Microbiome Informatics and Therapeutics, and Crohn’s and Colitis Foundation to R.J.X and from the Deutsche Forschungsgemeinschaft (German Research Foundation, grant no. 426120468) to M.S.

Author information

Author notes

  1. Daniel F. Rojas-Tapias

    Present address: Department of Agricultural Microbiology, Colombian Corporation for Agricultural Research—Agrosavia, Bogotá, Colombia

  2. These authors contributed equally: Daniel F. Rojas-Tapias, Eric M. Brown.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Daniel F. Rojas-Tapias, Eric M. Brown, Emily R. Temple, Michelle A. Onyekaba, Ahmed M. T. Mohamed, Kellyanne Duncan, Melanie Schirmer, Rebecca L. Walker, Toufic Mayassi, Hera Vlamakis & Ramnik J. Xavier

  2. Center for Computational and Integrative Biology and Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, USA

    Daniel F. Rojas-Tapias, Eric M. Brown, Ahmed M. T. Mohamed, Kellyanne Duncan, Toufic Mayassi & Ramnik J. Xavier

  3. Emmy Noether Group, ZIEL—Institute for Food and Health, Technical University of Munich, Freising, Germany

    Melanie Schirmer

  4. Metabolomics Platform, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Kerry A. Pierce, Julián Ávila-Pacheco & Clary B. Clish

  5. Center for Microbiome Informatics and Therapeutics, Massachusetts Institute of Technology, Cambridge, MA, USA

    Hera Vlamakis & Ramnik J. Xavier

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Contributions

D.F.R.-T., H.V. and R.J.X. conceived the study. D.F.R.-T., E.M.B., E.R.T., K.A.P. and J.A.-P. provided the methodology. D.F.R.-T., M.S. and R.L.W. did the computational analyses. D.F.R.-T., E.M.B., E.R.T., M.A.O., A.M.T.M., K.D., T.M. and K.A.P. carried out the investigations. D.F.R.-T., E.M.B., H.V. and R.J.X. wrote the manuscript. All authors edited the manuscript. C.B.C., H.V. and R.J.X. supervised the study.

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Correspondence to Ramnik J. Xavier.

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Extended data

Extended Data Fig. 1 Phylogenetic analysis of nitrate reductases.

Analysis of the Nar, Nap, and Nas nitrate reductase families in human (a) gut and (b) oral microbes, using the NarG and NapA protein sequences of E. coli, and NasA of K. pneumoniae as templates. The sequences were searched in the assembled gene catalog from Pasolli et al. (2019)55. (c) Phylogenetic tree of NarG in gut microbes reveals that Veillonella is one of the few genera within the Firmicutes with a putative nitrate reductase. Genes with at least 45% amino acid similarity were used for analysis.

Extended Data Fig. 2 Nitrate-stimulated growth of various Veillonella strains, and narI::chl complementation experiments.

(a) The growth of V. parvula SKV38 was assessed in different complex media supplemented or not with 40 mM nitrate (n = 4 biological replicates). (b) Growth curves of WT and narI::chl strains in SK, SKL, SKN, and SKLN media. Cells lacking the NarI protein are unable to grow in SKN, and nitrate does not stimulate its growth when lactate is present (n = 3 biological replicates). (c) The growth of various Veillonella strains was assessed in SK, SKL, SKN, and SKLN media (n = 3 biological replicates). Growth rates (µmax) were calculated for each growth curve. Data (a-c) represent mean ± SEM, analyzed by one-way ANOVA and Tukey’s HSD test. NS, not significant.

Source data

Extended Data Fig. 3 Nitrate metabolism in Veillonella.

(a) Levels of nitrate and nitrite were measured at the mid-exponential phase in cells growing on SKN medium supplemented or not with lactate as the carbon source (n = 3 biological replicates). The measured concentrations of nitrate and nitrite in the uninoculated SK medium were 43.161 ± 0.410 mM and <0.050 mM, respectively (n = 3 biological replicates). (b) Ammonium (n = 6 biological replicates) or (c) nitrite (n = 4 biological replicates) were used to determine if the positive effect of nitrate on growth of V. parvula was due to nitrate being used as a source of nitrogen or an electron acceptor, respectively. Growth rates (µmax) were calculated for each growth curve. Data represent mean ± SEM, analyzed by one-way ANOVA and Tukey’s HSD test (a, b) or Dunnett’s test (c). NS, not significant.

Source data

Extended Data Fig. 4 Amino acids and peptides stimulate the growth of different Veillonella strains.

(a) The growth of various Veillonella strains was studied in SKbN with 0.5% yeast extract, and with and without 1% casitone, 1% casamino acids, or 50 mM L-aspartic acid (n = 3 biological replicates). (b) The addition of lactate or malate stimulates growth of V. parvula in SKb medium (n = 4 biological replicates). Growth rates (µmax) were calculated for each growth curve. Data represent mean ± SEM, analyzed by one-way ANOVA and Tukey’s HSD test (a) or Dunnett’s test (b). NS, not significant.

Source data

Extended Data Fig. 5 LC-MS analysis of amino acids in unspent and spent media.

LC-MS analysis of the unspent (n = 6 biological replicates) and spent SKL, SKN, and SKLN media (n = 3 biological replicates) was used to study amino acid consumption. Supernatants were collected at the (a) mid-exponential and (b) late exponential phases. Data represent mean ± SEM, analyzed by one-way ANOVA and Tukey’s HSD test. NS, not significant.

Source data

Extended Data Fig. 6 Role of the PMF in the production of ATP and transport, and phenotype of the L-lactate permease (lldP::chl) mutant.

(a) Measurement of the proton motive force (PMF) in WT and narG::tet cells growing on SK with and without lactate or malate, and with or without CCCP. The PMF was measured before and 40 min after addition of 40 mM nitrate. CCCP was added to confirm that the observed changes were associated with the proton gradient (n = 3 biological replicates). (b) Lactate (n = 3 biological replicates) and aspartate (n = 2 biological replicates) consumption were measured in resting WT cells resuspended in resting cell (RC) buffer with or without nitrate. (c) Lactate consumption (n = 4 biological replicates) was measured in growing atp::tet cells with or without nitrate. Both metabolites were measured in the supernatant using colorimetric techniques. (d) Growth experiments with WT Veillonella and a strain lacking LldP (lldP::chl) in SK, SKL, SKN, and SKLN media (n = 4 biological replicates). Cells lacking the LldP transporter are nearly unable to grow with lactate as the carbon source. Growth rates (µmax) were calculated for each growth curve. Data (a–d) represent mean ± SEM, analyzed by two-sided t-test (a, b, and c), or one-way ANOVA and Tukey’s HSD test (growth rates in (d). NS, not significant.

Source data

Extended Data Fig. 7 Phenotype of cells lacking components of the ATP synthase, and expression analysis of the atp operon.

(a) Growth experiments with WT Veillonella and the AtpC (atpC::chl) and AtpD-AtpC (atpDC::chl) deletion strains in SK, SKL, SKN, and SKLN media (n = 4 biological replicates). These deletion strains exhibited a phenotype similar to the one seen in the atp::tet strain. Growth rates (µmax) were calculated for each growth curve. (b) Transcriptional analysis of the atpA gene in cells growing in SKL, SKN, and SKLN media (n = 3 biological replicates). Data represent mean ± SEM, analyzed by one-way ANOVA and Tukey’s HSD test. NS, not significant.

Source data

Extended Data Fig. 8 Validation of the DSS-treated mouse model.

(a) Measurements of IFN-γ, TNF, IL-6, IL-17A, and IL-10 in the mesenteric lymph nodes of WT-colonized ASF mice without (n = 4) and with (n = 5) DSS treatment. (b) Lamina propria cells were isolated from colonic tissue and analyzed via flow cytometry. From left to right: Cells were first gated for positive expression of the pan immune cell marker CD45 while excluding dead cells staining for the viability dye Zombie UV. Next, gating was further cleaned by gating on FSC-A to exclude debris and FSC-A versus FSC-H to exclude doublets. Finally neutrophils were gated based on double positive expression of the two surface protein markers Ly6G and Ly6C. c) Left: representative contour plots showing the proportion of neutrophils based on positive expression of Ly6C and Ly6G on live CD45+ lymphocytes isolated from mouse colonic lamina propria. Right: Frequency of neutrophils among total CD45+ colonic lamina propria lymphocytes. (d) Total number of neutrophils isolated from mouse colons based on gating in c. The data in a, c, and d are representative of two independent experiments. For c and d, boxplots display the first and third quartiles with a thick line representing the median; n = 4 mice without DSS, and n = 5 mice with DSS treatment. A Student’s t-test was used in a, c, and d for statistical analyses.

Source data

Extended Data Fig. 9 Colonization of Veillonella in a mouse model of colitis.

(a) Raw Ct values of bacterial 16 S rRNA gene measured by qPCR in the stool of WT-colonized ASF mice with and without DSS treatment (n = 8–11 mice). (b) Absolute abundance of V. parvula in the stool of ASF mice as measured by CFU per mg of stool on selective SKLN media (n = 3 mice). (c) Absolute abundance of V. parvula in the colonic tissue of ASF mice as measured by CFU per mg of tissue on selective SKLN media (n = 4 mice). (d) Concentration of lactate in the gut of control- and DSS-treated ASF mice colonized with V. parvula (n = 8 mice). (e) Relative expression of the IIdD, lldP, hisA and argB genes in WT Veillonella colonizing the stool of mice with and without DSS treatment (n = 3–4). The V. parvula gyrA gene was used as an internal control. (f) Colon lengths of WT and iNOS KO SPF mice (n = 4–7). Data represent two independent experiments and show mean (a, d–f) ± SEM (d–f) or median (b, c) values. Data were analyzed by one-way ANOVA and Tukey’s HSD test or Mann-Whitney U-test.

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Supplementary information

Supplementary Information

Supplementary Fig. 1 and Table 1.

Reporting summary

Supplementary Tables

Supplementary Table 2 Resistance genes used for V. parvula mutants. Supplementary Table 3 Primers for mutants. Supplementary Table 4 Primers for qPCR.

Supplementary Data File 1

LC–MS data.

Supplementary Data File 2

LC–MS analysis of unspent and spent SKL, SKN and SKLN media at the late exponential phase used to study dipeptide consumption.

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Rojas-Tapias, D.F., Brown, E.M., Temple, E.R. et al. Inflammation-associated nitrate facilitates ectopic colonization of oral bacterium Veillonella parvula in the intestine. Nat Microbiol 7, 1673–1685 (2022). https://doi.org/10.1038/s41564-022-01224-7

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