In this study, we use an untargeted mass spectrometry-based approach to characterize VOCs from E. faecalis, C. sporogenes, and E. lenta and identify previously uncharacterized metabolites of bacterial levodopa metabolism. Many human microbiome studies seeking to understand the metabolic capacity of complex communities have highlighted the need to elucidate the molecular mechanisms contributing to the gut’s biochemical diversity [14,15,16,17]. Such efforts have prompted more targeted analyses of microbial metabolism to characterize the pathways encoded by individual strains and determine their biological significance. The discovery of gut microbial-derived VOCs has become a growing area of research that can provide major mechanistic insights into the biological processes underlying host-microbiome interactions with applications for personalized medicine. The main advantage of identifying characteristic VOCs over traditional biomarkers of drug metabolism is the reduction of analytical complexity. While the presence of levodopa-metabolizing enzymes can gauge potential gut microbial activities, the high implementation costs to extract and analyze these markers limits their routine utilization as indicators of off-target metabolism. Hence, VOC signatures of levodopa metabolism offer a robust, microbiome-centered approach to assess individual changes in its pharmacokinetics.

Studies have demonstrated a positive correlation between tyrDC gene expression, substrate concentration, and growth of E. faecalis, indicating that higher tyrDC activity enhances bacterial growth [24,25,26,27]. Importantly, the VOCs associated with levodopa decarboxylation correspond to metabolic activities tied to cell growth. These effects are further supported by functional analyses at the transcriptional level, which reveal that an increased tyrDC substrate concentration leads to enhanced pathways for purine and pyrimidine catabolism for DNA biosynthesis, along with an activation of amino-sugars metabolism in E. faecalis [27]. Furthermore, the accumulation of reactive α-hydroxycarbonyl and α-aminocarbonyl intermediates during the growth phase of various bacteria has been correlated with an increased production of pyrazine VOCs, which are widely associated with lactic acid bacteria [28,29,30,31,32]. These findings suggest that biological pathways corresponding to energy metabolism and growth may potentially give rise to the increased production of 2,6-dimethylpyrazine and 4,6-dimethylpyrimidine. The presence of these levodopa-induced diazines could potentially indicate a shift in E. faecalis metabolic activity in response to increased tyrDC substrate abundance. Notably, the results show that inhibiting levodopa decarboxylation with AFMT prevents the production of 2,6-dimethylpyrazine and 4,6-dimethylpyrimidine. The simultaneous inhibition of these compounds with AFMT suggests that these VOCs indicate a shift in E. faecalis metabolic activity in response to levodopa treatment and increased activity towards tyrDC substrates. Taking into account the low concentration of 2,6-dimethylpyrazine and 4,6-dimethylpyrimidine produced during levodopa decarboxylation by E. faecalis, follow-up analyses using complimentary methods with increased resolution and sensitivity, such as proton transfer traction mass spectrometry (PTR-MS) or selected ion flow tube mass spectrometry (SIFT-MS), would entail the selective detection and accurate quantification of these VOCs [33,34,35]. Parallel applications of such methods for quantitative determination of these compounds from complex microbiota samples would further their utility to potentially assess variable responses to levodopa treatment across PD patients. The previously established relationship between tyrDC presence and levodopa decarboxylation in PD patients demonstrates the direct contribution of E. faecalis in observed interindividual variability in treatment response [4]. While the abundance of tyrDC has been proposed as a predictive biomarker of levodopa metabolism in PD patient microbiotas, the complex nature of the gut makes VOCs a robust alternative for the non-invasive assessment of metabolic heterogeneity. Given that tyrDC abundance has been shown to positively correlate with levodopa dosages in PD patient samples [4], future studies also analyzing the abundance of 2,6-dimethylpyrazine and 4,6-dimethylpyrimidine from complex samples could explore their potential as indicative markers of levodopa metabolism.

We also detect a shift in the volatile metabolome of C. sporogenes during the bioconversion of levodopa to DHPPA that corresponds to an increased abundance of VOCs classified as fatty acid esters. These energy-rich compounds are associated with pathways of cell signaling, membrane formation, and fatty acid biosynthesis. Moreover, fatty acid esters are associated with the distinct metabolite profile detected from C. sporogenes during anaerobiosis, but not aerobiosis [36]. The production of short and medium chain fatty acids is well implicated across Clostridia species [37,38,39], with various metabolic pathways producing a variety of fatty acid products via chain elongation mechanisms using coenzyme A [37, 40]. Similarly, the fldABC protein complex responsible for DHPPA production uses the same CoA-dependent transfer mechanism to deaminate levodopa [5]. These processes generate an accumulation of ester precursors that enables their production. Although we did not observe statistically significant differences in VOC abundances between C. sporogenes cultured with and without levodopa, we report previously uncharacterized compounds that reflect the strain’s unique metabolic activity. While these VOCs have been implicated in cellular processes underlying energy production, further investigations are needed to link the underlying biological pathways leading to their production.

We next sought to characterize VOCs associated with the dehydroxylation of the levodopa intermediate DHPPA. E. lenta performs catechol dehydroxylation using distinct molybdenum-dependent enzymes that are variably distributed across individual strains [4, 23]. The prevalence of genes encoding these specialized enzymes correlates with the metabolism of their specific substrates, with hcdh activity being the most prevalent across closely related gut microbiota [23, 41]. As such, there is a growing interest to elucidate the chemical mechanisms exerted by these enzymes to mediate primary and secondary metabolism in the gut. To better understand the effects of catechol dehydroxylation, we identified significant changes in VOCs detected during DHPPA metabolism by hcdh. During the dehydroxylation of DHPPA to 3-HPPA, we detect a significant increase in 4,5-dimethylpyrazine and a putative dimethylpyrimidine isomer from E. lenta. With the mechanisms of catechol dehydroxylase enzymes remaining poorly understood, it is difficult to decipher their biological roles and pathway involvements. However, additional work from Rekdal et al. demonstrated that the dehydroxylation of dopamine, the other levodopa intermediate, provided a growth advantage to E. lenta and potentially serves as an alternative electron acceptor [4, 23]. Furthermore, metabolite profiles recently generated by Noecker et al. revealed that nucleotide and cell wall metabolites comprised a large proportion of the E. lenta metabolome, along with nucleic acid intermediates [42]. Based on these findings, we suspect the production of 4,5-dimethylpyrimdine and the other DHPPA-associated VOC could be a result of the potential growth-promoting effects of DHPPA metabolism. These studies also show that the addition of tungstate to the growth medium blocks the molybdenum-dependent dehydroxylase activity and inhibits the growth increase of E. lenta [4]. Interestingly, supplementing tungstate to E. lenta cultures suppressed the production of 4,5-dimethylpyrimidine and the putative dimethylpyrimidine isomer, providing evidence for endogenous production and further suggesting these VOCs may arise from increased growth tied to catechol dehydroxylation. While further investigations are necessary to characterize the dimethylpyrimidine isomer and confirm the biological origins of these VOCs, previous works have demonstrated the role of pyrimidines and their derivatives for bacterial growth and sensing mechanisms, implicating metabolic pathways for cell growth and signal processing as potential sources for VOCs [43,44,45]. Taken together, these findings open the door to improve our understanding of the relationship of dimethylpyrimidine VOCs and bacteria growth to improve our understanding of the effects of catechol dehydroxylation on gut microbiota metabolism.

VOCs can be generated across a wide range of biosynthetic pathways and are thought to diffuse through cell membranes- giving them the potential to modulate gene expression and influence physiology [28]. Thus, an emerging area of research is focused on understanding the molecular mechanisms underlying VOC production. While little information is known regarding the genes and pathways that interact with VOCs, the main biological responses reported for microbial VOCs include biofilm formation, virulence, secondary metabolite production, and growth [28]. In line with these findings, many of the VOCs identified in this study are implicated in those cellular processes. In this study, we detected unique volatile signatures from E. faecalis, C.sporogenes, and E. lenta. Functional group analyses revealed diverse chemical compositions within the VOC profile of each strain, whose relative abundances reflect different biological processes. The VOC profile of E. faecalis was mainly composed of heteroaromatic compounds, followed closely by aldehydes and VOCs classified as “other.” Additionally, we identified analyte 1 from E. faecalis as an endogenous VOC. We suspect that these compounds originate from primary metabolic pathways encoded in lactic acid bacteria and are likely products of carbohydrate fermentation and amino acid degradation. The VOC profile of E. lenta showed a similar chemical composition after performing functional group analyses. Little is known about the metabolic activity of E. lenta and the biosynthetic pathways leading to VOC production. Interestingly, the VOCs we detected from E. lenta are implicated in processes for amino acid and nucleic acid metabolism. These findings are in agreement with previous studies using systems biology-based approaches, which show that a large portion of E. lenta’s metabolic activity corresponds to cell growth and energy metabolism [41]. Collectively, these findings support that the distinct volatile profiles capture differences in metabolic activity across bacteria strains. Furthermore, the chemical signals encoded in these signatures can elicit a range of processes to coordinate behavior and respond to environmental stimuli. Thus, it is likely that the organism-specific VOC profiles detected in this study have distinct biological effects.

Here, we report differences in the VOC profiles of E. faecalis, C. sporogenes, and E. lenta and identify VOCs associated with levodopa metabolism. Using an untargeted, metabolomics approach, we report previously uncharacterized VOCs from each strain that furthers our understanding of metabolic processes harnessed by different gut bacteria. Collectively, these findings give insight into the metabolic activities of different gut microbiota, identify reproducible changes that occur during the breakdown of levodopa, and link 4,6-dimethylpyrimidine and 2,6-dimethylpyrazine to the endogenous bioconversion of levodopa by E. faecalis. Our consideration of species-specific VOCs shows diverse chemical classes within each organism’s volatilome that reflect interspecies differences in metabolic activity. The findings in this work open the door for further, targeted analyses of discriminant VOCs of levodopa metabolism and further exploration of their biological significance. In the future, functional interpretations of these VOCs will enable us to decode the molecular mechanisms by which gut bacteria interfere with PD treatment, offering novel strategies to assess treatment efficacy and improve patient outcomes.