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Vitamin B12 produced by gut bacteria modulates cholinergic signalling

Nature Cell Biology volume 26, pages 72–85 (2024)Cite this article

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Abstract

A growing body of evidence indicates that gut microbiota influence brain function and behaviour. However, the molecular basis of how gut bacteria modulate host nervous system function is largely unknown. Here we show that vitamin B12-producing bacteria that colonize the intestine can modulate excitatory cholinergic signalling and behaviour in the host Caenorhabditis elegans. Here we demonstrate that vitamin B12 reduces cholinergic signalling in the nervous system through rewiring of the methionine (Met)/S-adenosylmethionine cycle in the intestine. We identify a conserved metabolic crosstalk between the methionine/S-adenosylmethionine cycle and the choline-oxidation pathway. In addition, we show that metabolic rewiring of these pathways by vitamin B12 reduces cholinergic signalling by limiting the availability of free choline required by neurons to synthesize acetylcholine. Our study reveals a gut–brain communication pathway by which enteric bacteria modulate host behaviour and may affect neurological health.

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Fig. 1: B12 produced by gut bacteria suppresses the hyperactive behaviour of unc-2(gof) mutants.
Fig. 2: C. aquatica colonizes the C. elegans intestine and modulates behaviour.
Fig. 3: B12 inhibits cholinergic signalling.
Fig. 4: The B12-dependent Met/SAM cycle acts in the intestine and hypodermis to modulate behaviour.
Fig. 5: B12modulates excitatory cholinergic signalling through metabolic crosstalk between the Met/SAM cycle and the choline-oxidation pathway.
Fig. 6: Betaine can act as a methyl donor in the Met/SAM cycle.
Fig. 7: A neuronal choline transporter is required to mediate the effect of B12 on excitatory signalling.

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Data availability

All data files from this study have been uploaded to figshare and are freely available at https://doi.org/10.6084/m9.figshare.21197953. Source data are provided with this paper. All other data and reagents generated in this study are available from the corresponding author on reasonable request.

Code availability

The MATLAB scripts used in this study are available at https://github.com/jeremyflorman/Tracker_GUI.

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Acknowledgements

We thank the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440), for some of the worm and bacterial strains. We thank B. Samuel, H. Schulenburg, M. Shapira, V. Ambros and M. Treinin for bacterial strains; V. Budnik, A. Byrne, A. Walker and C. Vance for helpful discussions; and W. Joyce for technical support. This work was supported in part by National Institutes of Health grant numbers R01NS107475 and R01GM140480 (M.J.A.), DK068429 (A.J.M.W.), 1R35GM131877 (F.C.S.) and a grant from the Riccio Fund for Neuroscience (M.J.A. and A.J.M.W.). F.C.S. is a Faculty Scholar of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Department of Neurobiology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Woo Kyu Kang, Jeremy T. Florman, Antonia Araya, Andrea Thackeray & Mark J. Alkema

  2. Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY, USA

    Bennett W. Fox & Frank C. Schroeder

  3. Department of Systems Biology, University of Massachusetts Chan Medical School, Worcester, MA, USA

    Albertha J. M. Walhout

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  1. Woo Kyu Kang
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  2. Jeremy T. Florman
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  3. Antonia Araya
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  4. Bennett W. Fox
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  7. Albertha J. M. Walhout
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  8. Mark J. Alkema
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Contributions

W.K.K., J.T.F., B.W.F., A.A. and A.T. performed the experiments and analysed the data. W.K.K., J.T.F., B.W.F., F.C.S., A.J.M.W. and M.J.A. designed the experiments. W.K.K. and M.J.A. conceived the study and wrote the paper.

Corresponding author

Correspondence to Mark J. Alkema.

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The authors declare no competing interests.

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Extended data

Extended Data Fig. 1 Effects of different bacterial diets on growth, immune response and acdh-1 expression of C. elegans.

a, Growth rate as indicated by body length of unc-2(gof) mutants grown on the indicated bacterial strains for 24 h (mean ± s.e.m., n = 3, one-way ANOVA with Dunnett’s multiple comparison). b, Fluorescence values of immune reporter Pirg-1::GFP after 24 h exposure to indicated bacterial strains. Pathogenic strain P. aeruginosa 14 was included as a positive control (mean ± s.e.m., n = 3, one-way ANOVA with Dunnett’s multiple comparison). c, Representative DIC and fluorescence images of Pacdh-1::GFP animals fed different bacterial diets for 24 h. Shades of green represent relative GFP expression levels, ‘High’ indicates strong fluorescence throughout the intestine as in the OP50 shown, ‘Low’ indicates barely detectable fluorescence as in the Comamonas shown, ‘Moderate’ indicates visible fluorescence but weaker compared to the GFP signal on OP50 (n = 3 biologically independent samples with similar results). Scale bar, 300 µm. d, Growth rate as indicated by body length of wild-type and unc-2(gof) mutants fed OP50, OP50 with 64 nM B12, Comamonas, or Comamonas cbiAΔ for 24 h (mean ± s.e.m., n = 3, one-way ANOVA with Dunnett’s multiple comparison). e, Reversals of wild-type and unc-2(gof) mutants fed OP50, Comamonas, Comamonas cbiAΔ, or Comamonas cbiAΔ with 64 nM B12 for 24 h (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). f, Growth rate as indicated by body length of wild-type and unc-2(gof) mutants fed live or heat-killed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., n = 3, one-way ANOVA with Tukey’s multiple comparison). Source numerical data are provided.

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Extended Data Fig. 2 Antibiotic susceptibility for C. aquatica gut bacteria elimination.

a, Bacterial CFU per animal from unc-2(gof) mutants grown on Comamonas treated with the indicated concentrations of kanamycin for 24 h (mean ± s.e.m., n = 3, one-way ANOVA with Dunnett’s multiple comparison). b, Reversal frequency of unc-2(gof) mutants grown on OP50 with indicated concentrations of kanamycin for 24 h (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). c, Bacterial colonies of OP50 and Comamonas isolated the worm gut. Bacterial colonies were isolated and identified by 16 S rRNA gene using 27 f and 1492r primers (Methods) (n = 21 independent experiments with similar results). Source numerical data are provided.

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Extended Data Fig. 3 B12 reduces cholinergic signalling especially under conditions of increased acetylcholine release.

a, Reversal frequency of unc-2(gof) and cat-1(ok411); unc-2(gof) mutants fed OP50 ± 64 nM B12 (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). b, Quantification of locomotion speed of wild-type and ace-1(p1000); ace-2(g72) mutants fed OP50, OP50 with 64 nM B12, Comamonas, or Comamonas cbiAΔ for 24 h (mean ± s.e.m., n = 4, two-way ANOVA with Tukey’s multiple comparison). c-f, Quantification of reversal frequency (c), locomotion speed (d), head bending (e), and body bending (f) of wild-type and unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). g, Quantification of paralysis percentage of wild-type animals fed OP50 on 1 mM aldicarb-containing NGM agar plates or M9 liquid buffer (mean ± s.e.m., n = 4 (crawling on agar), n = 5 (swimming on liquid), two-way ANOVA with Tukey’s multiple comparison). Source numerical data are provided.

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Extended Data Fig. 4 B12 regulates C. elegans behaviour and growth through Met/SAM cycle.

a, Reversals of unc-2(gof), sams-1(ok2946); unc-2(gof), cbs-2(ok666); unc-2(gof), pcca-1(ok2282) unc-2(gof), or mce-1(ok243); unc-2(gof) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). b, Growth rate of unc-2(gof), mmcm-1(ok1637); unc-2(gof), metr-1(ok521); unc-2(gof), sams-1(ok2946); unc-2(gof) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). c, Quantification of paralysis percentage of unc-2(gof) and sams-1(ok2946); unc-2(gof) mutants fed OP50 ± B12 on 1 mM aldicarb (mean ± s.e.m., n = 4, two-way ANOVA with Tukey’s multiple comparison). d, Quantification of acetylcholine in unc-2(gof) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). e, Quantification of acetylcholine in WT and metr-1(ok521) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). f, Quantification of methionine (Met) in wild-type, metr-1(ok521), unc-2(gof), metr-1(ok521); unc-2(gof) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). g, Growth rate of metr-1(ok521); unc-2(gof) mutants expressing metr-1 cDNA driven by the indicated tissue-specific promoter fed OP50 ± B12 for 24 h (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). h-i, Growth rate (h) and reversals (i) of unc-2(gof) mutants fed OP50 with the indicated metabolites for 24 h (mean ± s.e.m., n = 3 (h), n indicated (i), one-way ANOVA with Dunnett’s multiple comparison). j,k, Quantification of homocysteine (Hcy) (j), S-adenosylhomocysteine (SAH) (k) in wild-type, metr-1(ok521), unc-2(gof), metr-1(ok521); unc-2(gof) mutants fed OP50 ± B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). Source numerical data are provided.

Source data

Extended Data Fig. 5 Choline metabolism is linked to the Met/SAM cycle.

a, Growth rate as indicated by body length of unc-2(gof) mutants fed OP50 ± 64 nM B12 with 30 mM choline for 24 h (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). b, Expression pattern of Palh-9::GFP in the intestine, hypodermis, and RIM neurons (n = 3 biologically independent samples with similar results). Scale bar, 100 µm. c, Growth rate as indicated by body length of unc-2(gof) mutants subjected to RNAi knockdown of chdh-1 or alh-9 fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are provided.

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Extended Data Fig. 6 In vitro activity assay of METR-1.

a, SDS–PAGE of purified METR-1 proteins. Purity of immunopurified GFP-tagged METR-1 protein was analysed with 4–10% polyacrylamide gels electrophoresis under denaturing conditions. Panels show silver staining (left) and western blot stained with a GFP antibody (right). Arrowhead indicates METR-1::GFP protein (molecular weight ~ 166 kDa). (n = 3 independent experiments with similar results) b, Methionine synthase activity of the purified METR-1::GFP protein was assayed in the presence of 5-methyltetrahydrofolate (5-meTHF) or betaine as a methyl donor ± SAM and DTT. Enzyme reaction was performed in 50 mM Tris-HCl buffer (pH 7.5) at 25 °C for 6 h (Methods) (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). Source numerical data and source blot images are provided.

Source data

Extended Data Fig. 7 A neuronal choline transporter is required to mediate the effect of B12 on excitatory transmission.

a, Reversal frequency of unc-2(gof), acr-23(ok2804); unc-2(gof), or lgc-41(sy1494) unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). b, Reversal frequency of unc-2(gof) or deg-3(u701); unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). c, Reversal frequency of unc-2(gof) mutants fed OP50, OP50 with B12 (64 nM), OP50 with betaine (75 mM), or OP50 with both B12 and betaine (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). d, Growth rate as indicated by body length of unc-2(gof) and cho-1(tm373); unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are provided.

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Kang, W.K., Florman, J.T., Araya, A. et al. Vitamin B12 produced by gut bacteria modulates cholinergic signalling. Nat Cell Biol 26, 72–85 (2024). https://doi.org/10.1038/s41556-023-01299-2

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