Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism

Abstract

The human gut microbiota metabolizes the Parkinson's disease medication Levodopa (l-dopa), potentially reducing drug availability and causing side effects. However, the organisms, genes, and enzymes responsible for this activity in patients and their susceptibility to inhibition by host-targeted drugs are unknown. Here, we describe an interspecies pathway for gut bacterial l-dopa metabolism. Conversion of l-dopa to dopamine by a pyridoxal phosphate-dependent tyrosine decarboxylase from Enterococcus faecalis is followed by transformation of dopamine to m-tyramine by a molybdenum-dependent dehydroxylase from Eggerthella lenta These enzymes predict drug metabolism in complex human gut microbiotas. Although a drug that targets host aromatic amino acid decarboxylase does not prevent gut microbial l-dopa decarboxylation, we identified a compound that inhibits this activity in Parkinson's patient microbiotas and increases l-dopa bioavailability in mice.

Fig. 1

E. faecalis metabolizes L-dopa using a PLP-dependent tyrosine decarboxylase. (A) Proposed major pathway for L-dopa metabolism by the human gut microbiota and potential for interaction with host-targeted drugs. (B) Phylogenetic distribution of TyrDC in the human microbiota. Human Microbiome Project reference genomes were queried by means of BLASTP for homologs of the L. brevis TyrDC, and the results are visualized on a cladogram of phylogeny [based on 16S ribosomal RNA (rRNA) alignment]. TyrDC homologs found sporadically within Lactobacillus spp. (Lb) are widely distributed among Enterococcus (Ec; average amino acid identity 67.8% over 97.6% query length). (C) Testing representative gut microbial strains encoding TyrDC reveals that E. faecalis strains reproducibly convert L-dopa to dopamine. Strains were cultured for 48 hours anaerobically. Bar graphs represent the mean ± SEM of three biological replicates. (D) Deletion of tyrDC abolishes L-dopa decarboxylation by E. faecalis. Dopamine was detected in culture supernatants after 48 hours of anaerobic growth with 0.5 mM L-dopa. Bar graphs represent the mean ± SEM of three biological replicates. (E) Kinetic analysis of E. faecalis TyrDC reveals a preference for tyrosine. Error bars represent the mean ± SEM of three biological replicates. ND, not detected. (F) L-dopa and tyrosine are simultaneously decarboxylated in anaerobic cultures of E. faecalis MMH594 grown at pH 5 with 1 mM L-dopa and 0.5 mM tyrosine. Bar graphs represent the mean ± SEM of three biological replicates.

Fig. 2

E. lenta dehydroxylates dopamine using a molybdenum-dependent enzyme. (A) RNAsequencing identifies a putative molybdenum (moco)–dependent dopamine dehydroxylase (Dadh) in E. lenta A2. Differentially expressed candidate genes (false discovery rate < 0.1 and fold change > j2j) are plotted as a function of genome position, revealing three discrete loci of differentially expressed genes. (Inset) Analysis of the largest cluster of differentially expressed genes at 0.665 Mbp in the scaffolded assembly (190 kg base pairs in the reference contig) revealed that a putative dadh was up-regulated by 2568-fold in response to dopamine. (B) Tungstate treatment inhibits dehydroxylation of dopamine by E. lenta A2. Cultures were grown anaerobically with tungstate (WO4^2–) or molybdate (MoO4^2–) for 48 hours with 0.5 mM dopamine. Bar graphs represent the mean ± SEM of three biological replicates. (C) In vitro activity of Dadh-containing fractions purified from E. lenta A2. Extracted LC-MS/MS ion chromatograms for simultaneous detection of dopamine and m-tyramine after 12 hours of anaerobic incubation of enzyme preparation with 500 mM dopamine and artificial electron donors at room temperature. Peak heights show the relative intensity of each mass, and all chromatograms are shown on the same scale. (D) A single amino acid variant predicts dopamine metabolism by E. lenta and related strains (P = 0.013 Fisher’s exact test) and does not correlate with phylogeny. Strains were cultured anaerobically with 500 mM dopamine for 48 hours (El, E. lenta; Es, Eggerthella sinensis; Gs, Gordonibacter sp.; and Gp, Gordonibacter pamelaeae; Ph, Paraeggerthella hongkongensis). High (100% conversion) and low (<11% conversion) metabolizers are denoted in red and blue. For each strain, data points represent biological replicates (P *< 0.05 analysis of variance with Dunnett’s test versus sterile controls).

Fig. 3

E. faecalis and E. lenta Dadh predict L-dopa metabolism in complex human gut microbiotas. (A) Metabolismof L-dopa by cocultures of E. faecalis and E. lenta strains cocultured for 48 hours with 1mMd3-phenyl-L-dopa or 1 mM dopamine. Results are mean ± SEM (n = 3 replicates). (B) Metabolism of d3-phenyl-L-dopa by 19 unrelated human gut microbiota samples ex vivo. Samples were cultured anaerobically with d3-phenyl-L-dopa (1 mM) for 72 hours. Results are mean concentration ± SEM (n = 3 replicates). (C) The abundance of tyrDC predicts L-dopa decarboxylation in human gut microbiota samples. Data represent the average tyrDC abundance (as assessed with qPCR) across the three replicates for samples in (B). Results are mean ± SEM (****P < 0.0001, one tailed Mann-Whitney test). (D) The abundance of E. faecalis (as assessed with qPCR) predicts L-dopa decarboxylation in human gut microbiota samples. Each data point is the average abundance across three biological replicates for each sample shown in (B). Results are mean ± SEM (****P < 0.0001, one-tailed Mann-Whitney test). (E) Dopamine dehydroxylation by gut microbiota samples of 15 unrelated individuals. Samples were cultured for 48 hours with 0.5 mM dopamine. Bars are mean ± SEM of n = 6 for low reducers (<50%) and n = 9 for high reducers (>50%) (*** P = 0.0002, one tailed Mann-Whitney test). (F) Dadh abundance does not correlate with dehydroxylation by human gut microbiotas. Data represent qPCR with Dadh-specific primers. Each data point is the dadh abundance in each sample shown in (E). Bars represent the mean and SE. (G) Dadh sequence variants predict dopamine dehydroxylation ex vivo. Full-length dadh from each culture in (E) was sequenced by using primers specific for the region containing position 506. Samples in which a mix of variants were present (n = 5) were removed. Bars represent the mean and SEM [n = 3 for samples encoding the Arg506 Dadh variant, n = 7 for samples encoding the Ser506 Dadh variant., n = 3 for DSM2243, and n = 3 for A2] (** P = 0.0083, one-tailed Mann-Whitney test, CGC samples versus AGC samples).

Fig. 4

L-dopa decarboxylation by E. faecalis is inhibited by AFMT but not the host-targeted drug carbidopa. (A) Carbidopa and AFMT. (B) Carbidopa preferentially inhibits human AADC over TyrDC. AADC or TyrDC were incubated with inhibitor, and reaction rates were measured with LC-MS/MS. “% Activity” represents the rate relative to a no inhibitor (vehicle) control. Results are mean ± SEM (n = 3 replicates). (C) Activity of carbidopa and AFMT in cultures of E. faecalis grown for 16 hours anaerobically with 0.5 mM L-dopa. Error bars represent the mean ± SEM for three biological replicates. (D) Activity of carbidopa in a human fecal microbiota from a Parkinson’s patient. The sample was cultured anaerobically with carbidopa and 1 mM d₃-phenyl-L-dopa for 72 hours. Error bars represent the mean ± SEM for three biological replicates. (E) AFMT preferentially inhibits TyrDC over AADC in vitro. AADC or TyrDC were incubated with inhibitor, and reaction rates were measured with LC-MS/MS. “% Activity” represents the rate relative to a no inhibitor (vehicle) control. Error bars represent the mean ± SEM for three biological replicates. (F) Detection of an AFMT-PLP covalent adduct after incubation of TyrDC or AADC with AFMT for 1 hour. The data shown is the extracted ion chromatogram of the mass of the predicted covalent adduct. (G) Action of AFMT in human fecal microbiotas from Parkinson’s patients incubated anaerobically with AFMT and 1 mM d₃-phenyl-L-dopa for 72 hours. Error bars represent the mean ± SEM for three biological replicates. (H) Pharmacokinetic analysis in gnotobiotic mice colonized with E. faecalis and given L-dopa + carbidopa + AFMT demonstrates higher serum L-dopa relative to vehicle controls. Error bars represent the mean ± SEM. (I) The maximum serum concentration (C_max) of L-dopa is significantly higher with AFMT relative to vehicle controls. In (H) and (I), *P < 0.05, Mann-Whitney U test; n = 4 to 5 mice per group.