Vitamin B12 produced by gut bacteria modulates cholinergic signalling

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.

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, number of animals indicated, 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, number of animals indicated, 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 is 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, number of animals indicated, 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., n and number of animals indicated, 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, number of animals indicated, one-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Extended Data Fig. 2. Antibiotic susceptibility for Comamonas 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, number of animals indicated, 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., n and number of animals indicated, 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 16S rRNA gene using 27f and 1492r primers (Methods) (n = 21 independent experiments with similar results). Source numerical data are available in source data.

Extended Data Fig. 3. B12 reduces cholinergic signaling 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., n and number of animals indicated, 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, number of animals indicated, 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., n and number of animals indicated, 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), number of animals indicated, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Extended Data Fig. 4. B12 regulates C. elegans behavior 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., n and number of animals indicated, 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, number of animals indicated, 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, number of animals indicated, 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., n indicated, 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., n indicated, 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., n and number of animals indicated, 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, number of animals indicated, 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), number of animals indicated, 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., n and number of animals indicated, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in 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, number of animals indicated, 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 is 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, number of animals indicated, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

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 analyzed 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., n and number of animals indicated, one-way ANOVA with Dunnett’s multiple comparison). Source numerical data and source blot images are available in 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., n and number of animals indicated, 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., n and number of animals indicated, 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., n and number of animals indicated, 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, number of animals indicated, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Fig. 1:. B12 produced by gut bacteria suppresses hyperactive behavior of unc-2(gof) mutants.

a, Reversal frequency of unc-2(gof) mutants fed the indicated bacterial strains for 24 h (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). b, GFP expression of Pacdh-1::GFP animals fed the indicated bacterial strains 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, see Extended Data Fig. 1c). c, Reversal frequency of wild-type and unc-2(gof) mutants fed OP50, Comamonas, Comamonas cbiAΔ (B12-), or OP50 supplemented with 64 nM B12 (mean ± s.e.m., two-way ANOVA with Sidak’s multiple comparison). d, Reversal frequency of unc-2(gof) mutants fed OP50 supplemented with the indicated concentrations (nM) of B12 (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). e, Reversal frequency of unc-2(gof) mutants fed OP50 supplemented with 64 nM B12 for the indicated time (2G, 2 generations) and after transfer to OP50 (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). f, Reversal frequency of unc-2(gof) mutants fed live and heat-killed OP50 ± 64 nM B12 (mean ± s.e.m., two-way ANOVA with Sidak’s multiple comparison). Source numerical data are available in source data.

Fig. 2:. Comamonas colonizes the C. elegans intestine and modulates behavior.

a, Bacterial colony forming units (CFU) per animal from unc-2(gof) mutants fed OP50, P. aeruginosa PA14, or Comamonas (mean ± s.e.m., two-tailed unpaired t-test). b, Bacterial colony forming units (CFU) per animal from unc-2(gof) mutants fed Comamonas and Comamonas cbiAΔ (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). c, Bacterial CFU per animal from unc-2(gof) mutants fed Comamonas after transfer to OP50, measured over multiple days, as indicated (mean ± s.e.m., two-tailed unpaired t-test). d, Reversal frequency of unc-2(gof) mutants fed OP50 with 64 nM B12, Comamonas, or dead Comamonas after transfer to OP50, measured over multiple days, as indicated (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). e, Antibiotics susceptibility of E. coli and C. aquatica treated for 24 h with the indicated antibiotics (1x concentrations: 50 μg/ml Ampicillin, 100 μg/ml Carbenicillin, 50 μg/ml Kanamycin, 50 μg/ml Chloramphenicol, 100 μg/ml Streptomycin, and 12.5 μg/ml Tetracycline). f, Bacterial CFU per animal from unc-2(gof) mutants fed Comamonas treated with 200 μg/ml kanamycin for the indicated time (mean ± s.e.m., one-way ANOVA with Dunnett’s multiple comparison). g, Reversal frequency of unc-2(gof) mutants colonized with Comamonas after kanamycin treatment for 24 h (mean ± s.e.m., two-way ANOVA with Sidak’s multiple comparison). Source numerical data are available in source data.

Fig. 3:. B12 inhibits cholinergic signaling.

a, Percentage of paralyzed wild-type and unc-2(gof) mutants fed OP50 ± 64 nM B12 on 1 mM aldicarb (mean ± s.e.m., n = 4, two-way ANOVA with Tukey’s multiple comparison). b,c, 10 min tracks (b) and quantification of path length (c) of single young adult animal of wild-type and ace-1(p1000);ace-2(g72) double 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). Scale bar, 5 mm. d, Quantification of convulsion phenotype of acr-2(n2420gf) mutants fed OP50, OP50 with 64 nM B12, Comamonas, or Comamonas cbiAΔ for 24 h (n = 5, one-way ANOVA with Dunnett’s multiple comparison). In violin plots, middle-dotted line shows median, upper and lower lines represent 1st and 3rd quartiles. e, Calcium transient in muscle of a freely moving animal expressing a Pmyo-3::GCaMP6 transgene. Pixel intensity (arbitrary units) and corresponding colormap are depicted in the color bar. f, Quantification of the mean GCaMP6 fluorescence in body wall muscles of wild-type, unc-2(gof), and acr-2(n2420gf) mutants fed OP50 ± 64 nM B12 (mean ± s.e.m., n = 4, two-way ANOVA with Tukey’s multiple comparison). g, Percentage of paralyzed wild-type animals fed OP50 ± 64 nM B12 on 0.01 mM aldicarb during swimming (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). h, B12 reduces swimming-induced quiescence. Fraction of wild-type animals fed OP50 ± 64 nM B12 in quiescence (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Fig. 4:. B12 dependent Met/SAM cycle acts in intestine and hypodermis to modulate behavior.

a, B12-dependent metabolic pathways, Met/SAM cycle and propionyl-CoA breakdown pathway are highly conserved in C. elegans. b, Reversal frequency of metr-1(ok521);unc-2(gof) or mmcm-1(ok1637);unc-2(gof) mutants fed OP50 ± 64 nM B12 (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). c, Percentage of paralyzed metr-1(ok521);unc-2(gof) mutants fed OP50 ± 64 nM B12 on 1 mM aldicarb (n = 3 (OP50) and n = 4 (OP50 +B12), two-way ANOVA with two-way ANOVA with Tukey’s multiple comparison). d, Percentage of quiescence of wild-type and metr-1(ok521) mutants fed OP50 ± 64 nM B12 (n = 3, two-way ANOVA with two-way ANOVA with Tukey’s multiple comparison). e, Quantification of acetylcholine in unc-2(gof) and metr-1(ok521);unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). f, Expression pattern of Pmetr-1::GFP. Upper panel: confocal slice showing strong expression in intestine and hypodermis (arrow). Lower panels: Z-projection of the head (left) and the tail (right) showing no detectable signal in neurons (n = 3 biologically independent samples with similar results). Scale bars, 25 μm. g, Reversal frequency of metr-1(ok521);unc-2(gof) mutants expressing metr-1 cDNA driven by Pmetr-1 (endogenous), Pelt-2 (intestinal), Pdpy-7 (hypodermal), Pmyo-3 (muscle), or Ptag-168 (pan-neuronal) promoter fed OP50 ± 64 nM B12 (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Fig. 5:. B12 modulates excitatory cholinergic signaling through metabolic crosstalk between the Met/SAM cycle and the choline-oxidation pathway.

a, Metabolic network of Met/SAM cycle and choline metabolism. Red and blue indicate C. elegans and human metabolic enzymes involved, respectively. b, Reversal frequency of unc-2(gof) mutants fed OP50 ± 64 nM B12 with indicated concentrations of choline (mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparison). c, Percentage of quiescence of wild-type animals fed OP50, OP50 with choline (30 mM), OP50 with B12 (64 nM), or OP50 with both B12 and choline (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). d, Quantification of free choline of unc-2(gof) mutants fed OP50 ± 64 nM B12 for 24 h (mean ± s.e.m., two-tailed unpaired t-test). e, Co-expression pattern of Pmetr-1::GFP and Pchdh-1::mCherry in the intestine and hypodermis. chdh-1 is expressed in the intestine, hypodermis, and RIM neurons (n = 3 biologically independent samples with similar results). Scale bar is 100 μm. f, Reversal frequency of unc-2(gof) mutants subjected to RNAi knockdown of chdh-1 or alh-9 fed OP50 ± 64 nM B12 (mean ± s.e.m., two-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Fig. 6:. Betaine can act as a methyl donor in the Met/SAM cycle.

a, Schematic representation of domain architectures in methyltransferase proteins. HMT, homocysteine methyltransferases; BHMT, betaine-homocysteine methyltransferase; MS, Methionine synthase. b, Phylogenetic analysis of the methyltransferase enzymes. The tree was constructed with amino acid sequences of the homocysteine binding domains methyltransferase enzymes from bacteria, yeast, fly, plant and mammals (Methods). Black, blue, and red lines indicate HMT, BHMT, and MS enzymes, respectively. Maximum likelihood bootstrap values are shown at the relevant branches. c, in vitro enzyme activity of the purified METR-1 measured by the production of methionine from homocysteine in the presence of 5-methyltetrahydrofolate (5-meTHF) or betaine as a methyl donor (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison) (Methods). d, Growth rate as indicated by body length of wild-type fed OP50with the indicated concentrations of betaine (mean ± s.e.m., n = 3, one-way ANOVA with Tukey’s multiple comparison). e, Growth rate as indicated by body length of wild-type and metr-1(ok521) mutants fed OP50 with the indicated concentrations of betaine (mean ± s.e.m., n = 3, two-way ANOVA with Tukey’s multiple comparison). f, Percentage of mthf-1(gk465);bli-2(e768) mutants that reached adulthood on OP50 ± 75 mM betaine (mean ± s.e.m., two-tailed unpaired t-test). g, Percentage of mthf-1(gk465);bli-2(e768) mutants that reached adulthood on OP50, OP50 with B12 (64 nM), OP50 with betaine (75 mM), or OP50 with both B12 and betaine (mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparison). Source numerical data are available in source data.

Fig. 7:. A neuronal choline transporter is required to mediate the effect of B12 on excitatory signaling.

a, Reversal frequency of unc-2(gof) and cho-1(tm373);unc-2(gof) mutants fed OP50 ± 64 nM B12 (mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparison). b, Quantification of paralysis percentage of unc-2(gof) and cho-1(tm373);unc-2(gof) mutants fed OP50 ± 64 nM B12 on 1 mM aldicarb (mean ± s.e.m., n = 3, one-way ANOVA with Tukey’s multiple comparison). c, Reversal frequency of unc-2(gof) and cho-1(tm373);unc-2(gof) mutants fed OP50, OP50 with B12 (64 nM), or OP50 with both B12 (64 nM) and choline (30 mM) (mean ± s.e.m., one-way ANOVA with Tukey’s multiple comparison). d, Model: B12 produced by gut bacteria modulates excitatory neurotransmission of the host C. elegans. Metabolic crosstalk between the B12 dependent Met/SAM cycle and the choline-oxidation pathway in the intestine and hypodermis lowers the levels of free choline. The reduced choline availability by B12 limits acetylcholine synthesis in the neurons particularly under conditions of elevated acetylcholine release. Source numerical data are available in source data.