Discovery and Inhibition of an Interspecific Intestinal Bacterial Pathway for Levodopa Metabolism



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The dope on lmetabolism of dopa

The effectiveness of lDopa treatment for Parkinson's disease varies greatly from one individual to another, depending on the composition of their microbiota. l-Dopa is decarboxylated to active dopamine, but if the intestinal microbiota is metabolized l-dopa before crossing the blood-brain barrier, the drugs are ineffective. Maini Rekdal et al. found that different species of bacteria are involved in lmetabolism of dopa (see O Neill's Perspective). Tyrosine decarboxylase (TDC) from Enterococcus faecalis and dopamine dehydroxylase (Dadh) from Eggerthella lenta A2 metabolized sequentially l-dopa en m-tyramine. The microbial l-dopa decarboxylase can be inactivated byS) -α-fluoromethyltyrosine (AFMT), which indicates the possibilities of developing combinations of Parkinson's drugs to circumvent microbial inactivation.

Science, this number p. eaau6323; see also p. 1030

Structured abstract

INTRODUCTION

Parkinson's disease is a debilitating neurological condition that affects more than 1% of the world's population aged 60 and over. The main drug used to treat Parkinson's disease is levodopa (l-dopa). To be efficient, l-dopa must enter the brain and be converted into dopamine neurotransmitter by the human enzyme, aromatic amino acid decarboxylase (AADC). However, the gastrointestinal tract is also a major site for l-dopa decarboxylation, and this metabolism is problematic because dopamine generated at the periphery can not cross the blood-brain barrier and causes undesirable side effects. So, l-dopa is co-administered with drugs that block peripheral metabolism, including carbidopa, an inhibitor of AADC. Even with these drugs, up to 56% of l-dopa can not reach the brain. In addition, the effectiveness and side effects of lDopa treatment is extremely heterogeneous in patients with Parkinson's, and this variability can not be fully explained by differences in host metabolism.

REASONING

Previous studies in humans and animal models have shown that gut microbiota can metabolize l-dopa. The proposed main route involves an initial decarboxylation of l-dopa to dopamine, followed by conversion of dopamine to m-tyramine by means of a distinctly microbial dehydroxylation reaction. Although these metabolic activities were found in complex intestinal microbiota samples, the specific organisms, the gene, and the responsible enzymes were unknown. The effects of host inhibitors, such as carbidopa, on microbial intestinal bacteria lThe metabolism of dopa was not clear either. As a first step towards understanding the effect of the gut microbiota on the treatment of Parkinson's disease, we sought to elucidate the molecular basis of the gut microbiota. lmetabolism of dopamine and dopamine.

RESULTS

Make the assumption that ldecarboxylation -dopa would require a pyridoxal phosphate-dependent enzyme (PLP), we searched for bacterial genomes of the intestine for candidates and identified a conserved tyrosine decarboxylase (TyrDC) in Enterococcus faecalis. Genetic and biochemical experiments revealed that TyrDC simultaneously decarboxylates both l-dopa and its preferred substrate, tyrosine. Then, we used the enrichment culture to isolate a strain of dehydroxylation of dopamine. Eggerthella lenta, a species previously involved in drug metabolism. The transcriptomic bound this activity to a dopamine dehydroxylase (Dadh) enzyme dependent on a molybdenum cofactor. Unexpectedly, the presence of this enzyme in the bacterial genomes of the intestine did not correlate with the metabolism of dopamine; Instead, we identified a single-nucleotide polymorphism (SNP) in the dad gene that predicts the activity. The abundance of E. faecalis, tyrDCand the different SNPs of dad correlated with lDopamine and dopamine metabolism in complex intestinal microbiota of Parkinson's patients, indicating that these organisms, genes, enzymes and even nucleotides are relevant in this context.

We then tested whether carbidopa, an AADC inhibitor targeting the host, affected l-dopa decarboxylation by E. faecalis TyrDC. Carbidopa showed significantly reduced potency against bacteria and was totally ineffective in the complex intestinal microbiota of Parkinson's patients, suggesting that this drug probably does not prevent the lmetabolism of dopa in vivo. Identify a selective inhibitor of intestinal bacteria ldecarboxylation -dopa, we exploited our molecular understanding of the microbial intestine lmetabolism of dopa. In view of TyrDC's preference for tyrosine, we looked at tyrosine mimetics and found that (S) -α-fluoromethyltyrosine (AFMT) prevented l-dopa decarboxylation by TyrDC and E. faecalis as well as complex samples of gut microbiota from Parkinson's patients. Coadministering AFMT with l-dopa and carbidopa to mice colonized with E. faecalis also increased the maximum serum concentration of l-dopa. This observation is consistent with the inhibition of intestinal microbial bacteria. lmetabolism of dopa in vivo.

CONCLUSION

We characterized an interspecific pathway for intestinal bacteria lmetabolism of dopa and has demonstrated its relevance in human intestinal microbiota. Variations in these microbial activities could possibly contribute to heterogeneous responses to l-dopa observed in patients, including reduced efficacy and adverse effects. Our results will help to better understand the contribution of the gut microbiota to treatment outcomes and to highlight the possibility of developing therapies targeting both microbial drug metabolism in the gut and intestines.

Intestinal microbes metabolize Parkinson's drug l-dopa.

Decarboxylation of l-dopa by E. faecalis TyrDC and human AADC probably limit the availability of drugs and contribute to side effects. E. lenta dehydroxylated dopamine produced from l-dopa using a molybdenum-dependent enzyme. Carbidopa, a drug targeting the host, had no effect on intestinal bacteria. lDecarboxylation -dopa, AFMT inhibited this activity in complex intestinal microbiota.

Abstract

The human gut microbiota metabolizes the drug for Parkinson's disease, levodopa (l-dopa), potentially reducing the availability of the drug and causing side effects. However, the organisms, genes and enzymes responsible for this activity in patients and their susceptibility to inhibition by drugs targeting the host are unknown. Here we describe an interspecific pathway for intestinal bacteria lmetabolism of dopa. Converting l-dopa dopamine by tyrosine decarboxylase dependent pyridoxal phosphate Enterococcus faecalis is followed by the transformation of dopamine into m-tyramine by a molybdenum-dependent dehydroxylase Eggerthella lenta. These enzymes predict drug metabolism in complex intestinal microbiota. Although a drug that targets decarboxylase, an aromatic amino acid, does not prevent intestinal microbes ldecarboxylation, we have identified a compound that inhibits this activity in the microbiota of Parkinson's patients and lbioavailability of dopa in mice.

A growing body of evidence links the trillions of microbes that populate the human gastrointestinal tract (the human gut microbiota) with neurological conditions, including the debilitating neurodegenerative disorder, Parkinson's disease (1, 2). Intestinal microbes of patients with Parkinson's disease exacerbate motor deficits when transplanted into mouse models without disease germs (2). This effect is reversed with antibiotic therapy, suggesting a causal role of intestinal microbes in neurodegeneration. Several studies have revealed differences in intestinal microbiota composition in patients with Parkinson's disease compared to healthy controls that may correlate with the severity of the disease (39). However, the influence of the human gut microbiota on the treatment of Parkinson's disease and other neurodegenerative diseases remains poorly understood.

The main treatment for Parkinson's disease is levodopa (l-dopa) (ten), which is prescribed to treat motor symptoms resulting from the loss of dopaminergic neurons in the substantia nigra. After crossing the blood-brain barrier, l-dopa is decarboxylated by the aromatic amino acid decarboxylase (AADC) to give dopamine, the active therapeutic agent. However, peripherally generated dopamine from the AADC can not cross the blood-brain barrier and only 1-5% of l-dopa reaches the brain, because of presystemic metabolism in the intestine by enzymes such as AADC (1113). Peripheral production of dopamine also causes gastrointestinal side effects, can lead to orthostatic hypotension via activation of vascular dopamine receptors and can induce cardiac arrhythmias (14, 15). To decrease the peripheral metabolism, l-dopa is coadministered with AADC inhibitors such as carbidopa. Despite this, 56% of l-dopa is metabolized at the periphery (16), and patients exhibit highly variable responses to the drug, including a loss of efficacy over time (17).

Several sources of data suggest that intestinal microbial interactions with l-dopa influences the results of treatment (18). The administration of broad-spectrum antibiotics improves l-dopa, suggesting that intestinal bacteria interfere with the effectiveness of the drug (19, 20). The gut microbiota can also metabolize l-dopa, potentially reducing its bioavailability and causing side effects (2124). The proposed main route involves an initial decarboxylation of l-dopa dopamine followed by a distinctly microbial dehydroxylation reaction that converts this neurotransmitter into m-ytyramine by selectively removing the para hydroxyl group from the catechol nucleus (Fig. 1A) (25, 26). When we began our work, the intestinal microbial species, genes, and enzymes involved in these transformations were unknown because previous studies had examined undefined and uncharacterized consortia. The clinical relevance of this route was also unclear, given the potential effects of co-administered inhibitors of peripheral host disease. lmetabolism -dopa on these intestinal microbial activities.

Fig. 1 E. faecalis metabolizes l-dopa using a PLP-dependent tyrosine decarboxylase.

(A) Main path proposed for ldopa metabolism by human gut microbiota and potential for interaction with host-targeted drugs. (B) Phylogenetic distribution of TyrDC in the human microbiota. The reference genomes of the Human Microbiome Project were queried using BLASTP to obtain counterparts from the L. brevis TyrDC, and the results are visualized on a cladogram of phylogeny[basésur16[basedon16[basésur16[basedon16S alignment of ribosomal RNA (rRNA)]. TyrDC homologues sporadically found in lactobacilli spp. (kg) are widely distributed among enterococcus (Ec; average amino acid identity 67.8% over a query length of 97.6%). (C) The representative intestinal microbial strains test for TyrDC reveals that E. faecalis reproducible strains converted l-dopa to dopamine. The strains were cultured for 48 hours under anaerobic conditions. Bar graphs represent the mean ± SEM of three biological replicates. (re) 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. The 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.

Human intestinal bacteria Enterococcus faecalis décarboxylates l-dopa

We sought to elucidate the genetic and biochemical bases of the gut microbiota. ldopa metabolism and understand how coadministered AADC inhibitors affect this pathway. Using a genome extraction approach, we first identified the strains that code for candidate genes. l-dopa decarboxylating enzymes. The decarboxylation of aromatic amino acids is generally performed by enzymes using pyridoxal-5'-phosphate (PLP), an organic cofactor providing an electron collector (27). PLP-dependent tyrosine decarboxylase (TyrDC) derived from the food-related strain Lactobacillus brevis It has been shown that CGMCC 1.2028 has an activity close to l-dopa in vitro (28). To locate TyrDC homologs in human intestinal bacteria, we performed a search for BLASTP (Local Protein Alignment Tool) on all available Human Microbiome Project (HMP) reference genomes. National Biotechnology Center (NCBI). The majority of the hits were found in the neighboring genre enterococcuswith some lesions in lactobacilli and proteobacteria (Fig. 1B, Fig. S1 and S1 data file). We selected 10 representative intestinal strains containing TyrDC homologs (29-100% amino acids) and examined their ability to decarboxylate. l-dopa in anaerobic culture. Although both Enterococcus faecalis and Enterococcus faecium displayed activity, only E. faecalis showed complete decarboxylation among all the strains tested (Figure 1C). All E. faecalis the strains tested share the same highly conserved gene tyrDC operon (Fig S2), and we found tyrDC in 98.4% of E. faecalis NCBI-deposited assemblies with a median amino acid identity of 99.8 (range 97.0 to 100). This high degree of sequence and prevalence conservation is consistent with the fact that decarboxylation of tyrosine is a common phenotypic trait of E. faecalis (29). We therefore chose this predominant and genetically treatable intestinal organism as a characterization model l-dopa decarboxylation (30).

Well lyophilized E. faecalis cell decarboxylate l-dopa (31) and the tyrDC the role of the operon in the decarboxylation of tyrosine E. faecalis is well characterized (32), the link between tyrDC and ldecarboxylation -dopa was unknown. We used in vitro genetic and biochemistry experiments to confirm that TyrDC is necessary and sufficient to l-dopa decarboxylation by E. faecalis. E. faecalis MMH594 mutants carrying a disruption of the 2 kb Tet cassette tyrDC could not decarboxylate l-dopa (Fig. 1D and Fig. S3) and showed no growth defect compared to the wild type (Fig. S4). The in vitro characterization of TyrDC revealed a catalytic efficiency five times higher with respect to L-tyrosine compared to l-dopa, suggesting that drug metabolism results from promiscuous enzymatic activity (Fig 1E, Fig S5 and Table S1). This selectivity contrasts sharply with that of AADC, which displays very low activity with respect to L-tyrosine (33). Although TyrDC's E. faecalis decarboxylate tyrosine and phenylalanine (3437), his ability to accept l-dopa had not been demonstrated. A recent independent report also corroborates this conclusion (38).

We then tested whether tyrosine, which is TyrDC's preferred substrate and is present in the small intestine, could interfere with l-dopa decarboxylation by E. faecalis (39, 40). In competition, purified TyrDC (Fig S6) and anaerobic E. faecalis decarboxylated cultures l-dopa and tyrosine simultaneously (tyrosine 500 μM, approximating the concentration of the small intestine at rest) (Fig. 1F and Fig. S7) (40). This observation contrasts sharply with previous research on phenylalanine metabolized by E. faecalis only when tyrosine is completely consumed (36). Simultaneous decarboxylation of l-dopa and tyrosine also occurred at E. faecalis MMH594 cultures with higher tyrosine concentrations (1.5 mM, approaching concentration in the small intestine after meal) (Figure S8) and in three human stool suspensions (Figure S9). As observed previously for tyrosine, lDecarboxylation of -dopa was produced more rapidly at a low pH among all the strains tested (Figs S7 and S8), suggesting that this metabolism is probably accelerated to the lower pH of the upper small intestine. (41, 42). Because the constant Michaelis (Km) from TyrDC for l-dopa (1.5 mM) is less than the estimated maximum in vivo of the small intestine leven at its lowest clinically administered dose (5 mM), these data strongly suggest that peripheral decarboxylation is performed by both bacterial enzymes from the host and the intestine.

Eggerthella lenta dopamine dehydroxylates using a molybdenum-dependent enzyme

Having identified an intestinal bacteria l-dopa decarboxylase, we then examined the conversion of dopamine into m-tyramine because this activity can affect peripheral adverse effects. l-dopa decarboxylation. E. faecalis did not further metabolize dopamine, indicating that this step was performed by another microorganism. Dopamine dehydroxylation was not reported for any bacterial isolates and a screening of 18 human intestinal strains did not reveal metabolizers. Therefore, we used the enrichment culture to obtain a dopamine-dehydroxylating organism. Recognizing the chemical parallels between this reductive dehydroxylation and this reductive dehalogenation of chlorinated aromatics, which allows the anaerobic respiration of certain bacteria (43), we inoculated a stool sample from a human donor into a minimal growth medium containing 0.5 mM dopamine as the sole electron acceptor (Figures S10 and S11). Transition over several generations enriched in active strains, evaluated by a colorimetric test for dehydroxylation of catechol (Fig. S11). This effort allowed to identify a strain of Actinobacterium intestine Eggerthella lenta (hereinafter referred to as strain A2) capable of selectively removing the para hydroxyl group from dopamine to give m-tyramine (Fig S12). Because E. lenta Digoxin, a drug of cardiac origin, is also inactivated; our results suggest a more important role of this digestive organism in the metabolism of the drug (44, 45).

Catechol dehydroxylation is a difficult chemical reaction that has no equivalent in synthetic chemistry and probably involves an unusual enzymology. To identify the dehydroxylating enzyme of dopamine, we first studied the E. lenta Genome A2 for the genes encoding homologs of the only aromatic compound characterized para-dehydroxylase, 4-hydroxybenzoyl-CoA reductase (46), but found no results. Tests with E. lenta The A2 cell lysates exhibited dopamine dehydroxylation that required anaerobic conditions and were induced by dopamine (Fig. S13). So we used the RNA sequencing of E. lenta A2 to identify dehydroxylase. This experiment revealed> 2500-fold up-regulation of three colocalized genes in response to dopamine (Fig 2A and Table S2). These genes encode a (moco) -cofactor (moco) -benzyme cofactor belonging to the predicted dimethylsulfoxide reductase family of bis-molybdopterin guanine dinucleotide. Moco-dependent enzymes catalyze a wide variety of oxygen transfer reactions, but these have not been shown to catalyze the dehydroxylation of catechol in vitro (47). We therefore hypothesized that this enzyme was a dopamine dehydroxylase (Dadh).

Fig. 2 E. lenta dopamine dehydroxylates using a molybdenum-dependent enzyme.

(A) RNA sequencing identifies a molybdenum-dependent dopamine dehydroxylase (Dadh) (moco) in E. lenta A2. Candidate genes expressed differentially (false discovery rate) < 0.1 and fold change > |2|) are plotted against the position of the genome, revealing three distinct loci of differentially expressed genes. (Box) Analysis of the largest group of genes differentially expressed at 0.665 Mbp in the scaffold assembly (190 kg base pair in the reference file) revealed dad was upregulated by 2568 times in response to dopamine. (B) Tungstate treatment inhibits the dehydroxylation of dopamine by E. lenta A2. The cultures were grown anaerobically with tungstate (WO42-) or molybdate (MoO42-) for 48 hours with 0.5 mM dopamine. Bar graphs represent the mean ± SEM of three biological replicates. (C) In vitro activity of fractions containing Dadh purified from E. lenta A2. Chromatograms on extracted LC-MS / MS ions for the simultaneous detection of dopamine and m-tyramine after 12 hours anaerobic incubation of an enzyme preparation with donors 500 μM dopamine and artificial electrons at room temperature. The peak heights show the relative intensity of each mass and all the chromatograms are represented on the same scale. (re) A single amino acid variant predicts the metabolism of dopamine in E. lenta and related strains (P = 0.013 Fisher's exact test) and does not correspond to phylogeny. Strains were cultured anaerobically with 500 μM dopamine for 48 hours (El, E. lenta; are, Eggerthella sinensis; gs, Gordonibacter sp. and Gp, Gordonibacter pamelaeae; Ph, Paraeggerthella hongkongensis). The high (100% conversion) and low (<11% conversion) metabolizers are indicated in red and blue. For each strain, the data points represent biological replicates (*P <0.05 analysis of variance with Dunnett's test versus sterile controls).

To evaluate the role of Dadh in the dehydroxylation of dopamine, we first examined whether this activity was dependent on molybdenum by growing E. lenta A2 in the presence of tungstate. The substitution of molybdate by tungstate during the biosynthesis of moco generates an inactive metallocofactor (Figure S14) (48). Treat the cultures of E. lenta Dehydroxylation of dopamine was inhibited by A2 with tungstate without affecting growth (Fig 2B and Fig S15), while incubation of cell lysates with tungstate had no effect, which is compatible with inhibition requiring active moco biosynthesis (Figure S16). We then confirmed the activity of Dadh in vitro. The heterologous expression of more than 20 constructs in multiple hosts failed to provide an active enzyme, prompting us to continue native purification. Anaerobic Activity – Guided Fractionation E. lenta The A2 cell lysates gave a dopamine dehydroxylation fraction containing four proteins, evaluated by SDS-polyacrylamide gel electrophoresis (Fig. 2C, Fig. S17 and Table S3). The dehydroxylation activity is correlated to a 115 kDa band confirmed by mass spectrometry as Dadh. Dadh was the only isolated protein that was positively regulated in the presence of dopamine (Tables S2 and S3). Together, these data strongly support the attribution of this enzyme.

We then assessed whether the presence of dad in microbial genomes correlated with the dehydroxylation of dopamine. A BLASTP search revealed that this enzyme is restricted to E. lenta and its close relatives Actinobacterial (Table S4), inviting us to examine a collection of 26 intestinal isolates of Actinobacterial (49) for their ability to dehydroxylate dopamine in anaerobic culture. Although Dadh appeared to be encoded by 24 of 26 strains (92-100% amino acid ID) (Fig S18 and Table S5), only 10 Eggerthella strains converted quantitatively into dopamine m-tyramine, with a weak metabolism (<11%) or zero in the others (Fig. 2D). This variability of dopamine metabolism at the strain level reinforces the fact that the identity of intestinal microbial species is often not predictive of metabolic functions (49, 50).

To better understand this variation, we first performed RNA sequencing experiments with metabolic methods (E. lenta 28B) and non-metabolizing (E. lenta DSM2243) in the presence and in the absence of dopamine. surprisingly, dad was upregulated in response to dopamine in both strains, indicating that lack of activity in E. lenta DSM2243 did not result from transcription differences (S6 and S7 tables). By aligning the protein sequences of Dadh, we have instead found a single amino acid substitution that almost perfectly predicts the metaboliser's status: position 506 is an arginine in the metabolizing strains and a serine in the inactive strains (Fig. 2D and Fig. S19). This change results from a single nucleotide polymorphism (SNP) at dad. The only exception, E. lenta W1BHI6, has arg506 variant and an additional substitution nearby (Cys500) (Fig. S19). Thus, amino acid residues in the enzyme Dadh, rather than the presence or transcription of dadpredict the dehydroxylation of dopamine among strains of intestinal bacteria. The variants of Dadh do not correlate with E. lenta phylogeny (Fig 2D), suggesting that this activity has been gained and / or lost several times.

E. faecalis and E. lenta metabolize l-dopa in human intestinal microbiota

After identifying the organisms and enzymes that perform the different stages of the l-dopa pathway, we then tested if E. faecalis and E. lenta generated m-tyramine in coculture. Wild type E. faecalis grown up with E. lenta A2 (Arg506) fully converted l-dopa to m-tyramine (Fig. 3A). Although a coculture containing the E. faecalis tyrDC mutant could not consume l-dopa, m-tyramine was produced when exogenous dopamine was added to this culture, revealing that E. lenta A2 was still metabolically active. Wild type incubation E. feacalis with the non-metabolizing E. lenta The strain DSM2243 (Ser506) only produced dopamine, indicating that this variant of Dadh is also inactive in a coculture environment (FIG. 3A).

Fig. 3 E. faecalis and E. lenta Dad predict ldopa metabolism in the complex microbiota of the human intestine.

(A) Metabolism of l-dopa by cocultures of E. faecalis and E. lenta strains co-cultured for 48 hours with 1 mM re3phenyll-dopa or 1 mM dopamine. The results are on average ± SEM (not = 3 replicates). (B) Metabolism of re3phenyll-dopa by 19 ex vivo samples of unrelated human intestinal microbiota. The samples were grown anaerobically with re3phenyll-dopa (1 mM) for 72 hours. The results are the mean concentration ± SEM (not = 3 replicates). (C) The abundance of tyrDC predicted l-dopa decarboxylation in samples of human intestinal microbiota. Data is the average tyrDC abundance (evaluated with qPCR) on the three replicates for (B) samples. The results are on average ± SEM (****P <0.0001, single-tailed Mann-Whitney test). (re) The abundance of E. faecalis (evaluated with qPCR) predicts l-dopa decarboxylation in samples of human intestinal microbiota. Each data point is the average abundance over three biological replicates for each sample presented in (B). The results are on average ± SEM (****P <0.0001, unilateral Mann-Whitney test). (E) Dehydroxylation of dopamine by gut microbiota samples from unrelated individuals. The samples were cultured for 48 hours with 0.5 mM dopamine. The bars are average ± SEM of not = 6 for low reducing agents (<50%) and not = 9 for high reducers (> 50%) (*** P = 0.0002, single-tailed Mann-Whitney test). (F) Dad abundance does not correspond to dehydroxylation by human intestinal microbiota. Les données représentent qPCR avec Papaamorces spécifiques. Chaque point de données est le dad abondance dans chaque échantillon indiqué en (E). Les barres représentent la moyenne et SE. (g) Les variants de séquence de Dadh prédisent la déshydroxylation de la dopamine ex vivo. Toute la longueur dad (E) a été séquence en utilisant des amorces spécifiques de la région contenant la position 506. Des échantillons dans lesquels un mélange de variants étaient présents (not = 5) ont été supprimés. Les barres représentent la moyenne et SEM[[[[not = 3 pour les échantillons codant pour la variante Arg506 Dadh, not = 7 pour les échantillons codant pour la variante Ser506 Dadh., not = 3 pour DSM2243 et not = 3 pour A2](** P = 0,0083, test de Mann-Whitney unilatéral, échantillons CGC versus échantillons AGC).

Pour rechercher si E. faecalis and E. lenta transform l-dopa dans le microbiote intestinal humain, nous avons évalué le métabolisme de l-dopa par suspensions fécales ex vivo. Alors que 7 des 19 échantillons ne montraient pas d&#39;épuisement détectable de l-dopa, les échantillons restants présentaient une variabilité importante du métabolisme, allant de la conversion partielle (25%) à la conversion presque complète (98%) l-dopa à m-la tyramine (Fig. 3B). Nous avons ensuite demandé si l’abondance de tyrDC métabolisme prévu dans ces échantillons. Dénombrement quantitatif par réaction en chaîne de la polymérase (qPCR) de tyrDC (51) et E. faecalis échantillons différenciés métabolisant et non métabolisant (P <0,0001, test de Mann-Whitney unilatéral) (Fig. 3, C et D). Par contre, E. lenta l&#39;abondance n&#39;a montré aucune association avec lmétabolisme de la dopa (fig. S20). Nous avons trouvé une forte corrélation linéaire entre tyrDC l&#39;abondance et E. faecalis abundance[coefficientdedétermination([coefficientofdetermination([coefficientdedétermination([coefficientofdetermination(R2) = 0,99, P <0.0001](fig. S21), ce qui reflète probablement la haute conservation des tyrDC in E. faecalis génomes. Ces données suggèrent également que E. faecalis est le microorganisme dominant responsable de l-dopa décarboxylation dans ces communautés microbiennes intestinales complexes. Conforme à cela, E. faecalis l&#39;abondance significativement corrélée avec tyrDC l’abondance dans 1870 microbiomes intestinaux humains (R2 > 0,812, P <2.2 × 10–16Corrélation de Pearson) (fig. S22).

Pour confirmer que E. faecalis pourrait décarboxyler lDans les microbiotes complexes intestinaux, nous avons ajouté cet organisme aux échantillons non métabolisants. Bien que l&#39;introduction de la tyrDCsouche déficiente n&#39;a pas changé lniveaux de dopamine, y compris la souche de type sauvage, ont conduit à l’épuisement complet du l-dopa (fig. S23, B à E). Dans certains échantillons, ajout de caractères sauvages E. faecalis était suffisante pour produire une production quantitative de m-la tyramine, indiquant la présence d’organismes déodroxylants de la dopamine dans ces communautés (fig. S23, B et D). Enfin, l&#39;ajout du type sauvage E. faecalis et la souche métabolisante E. lenta A2 aux échantillons non métabolisables ou à l&#39;ajout de E. lenta A2 seul à un échantillon de décarboxylation généré m-tyramine (fig. S23, A et C à E). Prises ensemble, ces données indiquent que l’abondance de E. faecalis et son encodé tyrDC prédit la variation interindividuelle considérable dans lmétabolisme de la dopa observé dans des échantillons complexes de microbiote intestinal humain.

Comme prévu dans nos expériences précédentes, ni l’abondance de E. lenta ni dad déshydroxylation de la dopamine prévue dans les communautés microbiennes intestinales complexes (Fig. 3, E et F, et fig. S24). Cependant, quand nous avons amplifié dad à partir de ces cultures et déterminé le statut SNP en position 506, nous avons trouvé des échantillons contenant le Arg506 variant quantitativement dopé dopamine, alors que l&#39;activité des échantillons qui ont porté le Ser506 variante ne pouvait pas être distinguée de la non métabolisante E. lenta Souche DSM2243 (Fig. 3G). Ces résultats indiquent qu&#39;un seul résidu d&#39;acide aminé dans une enzyme microbienne intestinale prédit le métabolisme de la dopamine dans les communautés complexes. Étant donné que dad prévalence élevée (> 70%) dans les microbiomes intestinaux de sujets humains et dans les deux dad Des variants sont présents dans cette population (fig. S22 et S25), nous supposons que les SNP pourraient influencer le métabolisme xénobiotique dans le contexte du génome de l&#39;hôte (52) et le microbiome intestinal humain (53).

Pour approfondir la pertinence clinique de nos résultats, nous avons évalué le métabolisme de la dopamine et l-dopa par des suspensions fécales de patients atteints de la maladie de Parkinson ex vivo. Comme chez les sujets témoins, ces individus présentaient une variabilité importante du métabolisme de l-dopa (fig. S26A). Les dosages qPCR ont révélé que tyrDC l&#39;abondance et E. faecalis l&#39;abondance discriminée entre l-dopa décarboxylant et non décarboxylant des échantillons (P <0,005, test de Mann-Whitney unilatéral) (fig. S26, C et D). Nous avons également observé une diminution de l-dopa sans production correspondante de dopamine ou m-tyramine dans trois échantillons (fig. S26A). Instead of, l-dopa a été convertie en acide hydroxyphénylpropionique (fig. S26B), voie considérée comme apportant une contribution mineure au métabolisme du médicament in vivo (22, 25, 26). Enfin, nous avons constaté que le dad La SNP prédit la déshydroxylation de la dopamine dans ces échantillons (fig. S27). Dans l’ensemble, ces données confirment le rôle des bactéries intestinales dans la grande variabilité interindividuelle des ldécarboxylation -dopa observée chez des patients atteints de Parkinson (13). Une étude récente a rapporté que les selles tyrDC l&#39;abondance est positivement corrélée avec l-dopa chez les patients (38) mais n&#39;a pas démontré de lien entre tyrDC and l-dopa décarboxylation dans ces échantillons. Nos résultats indiquent que cette activité métabolique peut en effet affecter l-dopa efficacité thérapeutique.

(S) -α-Fluorométhyltyrosine (AFMT) inhibe les microbes intestinaux lmétabolisme de la dopa

Ayant montré que E. faecalis and E. lenta les enzymes prédisent lmétabolisme de la dopa par des microbiotes complexes intestinaux de patients, nous avons ensuite examiné si cette voie interspécifique était susceptible d&#39;être inhibée par des médicaments l-dopa décarboxylation. Aux États-Unis, les patients atteints de Parkinson sont une carbidopa co-prescrite (Fig. 4A), une l-dopa imitante qui inhibe l&#39;AADC en formant une liaison hydrazone covalente et stable avec son cofacteur PLP (54). Nous avons trouvé que la carbidopa était 200 fois moins active vis-à-vis de la purification. E. faecalis TyrDC[concentrationinhibitricemoitiémaximale(IC[halfmaximalinhibitoryconcentration(IC[concentrationinhibitricemoitiémaximale(IC[halfmaximalinhibitoryconcentration(IC50) = 57 μM]related to H. sapiens AADC (IC50 = 0,21 µM) et n’a montré qu’environ 50% d’inhibition de la l-dopa décarboxylation par E. faecalis cultures at the solubility limit of 2 mM (Fig. 4, B and C, and table S8), which is consistent with recently reported findings (38). Additionally, carbidopa did not affect growth of E. faecalis or metabolism or growth of E. lenta (figs. S28 to S30). Given the maximum predicted gastrointestinal concentration of carbidopa (0.4 to 9 mM), these data suggest that this drug does not fully inhibit gut bacterial l-dopa decarboxylation in Parkinson’s patients. We found that 2 mM carbidopa did not alter the kinetics of l-dopa degradation (fig. S31) or endpoint m-tyramine production in stool samples from both Parkinson’s patients and neurologically healthy controls (Fig. 4D and fig. S32). These observations support previous findings that carbidopa administration does not affect m-tyramine production in patients (55).

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 (not = 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. (re) Activity of carbidopa in a human fecal microbiota from a Parkinson’s patient. The sample was cultured anaerobically with carbidopa and 1 mM re3-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 re3-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 et donné l-dopa + carbidopa + AFMT demonstrates higher serum l-dopa relative to vehicle controls. Error bars represent the mean ± SEM. (I) The maximum serum concentration (Cmax) of l-dopa is significantly higher with AFMT relative to vehicle controls. In (H) and (I), *P < 0.05, Mann-Whitney U test; not = 4 to 5 mice per group.

Our results also highlight the possibility of therapeutically targeting gut microbial l-dopa decarboxylation to increase l-dopa efficacy. To selectively manipulate gut bacterial TyrDC in complex microbiotas, we turned to α-fluoromethyl amino acids, which are known mechanism-based inhibitors of PLP-dependent decarboxylases (33). A survey of potential amino acid substrates revealed that TyrDC requires a p-hydroxyl group for robust activity, whereas AADC prefers a m-hydroxyl substituent (fig. S33), leading us to hypothesize that the L-tyrosine analog (S)-α-fluoromethyltyrosine (AFMT) (Fig. 4A) might selectively inhibit the microbial enzyme. In vitro, AFMT strongly inhibited l-dopa decarboxylation by TyrDC (IC50 = 4.7 μM) but not AADC (~20% inhibition at solubility limit of 650 μM) (Fig. 4E and table S8). Consistent with this selectivity, AFMT formed a covalent PLP adduct only in the presence of TyrDC (Fig. 4F). AFMT was also effective in E. faecalis cultures (EC50 = 1.4 μM) (Fig. 4C), outperforming carbidopa by 1000-fold without affecting growth (table S8 and fig. S29). It also reduced l-dopa decarboxylation by cocultures of E. faecalis and E. lenta without affecting growth or metabolism of E. lenta (figs. S29, S30, and S34). Last, AFMT completely inhibited l-dopa decarboxylation in gut microbiota samples from Parkinson’s disease patients and neurologically healthy control subjects (Fig. 4G and fig. S35) and was nontoxic to eukaryotic cells (fig. S36).

To investigate AFMT activity in vivo, we administered either AFMT (25 mg/kg) or a vehicle control in combination with l-dopa (10 mg/kg) and carbidopa (30 mg/kg) to gnotobiotic mice colonized with E. faecalis MMH594 (Fig. 4H). We found that AFMT significantly increased the peak serum concentration (Cmax) of l-dopa compared with vehicle (P < 0.05, two-tailed Mann Whitney test) (Fig. 4I), which is consistent with inhibition of first-pass gut microbial metabolism in the intestine. Although we cannot rule out the possibility that AFMT modulates additional, uncharacterized targets, this observation is consistent with our in vitro inhibition data. This result also aligns with a recent report that small intestinal tyrDC abundance negatively correlates with plasma l-dopa levels in conventional rats receiving l-dopa and carbidopa (38). Overall, these data suggest that AFMT could be a promising tool compound for the study of bacterial l-dopa metabolism (56) and highlight the promise of developing l-dopa–based combination therapies containing drugs that target both host and gut microbial decarboxylation.

conclusions

We have used chemical knowledge and interdisciplinary tools to decipher the molecular mechanisms by which gut bacteria interfere with the treatment of Parkinson’s disease. The decarboxylation of l-dopa by E. faecalis mirrors host drug metabolism and, together with human AADC, likely limits drug availability and contributes to interindividual variation in efficacy. Together with recent work dissecting host and gut microbial contributions to the antiviral drug brivudine (57), our findings show that gut bacterial metabolism need not be chemically distinct from host activities to alter drug efficacy and suggest that such interactions may be underappreciated. Moreover, carbidopa’s failure to prevent l-dopa decarboxylation by E. faecalis implies that additional host-targeted drugs may lack efficacy toward activities also present in the gut microbiota. Although a recent, independent study also characterized E. faecalis TyrDC’s role in l-dopa decarboxylation and its lack of susceptibility to carbidopa (38), it did not show that this activity occurs in human gut microbiotas or identify strategies for inhibiting the bacterial enzyme. By contrast, we demonstrate that TyrDC predicts drug metabolism in Parkinson’s patient microbiotas and use an understanding of its substrate specificity to identify a small molecule that prevents l-dopa decarboxylation in patient samples and increases l-dopa bioavailability in vivo. Through discovery of predictive biomarkers for l-dopa metabolism and identification of an inhibitor of this activity, this work will enable efforts to elucidate the contribution of the gut microbiota to drug availability, patient drug response, and treatment outcomes.

We also show that E. lenta further metabolizes the dopamine produced by l-dopa decarboxylation using a distinctly microbial reaction, catechol dehydroxylation. It is possible that this transformation influences the multiple side effects of l-dopa administration linked to dopamine production. This discovery also raises questions about the biological consequences of gut microbial metabolism of endogenous dopamine, which is present in the gastrointestinal tract and has been linked to phenotypes ranging from gut motility to pathogen colonization (5860). The biological activity of the gut microbial metabolite m-tyramine in the host and the benefits of this metabolism for E. lenta are also poorly understood. Our findings will enable further study of these phenomena. Given that gut microbes dehydroxylate catechol groups found in numerous aromatic drugs and dietary compounds (18, 6163), the discovery of Dadh will enable identification of additional catechol dehydroxylases and help to elucidate the biological role of this enigmatic transformation. Uncovering the unexpected effect of SNPs on gut microbial dopamine metabolism suggests that simply detecting functional genes may not accurately predict the activities encoded by the human gut microbiome and underscores the importance of studying enzymes from this community.

Materials and methods summary

Our methods for the identification and biochemical characterization of E. faecalis TyrDC; characterization of anaerobic l-dopa metabolism by E. faecalis and gut microbiota samples; enrichment culturing for dopamine dehydroxylating organisms; RNA-sequencing; culture-based assays; purification of Dadh; assays of anaerobic dopamine metabolism by Actinobacteria and complex gut microbiota samples; PCR and qPCR assays; liquid chromatography–MS (LC-MS) methods; and assays for evaluating inhibitors in vitro, ex vivo, and in vivo are provided in the supplementary materials. Additional information about our protocols, including references to the supplementary materials, can be found throughout the main text.

Acknowledgments: We acknowledge the Broad Institute Microbial Omics Core (MOC) for assistance with RNA and 16S rRNA gene sequencing analysis and experimental design, the Harvard Bauer Core Proteomics facility for assistance with proteomics, M. Wilson (Harvard University) for helpful discussions and input, M. Gilmore and E. Selleck (Massachusetts Eye and Ear, Harvard Medical School, and Broad Institute) for supplying the E. faecalis tyrDC mutant and helpful discussions, F. Lebreton (Massachusetts Eye and Ear and Harvard Medical School) for supplying E. faecalis and E. faecium strains, R. Nayak [University of California, San Francisco (UCSF)] and M. Krueger (UCSF) for help with sample collection from healthy control subjects, and the Biocollective for collection and provision of stool samples from Parkinson’s patients. We thank Merck for the gift of AFMT. Funding: This work was supported by the Packard Fellowship for Science and Engineering (2013-39267) (E.P.B), the Howard Hughes Medical Institute (HHMI)–Gates Faculty Scholars Program (OPP1158186) (E.P.B), the National Institutes of Health (R01HL122593) (P.J.T.), the Searle Scholars Program (SSP-2016-1352) (P.J.T), the UCSF-Stanford Arthritis Center of Excellence (supported in part by the Arthritis Foundation) (P.J.T), and the Rheumatology Research Foundation (P.J.T.). V.M.R. is the recipient of a National Science Foundation Graduate Research Fellowship, a Gilliam Fellowship from HHMI, and the Ardis and Robert James Graduate Research Fellowship from Harvard University and acknowledges support from a National Institutes of Health Training Grant (5T32GM007598-38). E.N.B. is a Howard Hughes Medical Institute fellow of the Life Sciences Research Foundation. J.E.B. has fellowship support from the Natural Sciences and Engineering Research Council of Canada. P.J.T. is a Chan Zuckerberg Biohub investigator and a Nadia’s Gift Foundation Innovator supported, in part, by the Damon Runyon Cancer Research Foundation (DRR-42-16). Author contributions: V.M.R. and E.P.B. conceived of the project. V.M.R. purified and characterized all proteins biochemically with and without inhibitors, performed assays for l-dopa and dopamine metabolism in pure cultures and complex microbiotas in the presence and absence of inhibitors, performed qPCR experiments and analysis, and performed RNA-sequencing experiments in E. lenta A2 and E. lenta 28B. E.N.B. performed RNA-sequencing experiments in E. lenta DSM2243, performed assays for dopamine metabolism across the Actinobacterial library, and contributed to the design of the ex vivo experiments and other culture-based assays. J.E.B. performed RNA sequencing analysis, comparative genomics, and metagenomic analysis and contributed to the design of and conducted in vivo AFMT experiments. V.M.R., E.P.B., E.N.B., J.E.B., and P.J.T. provided critical feedback on experiments. V.M.R., J.E.B., P.J.T., and E.P.B. wrote the manuscript. Competing interests: E.P.B. has consulted for Merck, Novartis, and Kintai Therapeutics; is on the Scientific Advisory Boards of Kintai Therapeutics and Caribou Biosciences; and is an Associate Member of the Broad Institute of Harvard and MIT. P.J.T. is on the scientific advisory board for Kaleido, Seres, uBiome, and WholeBiome. Data and materials availability: the E. lenta A2 genome has been deposited into GenBank (PRJNA412637). RNA-sequencing data has been deposited into the Sequence Read Archive available by way of BioProject PRJNA507796. The small-molecule AFMT was obtained under a materials transfer agreement with Merck.

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