Gut bacterial tyrosine decarboxylases restrict the bioavailability of levodopa, the primary treatment in Parkinson’s disease

Human gut bacteria play a critical role in the regulation of immune and metabolic systems, as well as in the function of the nervous system. The microbiota senses its environment and responds by releasing metabolites, some of which are key regulators of human health and disease. In this study, we identify and characterize gut-associated bacteria in their ability to decarboxylate L-DOPA (also known as Levodopa or L-3,4-dihydroxyphenylalanine) to dopamine via the tyrosine decarboxylases, which are mainly present in the class Bacilli. Although the bacterial tyrosine decarboxylases have a higher affinity for tyrosine compared to L-DOPA, this does not affect their ability to decarboxylate L-DOPA, nor does any inhibitor of the human decarboxylase. This study indicates that in situ bioavailability of L-DOPA is compromised by the gut bacterial tyrosine decarboxylase abundance in Parkinson’s patients. Finally, we show that the tyrosine decarboxylase abundance in the microbiota at the site of L-DOPA absorption, the proximal small intestine, significantly influences L-DOPA bioavailability in the plasma of rats. Our results highlight the role of microbial metabolism in drug bioavailability, and specifically, that small intestinal abundance of bacterial tyrosine decarboxylase can explain the highly variable L-DOPA dosage regimens required in the treatment of individual Parkinson’s patients. Highlights Small intestinal bacteria is able to convert L-DOPA to dopamine L-DOPA metabolism by gut bacteria reduce the bioavailability of L-DOPA in the body, thus is a significant explanatory factor of the highly variable L-DOPA dosage regimens required in the treatment of individual Parkinson’s patients. Inhibitors of the human DOPA decarboxylase are not potent inhibitors for bacterial tyrosine decarboxylases


Introduction
The complex bacterial communities inhabiting the mammalian gut have a significant impact on the health of their host (Kahrstrom et al., 2016). Numerous reports indicate that intestinal microbiota, and in particular its metabolic products, have a crucial effect on various health and diseased states. Host immune system and brain development, metabolism, behavior, stress and pain response have all been reported to be associated with microbiota disturbances humans, peripheral L-DOPA metabolism involves DOPA decarboxylase, which converts L-DOPA to dopamine, thus preventing the passage of L-DOPA to its site of action in the brain, as dopamine cannot pass the blood-brain barrier (Pinder, 1970). For this reason, Parkinson's patients are treated with a DOPA decarboxylase inhibitor (primarily carbidopa) in combination with L-DOPA to enhance the effectiveness of L-DOPA delivery to the brain (Deleu et al., 2002). Nonetheless, the pharmacokinetics of L-DOPA/carbidopa treatment varies significantly among patients, some are resistant to the treatment, others undergo fluctuating response towards the treatment over time, thus require increasing L-DOPA dosage regimen leading to increased severity of adverse effects like dyskinesia (Katzenschlager and Lees, 2002). What remains to be clarified is whether inter-individual variations in gut microbiota composition play a causative role in the variation of treatment efficacy.
Several amino acid decarboxylases have been annotated in bacteria. Tyrosine decarboxylase genes (tdc) have especially been encoded in the genome of several bacterial species in the genera Lactobacillus and Enterococcus (Perez et al., 2015;Zhu et al., 2016). Though tyrosine decarboxylase (TDC) is named for its capacity to decarboxylate L-tyrosine into tyramine, it might also have the ability to decarboxylate L-DOPA to produce dopamine (Zhu et al., 2016) due to the high similarity of the chemical structures of these substrates. This implies that TDC activity of the gut microbiota might interfere with L-DOPA bioavailability, which could be of clinical significance in the L-DOPA treatment of Parkinson's patients.
The aim of the present study is to parse out the effect of L-DOPA metabolizing bacteria, particularly in the proximal small intestine, where L-DOPA is absorbed. Initially, we established TDC present in small intestinal bacteria efficiently converted L-DOPA to dopamine, confirming their capacity to modulate the in situ bioavailability of the primary drug used in the treatment of Parkinson's patients. We show that higher relative abundance of bacterial tdc gene in fecal samples of Parkinson's patients positively correlates with higher daily L-DOPA dosage requirement and duration of disease. We further confirm our findings in rats orally administered a mixture of L-DOPA/carbidopa, illustrating that L-DOPA levels in plasma negatively correlate with the abundance of bacterial tdc gene in the proximal small intestine.

Proximal small intestinal bacteria of rodents convert L-DOPA to dopamine
To determine whether proximal small intestinal microbiota maintain the ability to metabolize L-DOPA, luminal samples from the jejunum of wild-type Groningen rats were incubated in vitro with L-DOPA and analyzed by High-Performance Liquid Chromatography with Electrochemical Detection (HPLC-ED). The chromatograms revealed that L-DOPA decarboxylation ( Figure 1A) coincides with the conversion of tyrosine to tyramine ( Figure   1B-E). In addition, no other metabolites derived from L-DOPA were detected. To support the ex vivo experiment results, the uptake of L-DOPA was quantified in plasma samples from specific pathogen free and germ-free female C57 BL/6J mice after oral gavage with L-DOPA.
HPLC-ED analysis revealed higher levels of L-DOPA and its metabolites dopamine and DOPAC (3,4-Dihydroxyphenylacetic acid) in plasma samples of germ-free mice compared to their conventional counterparts ( Figure S1). Taken together, the results suggest that TDC is involved in L-DOPA metabolism by gut bacteria, which may, in turn, interfere with L-DOPA uptake in the proximal small intestine.

Bacterial tyrosine decarboxylase is responsible for L-DOPA decarboxylation
The coinciding tyrosine and L-DOPA decarboxylation observed in the luminal content of jejunum was the basis of our hypothesis that TDC is the enzyme involved in both conversions. Species of the genera Lactobacillus and Enterococcus have been reported to harbor this enzyme (Perez et al., 2015;Zhang and Ni, 2014). To investigate whether genomes of these genera indeed represent the main TDC encoding bacterial groups of the small intestine microbiota, the TDC protein (EOT87933) from the laboratory strain Enterococcus faecalis v583 was used as a query to search the US National Institutes of Health Human Microbiome Project (HMP) protein database, to identify whether the genome of other (small intestinal) gut bacteria also encode tdc. This analysis exclusively identified TDC proteins in species belonging to the bacilli class, including more than 50 Enterococcus strains (mainly E. faecium and E. faecalis) and several Lactobacillus and Staphylococcus species ( Figure S2A).
Next, we aligned the genome of E. faecalis v583 with two gut bacterial isolates, E. faecium W54, and L. brevis W63, illustrating the conservation of the tdc-operon among these species (Figure 2A). Intriguingly, analysis of E. faecium genomes revealed that this species encodes a second, paralogous tdc gene ( P TDCEFM) that did not align with the conserved tdc-operon and was absent from the other species (Figure 2A, Figure S2A, Data file S1).
To support our in silico data, a comprehensive screening of E. faecalis v583, E. faecium W54, and L. brevis W63 and 77 additional clinical and human isolates of Enterococcus, including clinical isolates and strains from healthy subjects, was performed. All enterococcal isolates and L. brevis were able to convert tyrosine and L-DOPA into tyramine and dopamine, respectively ( Figure 2B-D, Table S1). Notably, our HPLC-ED analysis revealed considerable variability among the tested strains with regard to their efficiency to decarboxylate L-DOPA. E. faecium and E. faecalis were drastically more efficient at converting L-DOPA to dopamine, compared to L. brevis. Growing L. brevis in different growth media did not change the L-DOPA decarboxylation efficacy (Figure S2B, C). To eliminate the possibility that other bacterial amino acid decarboxylases are involved in L-DOPA conversion observed in the jejunal content we expanded our screening to include live bacterial species harboring PLP-dependent amino acid decarboxylases previously identified by Williams et al (Williams et al., 2014). None of the tested bacterial strains encoding different amino acid decarboxylases could decarboxylate L-DOPA ( Figure S2D-G, Table   S2).
To verify that the tdc gene is solely responsible for L-DOPA decarboxylation in Enterococcus, wild type E. faecalis v583 (EFS WT ) was compared with a mutant strain (EFS ΔTDC ) in which both the tdc gene (tdcA) and tyrosine transporter (tyrP) were deleted (14), ( Figure 2E). Overnight incubation of EFS WT and EFS ΔTDC bacterial cells with L-DOPA resulted in production of dopamine in the supernatant of EFS WT but not EFS ΔTDC (Figure 2F), confirming the pivotal role of these genes in this conversion. To rule out that deletion of tyrP alone could explain the observed result by an impaired L-DOPA import, cell-free protein extracts were incubated with 1 mM L-DOPA overnight at 37C. While EFS WT cell-free protein extract converted all supplied L-DOPA into dopamine, no dopamine production was observed in the EFS ΔTDC cell-free protein extracts ( Figure 2G). Collectively, results show that TDC is encoded on genomes of gut bacterial species known to dominate the proximal small intestine and that this enzyme is exclusively responsible for converting L-DOPA to dopamine by these bacteria, although the efficiency of that conversion displays considerable speciesdependent variability.

High levels of tyrosine do not prevent bacterial decarboxylation of L-DOPA
To test whether the availability of the primary substrate for bacterial tyrosine decarboxylases (i.e., tyrosine) could inhibit the uptake and decarboxylation of L-DOPA, the growth, metabolites, and pH that was previously shown to affect the expression of tdc (Perez et al., 2015), of E. faecium W54 and E. faecalis v583 were analyzed over time. 100 µM L-DOPA was added to the bacterial cultures, whereas approximately 500 µM tyrosine was present in the growth media. Remarkably, L-DOPA and tyrosine were converted simultaneously, even in the presence of these excess levels of tyrosine (1:5 L-DOPA to tyrosine), albeit at a slower conversion rate for L-DOPA ( Figure 3A-B). Notably, the decarboxylation reaction appeared operational throughout the exponential phase of growth for E. faecalis, whereas it is only observed in E. faecium when this bacterium entered the stationary phase of growth, suggesting differential regulation of the tdc expression in these species.
To further characterize the substrate specificity and kinetic parameters of the bacterial tyrosine decarboxylases, tdc genes from E. faecalis v583 (TDCEFS) and E. faecium W54 (TDCEFM and P TDCEFM) were expressed in Escherichia coli BL21 (DE3) and then purified.
Michaelis-Menten kinetics indicated each of the studied enzymes had a significantly higher affinity (Km) (Figure 3C-I) and catalytic efficiency (Kcat/Km) for tyrosine than for L-DOPA (Table 1). Despite the differential substrate affinity, our findings illustrate that high levels of tyrosine do not prevent the decarboxylation of L-DOPA in batch culture.

Carbidopa is a potent inhibitor of the human decarboxylase but not of bacterial decarboxylases
To assess the extent to which human DOPA decarboxylase inhibitors could affect bacterial decarboxylases, three human DOPA decarboxylase inhibitors (carbidopa, benserazide, and methyldopa) were tested on purified bacterial TDCs and on the corresponding bacterial batch cultures. Comparison of the inhibitory constants (Ki TDC /Ki DDC ) demonstrates carbidopa to be a 1.4-1.9 x 10 4 times more potent inhibitor of human DOPA decarboxylase than bacterial TDCs Parkinson's patients could be attributed to the abundance of tdc genes in the gut microbiota, fecal samples were collected from male and female Parkinson's patients (Table S4) on different doses of L-DOPA/carbidopa treatment (ranging from 300 up to 1100 mg L-DOPA per day). tdc gene-specific primers were used to quantify its relative abundance within the gut microbiota by qPCR ( Figure S5). Remarkably, Pearson r correlation analyses showed a strong positive correlation (r = 0.70, R 2 = 0.49, p value = 0.024) between bacterial tdc gene relative abundance and L-DOPA treatment dose ( Figure 5A) as well as with the duration of disease (Fig. 5B). At this stage, it is unclear whether the relative abundance of tdc genes in fecal samples reflects its abundance in the small intestinal microbiota. This is of particular importance because L-DOPA is absorbed in the proximal small intestine, and reduction in its bioavailability by bacterial TDC activity in the context of Parkinson's patients' medication regimens would only be relevant in that intestinal region. Still, the selective prevalence of tdc encoding genes in the genomes of signature microbes of the small intestine microbiota supports the notion that the results obtained from fecal samples are a valid representation of tdc gene abundance in the small intestinal microbiota. Moreover, the significant correlation of the relative tdc abundance in the fecal microbiota and the required L-DOPA dosage as well as disease duration strongly supports a role for bacterial TDC in L-DOPA bioavailability.

Tyrosine decarboxylase gene abundance in small intestine correlates with L-DOPA bioavailability in rats
To further consolidate the concept that tdc gene abundance in proximal small intestinal microbiota affects peripheral levels of L-DOPA in blood and dopamine/L-DOPA ratio in the jejunal luminal content, male wild-type Groningen rats (n=25) rats were orally administered 15 mg L-DOPA/3.75 mg carbidopa per kg of body weight and sacrificed after 15 minutes (point of maximal L-DOPA bioavailability in rats (Bredberg et al., 1994)). Plasma levels of L-DOPA and its metabolites dopamine and DOPAC were measured by HPLC-ED, while relative abundance of the tdc gene within the small intestinal microbiota was quantified by gene-specific qPCR ( Figure S5). Strikingly, Pearson r correlation analyses showed that the ratio between dopamine and L-DOPA levels in the proximal jejunal content positively correlated with tdc gene abundance (r= 0.78, R 2 = 0.61, P value = 0.0001) ( Figure 6A), whereas the absolute L-DOPA concentration in the proximal jejunal content was negatively correlated with the abundance of the tdc gene (r= −0.68, R 2 = 0.46, P value = 0.0021) ( Figure   6B). Moreover, plasma levels of L-DOPA displayed a strong negative correlation (r = −0.66, R 2 = 0.434, P value = 0.004) with the relative abundance of the tdc gene ( Figure 6C).
Findings indicate that L-DOPA uptake by the host is compromised by higher abundance of gut bacteria encoding for tdc genes in the upper region of the small intestine.

Discussion
Our observation of small intestinal microbiota able to convert L-DOPA to dopamine (        Table S4). Solid fecal samples were collected in anaerobic fecal bags and kept sealed in a cold environment until brought to the hospital where they were immediately stored at −80°C until analysis.

Cloning and heterologous gene expression
The human DOPA decarboxylase gene cloned in pET15b was ordered from GenScript (Piscataway, USA) (

Incubation experiments of jejunal content
Luminal contents from the jejunum of wild-type Groningen rats (n=5) were suspended in EBB (5% w/v) containing 1 mM L-DOPA and incubated for 24 hours in an anaerobic chamber at 37 °C prior to HPLC-ED analysis (DC amperometry at 0.8 V).

DNA extraction
DNA was extracted from fecal samples of Parkinson's patients and jejunal contents of rats using QIAGEN (Cat no. 51504) kit-based DNA isolation as previously described (Zoetendal et al., 2006) with the following modifications: fecal samples were suspended in 1 mL inhibitEX buffer (1:5 w/v) and transferred to screw-caped tubes containing 0.5 g of 0.1 mm and 3 mm glass beads. Samples were homogenized 3 × 30 sec with 1-minute intervals on ice in a mini bead-beater (Biospec, Bartlesville, USA) 3 times.

Quantification of bacterial tyrosine decarboxylase
To identify bacterial species carrying the tdc gene, a broad range of tdc genes from various bacterial genera were targeted as previously described (Torriani et al., 2008) (Figure S5).
Quantitative PCR (qPCR) of tdc genes was performed on DNA extracted from each fecal sample of Parkinson's patients and rats' jejunal content using primers (Dec5f and Dec3r) targeting a 350bp region of the tdc gene. Primers targeting 16sRNA gene for all bacteria (Eub338 and Eub518) were used as an internal control (Table S7) Samples were adjusted to pH 8.6 with 200−800µl TE buffer (2.5% EDTA; 1.5 M Tris/HCl pH 8.6) and 5-10 mg of alumina was added. Suspensions were mixed on a roller shaker at room temperature for 15 min and were thereafter centrifuged for 30s at 20000 × g and washed twice with 1 mL of H2O by aspiration. L-DOPA and its metabolites were eluted using 0.7% HClO4 and filtered before injection into the HPLC-ED-system as described above (DC amperometry at 0.8 V).

QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical tests and (non)linear regression models were performed using GraphPad Prism 7. Statistical tests performed are unpaired T-tests, 2-way-ANOVA followed by a Fisher's LSD test. Specific tests and significance are indicated in the figure legends.

Screening for Enterococcus strains, isolated from healthy subjects and clinical isolates
Enterococcus strains were isolated from fecal or urine samples from clinical setting or from healthy volunteers (aged 2 to 79 years). All samples were collected between January 2008 and 2018 in Beni-Suef City, Egypt. A total of 77 Enterococcus spp. were isolated on bile esculin agar and observed microscopically after gram staining. The screening for decarboxylase activity was performed as described previously by Bover-Cid and Holzapfel (Bover-Cid and Holzapfel, 1999) with L-DOPA or tyrosine added as a substrate to a final concentration of 1% to screen for production of dopamine and tyramine. All Enterococcus strains were spot inoculated on agar plates containing the substrates of interest and on control plates without any substrate. The plates were duplicated, either aerobically or anaerobically at 37°C. Plates were checked daily for 4 days to record the change in the indicator color from yellow to violet, indicative of production of tyramine and dopamine, respectively (Table S1).

Fecal samples from patients with Parkinson's disease
All study subjects consented to the use of their samples for research. Parkinson's disease was