A neurotransmitter produced by gut bacteria modulates host sensory behaviour

Abstract

Animals coexist in commensal, pathogenic or mutualistic relationships with complex communities of diverse organisms, including microorganisms¹. Some bacteria produce bioactive neurotransmitters that have previously been proposed to modulate nervous system activity and behaviours of their hosts^2,3. However, the mechanistic basis of this microbiota-brain signalling and its physiological relevance are largely unknown. Here we show that in Caenorhabditis elegans, the neuromodulator tyramine produced by commensal Providencia bacteria, which colonize the gut, bypasses the requirement for host tyramine biosynthesis and manipulates a host sensory decision. Bacterially produced tyramine is probably converted to octopamine by the host tyramine β-hydroxylase enzyme. Octopamine, in turn, targets the OCTR-1 octopamine receptor on ASH nociceptive neurons to modulate an aversive olfactory response. We identify the genes that are required for tyramine biosynthesis in Providencia, and show that these genes are necessary for the modulation of host behaviour. We further find that C. elegans colonized by Providencia preferentially select these bacteria in food choice assays, and that this selection bias requires bacterially produced tyramine and host octopamine signalling. Our results demonstrate that a neurotransmitter produced by gut bacteria mimics the functions of the cognate host molecule to override host control of a sensory decision, and thereby promotes fitness of both the host and the microorganism.

Extended Data Fig. 1.. Octanol modulation by Providencia requires ingestion of bacteria and is not mediated by nutritive cues.

a, Cartoon and data from long-range chemotaxis assays of C. elegans grown on the indicated bacterial strains to the indicated attractive odors. Chemotaxis index (CI) = (animals at odorant – animals at control)/total animals. Dots, CI from single assays of approximately 100 animals. Horizontal line, median; errors, 1^st and 3^rd quartiles. Numbers in parentheses, number of assays performed over at least 3 days. b, Cartoon and data from osmotic ring avoidance assay. Dots, single assays of 10 animals. Numbers in parentheses, number of assays over at least 3 independent days. Y-axis is proportion of animals leaving an osmotic ring barrier of 8M glycerol after 10 minutes. P-value represents difference of means relative to JUb39-grown animals from a GLMM. Errors are SEM. Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals, respectively. c, Isolation of nematode-associated bacteria. Nematodes were isolated from residential compost in Massachusetts. Worms were allowed to crawl onto NGM plates from which they were picked to clean plates. Resulting bacterial colonies were isolated, grown on LB media and characterized via 16S rRNA sequencing. d, Expression of a tph-1p::gfp fluorescent reporter in indicated head neurons of young adult animals grown on either OP50 or JUb39. Dots, mean fluorescence of the soma of single neurons. Horizontal bar is mean; errors are SEM. Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals, respectively. P-values are from two-way ANOVA. e-f, Modulation index of worms grown on the indicated bacterial strains to 100% octanol, under the shown conditions. Animals were exposed to the indicated bacteria on the plate lid (e) for one generation, or to NGM control or bacteria-conditioned NGM (f) for 2 hours prior to the assay. Numbers in parentheses, independent experiments over 2 days with approximately 100 animals each. Values are shown on a log-odds (logit) scale and are normalized to the values of wild-type animals grown on OP50 for each day, indicated with a gray dashed line. Positive numbers indicate reduced avoidance of octanol. Errors are SEM. Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals, respectively. P-values between the indicated conditions are post-hoc comparisons from a GLMM, with Tukey-type multivariate-t adjustment for (f). g, Survival analysis of worms grown on the indicated strains. Dots, average proportion of surviving worms from 12 plates of 20 worms each on the indicated days. Error bars are SEM. Shaded blue and gray curves indicate 95% confidence intervals derived from 1000 bootstrap Gompertz function non-linear least squares (nls) fits of the indicated data. Top blue and gray distributions show bootstrapped median survival of the indicated strains.

Extended Data Fig. 2.. Detection of N-succinyl TA.

Major fragmentation reactions and resulting fragment ions are indicated in mass spectra of N-succinyl TA obtained in ESI− (a) and ESI+ (b) HPLC-MS2. Representative data are shown from at least three biologically independent experiments.

Extended Data Fig. 3.. Complementation of TA-containing metabolites in tdc-1 mutants via Providencia TDC-encoding genes.

a, Overview of MS2 network obtained in positive ion mode. Each node represents a unique feature, and edges between nodes indicate similarity between MS2 spectra. Red circle highlights the sub-network containing the majority (16 out of 24 features, representing 10 out of 15 differential compounds) of differential TA-containing metabolites. This sub-network contains glycosylated TA-derivatives, N-succinyl-TA is represented by a node in a different sub-network (red arrow). b, Magnified view of sub-network highlighted in (a). The TA-containing features restored upon growth of tdc-1 worms on wild-type JUb39, and abolished when grown on JUb39 ΔtyrDC::cmR ΔadcA, are highlighted in light red. MS2 spectra for two example compounds at m/z 499.1474 and m/z 604.1689 (circled in magenta) are shown in Extended Data Figs. 4 and 5.

Extended Data Fig. 4.. MS2 analysis and quantitation of TA-containing metabolite m/z 499.1474.

a, Major fragmentation reactions of m/z 499.1474 and resulting fragment ions. The shown putative structure is based on fragmentation and stable isotope incorporation. Stereochemistry and exact substitution pattern are unknown. b-c, MS2 spectra obtained in (b) ESI- and (c) ESI+ mode. Representative data are shown from at least three biologically independent experiments. d, Quantification of m/z 499.1474 in wild-type and tdc-1 worms fed the indicated bacterial strains as determined by positive-ion ESI+ HPLC-MS. Dots, independent samples from n=3 experiments. Errors are SEM.

Extended Data Fig. 5.. MS2 analysis and quantitation of TA-containing metabolite m/z 604.1689.

a, Major fragmentation reactions of m/z 604.1689 and resulting fragment ions. Stereochemistry and exact substitution pattern are unknown. The shown structure is based on fragmentation and stable isotope incorporation. b-c, MS2 spectra obtained in (b) negative- and (c) positive-ion mode. Representative data are shown from at least three biologically independent experiments. d, Quantification of m/z 604.1689 in wild-type and tdc-1 worms fed the indicated bacterial strains as determined by positive-ion ESI HPLC-MS. Dots, independent samples from n=3 experiments. Errors are SEM.

Extended Data Fig. 6.

Quantification of N-acetyl serotonin in worms fed the indicated bacteria, or in bacterial cultures alone, as determined by ESI+ HPLC-MS. Dots, independent samples from n=3 experiments. Errors are SEM.

Extended Data Fig. 7.. L-Tyr supplementation enhances octanol modulation.

a-b, Reversal response times of animals of the indicated genotypes grown on the indicated bacteria in control conditions (a) or supplemented with 0.5% L-Tyr (a,b) to 100% octanol using SOS assays. Dots, response time of single worms. Y-axis is log₁₀-scaled for these log-normal distributed data, and normalized to the indicated control group for each experimental day. Numbers in parentheses, number of worms tested in assays over at least 3 independent days. Boxplot, median and quartiles, whiskers, data range (excluding outliers). Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals for the difference of means, respectively. P-values indicating comparisons of means relative to the OP50 control for each conditions are from a LMM with Tukey-type multivariate-t adjustment. P-value in red indicates Wald F-statistic (a) or Wald t-statistic (b) for the effect of L-Tyr supplementation (a) or genotype (b) on the magnitude of the JUb39 effect. Wild-type data in (b) are also shown in Fig. 2g.

Extended Data Fig. 8.. Phylogenetic analysis of group II decarboxylase genes in Gammaproteobacteria.

a, Neighbor-joining unrooted tree based on sequences identified via a BLAST search using Enterococcus faecalis TyrDC and C. elegans TDC-1. Initial tree indicates 3 major groups. Representative enzymes and operon structures for each group are indicated by colored boxes. b, Bootstrapped maximum likelihood phylogeny using PhyML and Phylomizer pipeline. Maximum of two highly similar sequences per genus were included after each BLAST search. Genera are indicated to the right. Numbers on branch-points matching this tree out of 100 bootstrap replicates are indicated at values >60. Group representatives from (a) are indicated in corresponding colors. Providencia and C. elegans sequences discussed in this work are indicated in bold. Accession numbers and BLAST metrics are listed in SI Table 1. c, Presence of tyrDC and adcA among complete genomes in Gammaproteobacteria. Linked boxes indicate organization in an operon. Hatched shading indicates variable presence among genera. Colored triangles indicate taxa of interest. d, Homology-based model of the TyrDC catalytic domain in Providencia based on the Lb-TyrDC crystal structure using SWISS-MODEL (https://swissmodel.expasy.org). Residues in magenta, green and yellow are from Lb-TyrDC, JUb39-TyrDC, and JUb39-AdcA, respectively. PLP is depicted in red and L-Tyr (manually docked for illustration) is indicated in light blue. Position of A600/S586 in JUb39 TyrDC and Lb-TyrDC are indicated.

Extended Data Fig. 9.. Disruption of JUb39 TA production or host OA receptor signaling affects octanol modulation without altering intestinal bacterial cell numbers.

a, Presence of mCherry-expressing bacteria in the posterior intestines of young adult wild-type or octr-1 mutant animals. Bars show proportion of animals with the indicated distribution of bacterial cells present in animals grown on shown bacteria. Numbers in parentheses, number of animals. P-value is derived from an ordinal regression. b, Reversal response latency of animals of the indicated genotypes grown on the shown bacteria in control conditions of NGM + 0.5% L-Tyr to 100% octanol using SOS assays. Dots, response time of single worms. Y-axis is log₁₀-scaled for these log-normal distributed data, and normalized to the indicated control group for each experimental day. Numbers in parentheses, number of worms tested in assays over at least 3 independent days. Boxplot, median and quartiles, whiskers, data range (excluding outliers). Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals for the difference of means, respectively. P-values between indicated conditions are from a LMM with Tukey-type multivariate-t adjustment. P-values in red indicate Wald t-statistic representing the genotype x food interaction effect relative to WT.

Extended Data Fig. 10.. Providencia-derived TA complements the egg-laying defects of C. elegans tdc-1 mutants.

Quantification of the age of eggs in utero of animals of the indicated genotypes grown on the shown bacterial strains. Dots, proportion of eggs at or older than the 4-cell stage in individual animals. Numbers in parentheses, total number of animals scored in three independent experiments. Boxplot, median and quartiles, whiskers, data range (excluding outliers). P-values between the indicated conditions are from a binomial generalized linear mixed effects regression using a logit link function, with a post-hoc Tukey correction for multiple comparisons.

Fig. 1.. Providencia colonizes the C. elegans intestine and modulates octanol avoidance.

a, Long-range chemotaxis assays (Extended Data Fig. 1a) of C. elegans grown on the indicated bacterial strains to aversive odors. Chemotaxis index (CI) = (animals at odorant – animals at control)/total animals. Dots, CI from single assays of approximately 100 animals. Horizontal line, median; errors, 1^st and 3^rd quartiles. P-value is derived from z-statistic from a binomial general linearized mixed-effects model (GLMM) with random intercepts for assay plate and date and with false discovery rate (FDR) for post-hoc comparisons (two-sided test). n = 5 and 6 independent experiments for octanol and nonanone assays, respectively, over 3 days. b-c, Modulation index of worms in response to 100% octanol. ‘Dead’ (c), bacteria pre-treated with gentamicin. Modulation index, the log odds-ratio of the proportion of worms at octanol vs control of each condition normalized to the OP50-grown condition per independent day (gray dashed line). Positive numbers, reduced octanol avoidance. Errors are SEM. Gray vertical bars, Bayesian 95% and 66% credible intervals, respectively. P-values, two-sided GLMM with Dunnett-type (b), and Tukey-type multivariate-t adjustment (c). Numbers in parentheses, independent experiments over 3-5 days with approximately 100 animals each (also see Extended Data Fig. 9a). d, mCherry-expressing bacteria in the posterior intestines of young adults. Arrows, intact cells, asterisk, diffuse intestinal fluorescence, dashed lines, intestinal boundary. Anterior at left. Scale bar, 10 μm. e, Intestinal bacterial load in animals grown on the indicated bacterial strains. Dots, estimation of bacterial load in colony forming units (cfu) of 10 worms; n = 4 independent samples (see Methods). Data are mean ± SEM. f, Proportion of animals that migrated to 100% octanol or control in chemotaxis assays with indicated distribution of mCherry-expressing JUb39 cells. Numbers in parentheses, number of animals; 3 independent assays. P-value is derived from an ordinal regression z-statistic, using number of animals.

Fig. 2.. Providencia-produced TA compensates for loss of C. elegans tdc-1.

a, Biosynthesis of TA and OA in C. elegans. b, Modulation index of animals in response to 100% octanol. Dots, individual chemotaxis assays with approximately 100 animals each. Numbers in parentheses, number of independent assays over at least 3 independent days. Y-axis is log-odds (logit) scale, normalized to OP50 for each day (gray dashed lines). Errors are SEM. Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals, respectively. P-values, GLMM with Dunnett-type multivariate-t adjustment. P-value in red, Wald z-statistic for the magnitude of the JUb39 effect in tbh-1 compared to wild-type. c, Cartoon of SOS assay ^,. d, g, Reversal response latency of animals on NGM without (d) or with 0.5% L-Tyr (g) in response to 30% (d) or 100% octanol (g) in SOS assays. Dots, response time of single animals. Y-axis is log₁₀-scaled, normalized to the indicated control group for each experimental day. Numbers in parentheses, number of worms tested in assays over at least 3 independent days. Boxplot, median and quartiles, whiskers, data range (excluding outliers). Gray vertical bars, Bayesian 95% and 66% credible intervals for the difference of means, respectively. P-values, linear-mixed effects regression on log-transformed data (LMM). P-values in red (g), Wald F-statistic (left) or Wald t-statistic (right) for the effect of the indicated genotypes on the magnitude of the JUb39 effect. Data from wild type in (g, right) are repeated in Extended Data Fig. 7b. Alleles: tbh-1(ok1196) (g, left), tbh-1(n3247) (g, right). e, Quantification of N-succinyl TA. Dots, area under the curve (AUC) from three biologically independent experiments. Data are mean ± SEM. ND, not detected. f, HPLC-MS (ESI+) ion chromatograms for N-succinyl TA or D₂-N-succinyl TA in worms grown on JUb39 with the indicated amino-acid supplementation. Chromatograms for D₂-L-Tyr fed worms are scaled two-fold relative to L-Tyr feeding. Statistical comparisons are two-sided.

Fig. 3.. Two Providencia AADC enzymes act redundantly to modulate octanol avoidance.

a, Cartoons depicting the tyrDC locus in Lactobacillales, the adcA locus in Morganella (top), and corresponding loci including engineered muations in JUb39 (bottom). b, Presence of tyrDC, adcA, E.coli-type tyrP and Morganella-type tyt-1 at the family and genus level among Enterobacterales. Linked boxes indicate organization in an operon. c, f, Reversal response latency of wild-type C. elegans grown on the indicated bacterial genotypes in NGM + 0.5% L-Tyr (c) or supplemented with the indicated concentrations of TA (f) to 100% octanol using SOS assays. Dots, response time of single worms. Y-axis is log₁₀-scaled and normalized to the indicated control group for each experimental day (gray dashed line). Numbers in parentheses, number of worms tested in assays over at least 3 independent days. Boxplot, median and quartiles, whiskers, data range (excluding outliers). Gray thin and thick vertical bars at right, Bayesian 95% and 66% credible intervals for the difference of means, respectively. P-values between indicated conditions are from a LMM with Tukey-type multivariate-t adjustment. d, Quantification of N-succinyl TA in tdc-1 mutant animals grown on the indicated bacterial strains. OP50 and JUb39 data are repeated from Fig. 2e. Dots, area under the curve (AUC) from each of 3 biologically independent experiments. Data are mean ± SEM. ND, not detected. e, Heatmap showing mean abundance of TA-derived compounds of indicated molecular formula detected in N2 wild-type and tdc-1 mutant worms grown on the indicated bacterial strains, relative to N2 worms grown on OP50. Means are calculated from 3 independent experiments. Asterisks indicate D₂-containing compounds confirmed using D₂-L-tyrosine supplementation (Fig. 2f).

Fig. 4.. Modulation of octanol avoidance by Providencia requires the OCTR-1 OA receptor in the ASH sensory neurons.

a-b, Reversal response latency of animals of the indicated worm genotypes grown on the indicated bacteria on NGM + 0.5% L-Tyr (a) and supplemented with the indicated concentrations of TA (b) to 100% octanol using SOS assays. Dots, response time of single worms. Y-axis is log₁₀-scaled and normalized to the indicated control group (a) or to the wild-type control (b) for each experimental day. Numbers in parentheses, number of worms tested in assays over at least 3 independent days. Wild-type octr-1 sequences were expressed in ASH and ASI under the srv-11 and srg-47 promoters, respectively. Boxplot, median and quartiles, whiskers, data range (excluding outliers). Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals for the difference of means. P-values between indicated conditions are from a LMM with Tukey-type multivariate-t adjustment. P-values in red, Wald t-statistic representing the genotype x food interaction effect relative to WT. c, Cartoon depicting short-range bacterial choice assay. d-e, Relative preference index of wild-type or mutant animals grown on the indicated bacteria for the test bacteria JUb39. Data are normalized to the OP50 control condition for each genotype. Dots, single assays of at least 10 animals. Numbers in parentheses, number of assays over at least 5 independent days. Y-axis is on log-odds ratio (logit) scale. Errors are SEM. Gray thin and thick vertical bars, Bayesian 95% and 66% credible intervals, respectively. P-values represent difference of means relative to JUb39-grown animals from a GLMM with Dunnett-type multivariate-t adjustment. P-value in red, Wald z-statistic representing the genotype x food interaction effect relative to WT (e). Allele used: tbh-1(n3247) (e). f, Cartoon of working model.