Personalized Response of Parkinson's Disease Gut Microbiota to Nootropic Medicinal Herbs In Vitro: A Proof of Concept.

Parkinson's disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergic neurons. Although the etiology of PD remains elusive, it has been hypothesized that initial dysregulation may occur in the gastrointestinal tract and may be accompanied by gut barrier defects. A strong clinical interest in developing therapeutics exists, including for the treatment of gut microbiota and physiology. We previously reported the impact of healthy fecal microbiota anaerobic cultures supplemented with nootropic herbs. Here, we evaluated the effect of nootropic Ayurvedic herbs on fecal microbiota derived from subjects with PD in vitro using 16S rRNA sequencing. The microbiota underwent substantial change in response to each treatment, comparable in magnitude to that observed from healthy subjects. However, the fecal samples derived from each participant displayed unique changes, consistent with a personalized response. We used genome-wide metabolic reconstruction to predict the community's metabolic potential to produce products relevant to PD pathology, including SCFAs, vitamins and amino acid degradation products. These results suggest the potential value of conducting in vitro cultivation and analyses of PD stool samples as a means of prescreening patients to select the medicinal herbs for which that individual is most likely to respond and derive benefit.

Alteration of Community Metabolism by Prebiotics and Medicinal Herbs.

Several studies have examined the impact of prebiotics on gut microbiota and associated changes in host physiology. Here, we used the in vitro cultivation of human fecal samples stimulated with a series of chemically related prebiotics and medicinal herbs commonly used in Ayurvedic medicine, followed by 16S rRNA sequencing. We applied a genome-wide metabolic reconstruction of enumerated communities to compare and contrast the structural and functional impact of prebiotics and medicinal herbs. In doings so, we examined the relationships between discrete variations in sugar composition and sugar linkages associated with each prebiotic to drive changes in microbiota composition. The restructuring of microbial communities with glycan substrates alters community metabolism and its potential impact on host physiology. We analyzed sugar fermentation pathways and products predicted to be formed and prebiotic-induced changes in vitamin and amino acid biosynthesis and degradation. These results highlight the utility of combining a genome-wide metabolic reconstruction methodology with 16S rRNA sequence-based community profiles to provide insights pertaining to community metabolism. This process also provides a rational means for prioritizing in vivo analysis of prebiotics and medicinal herbs in vivo to test hypotheses related to therapeutic potential in specific diseases of interest.

Microbial liberation of N-methylserotonin from orange fiber in gnotobiotic mice and humans.

Plant fibers in byproduct streams produced by non-harsh food processing methods represent biorepositories of diverse, naturally occurring, and physiologically active biomolecules. To demonstrate one approach for their characterization, mass spectrometry of intestinal contents from gnotobiotic mice, plus in vitro studies, revealed liberation of N-methylserotonin from orange fibers by human gut microbiota members including Bacteroides ovatus. Functional genomic analyses of B. ovatus strains grown under permissive and non-permissive N-methylserotonin "mining" conditions revealed polysaccharide utilization loci that target pectins whose expression correlate with strain-specific liberation of this compound. N-methylserotonin, orally administered to germ-free mice, reduced adiposity, altered liver glycogenesis, shortened gut transit time, and changed expression of genes that regulate circadian rhythm in the liver and colon. In human studies, dose-dependent, orange-fiber-specific fecal accumulation of N-methylserotonin positively correlated with levels of microbiome genes encoding enzymes that digest pectic glycans. Identifying this type of microbial mining activity has potential therapeutic implications.

Short-Chain Fatty Acids Modulate Healthy Gut Microbiota Composition and Functional Potential.

Many studies have focused on the metabolic capacity of human gut microbiota to produce short-chain fatty acids and subsequent effects on host physiology. Given scarce data on how SCFAs produced by gut bacteria participate in cross-feeding to influence community structure and function, we evaluated the potential of SCFAs to modulate human gut microbiota in vitro. We employed anaerobic fecal cultivation in chemically defined medium supplemented with one of nine SCFAs to determine effects on both gut microbial community structure via 16S rRNA sequencing and function via genome reconstruction analysis. Each SCFA displayed significant and unique modulatory potential with respect to the relative abundance of bacterial taxa. Analysis of SCFA-supplemented communities revealed that alterations of individual closely related phylotypes displayed coherent changes, although exceptions were also observed which suggest strain-dependent differences in SCFA-induced changes. We used genome reconstruction to evaluate the functional implications of SCFA-mediated restructuring of fecal communities. We note that some SCFA-supplemented cultures displayed a reduction in the predicted abundance of SCFA producers, which suggests a possible undefined negative feedback mechanism. We conclude that SCFAs are not simply end-products of metabolism but also serve to modulate the gut microbiota through cross-feeding that alters the fitness of specified taxa. These results are important in the identification of prebiotics that elevate specific SCFAs for therapeutic benefit and highlight SCFA consumers as a salient part of the overall metabolic flux pertaining to bacterial fermentative processes.

Community Metabolic Interactions, Vitamin Production and Prebiotic Potential of Medicinal Herbs Used for Immunomodulation.

Historically, the health benefits and immunomodulatory potential of medicinal herbs have been considered an intrinsic quality of the herb itself. We have hypothesized that the health benefits of medicinal herbs may be partially due to their prebiotic potential that alter gut microbiota leading to changes in short chain fatty acids and vitamin production or biotransformation of herb encoded molecules and secondary metabolites. Accumulating studies emphasize the relationship between the gut microbiota and host immune function. While largely unknown, these interactions are mediated by secreted microbial products that activate or repress a variety of immune cell types. Here we evaluated the effect of immunomodulatory, medicinal Ayurvedic herbs on gut microbiota in vitro using 16S rRNA sequencing to assess changes in community composition and functional potential. All immunomodulatory herbs displayed substantial prebiotic potential, targeting unique taxonomic groups. Application of genome reconstruction and analysis of biosynthetic capacity of herb selected communities suggests that many of the 11 herbs tested altered the community metabolism as the result of differential glycan harvest and sugar utilization and secreted products including multiple vitamins, butyrate, and propionate that may impact host physiology and immune function. Taken together, these results provide a useful framework for the further evaluation of these immunomodulatory herbs in vivo to maintain immune homeostasis or achieve desired regulation of immune components in the context of disease.

Combined Prebiotic and Microbial Intervention Improves Oral Cholera Vaccination Responses in a Mouse Model of Childhood Undernutrition.

Undernourished children in low-income countries often exhibit poor responses to oral vaccination. Perturbed microbiota development is linked to undernutrition, but whether and how microbiota changes affect vaccine responsiveness remains unclear. Here, we show that gnotobiotic mice colonized with microbiota from undernourished Bangladeshi children and fed a Bangladeshi diet exhibited microbiota-dependent differences in mucosal IgA responses to oral vaccination with cholera toxin (CT). Supplementation with a nutraceutical consisting of spirulina, amaranth, flaxseed, and micronutrients augmented CT-IgA production. Mice initially colonized with a microbiota associated with poor CT responses exhibited improved immunogenicity upon invasion of bacterial taxa from cagemates colonized with a more "responsive" microbiota. Additionally, a consortium of five cultured bacterial invaders conferred augmented CT-IgA responses in mice fed the supplemented diet and colonized with the "hypo-responsive" community. These results provide preclinical proof-of-concept that diet and microbiota influence mucosal immune responses to CT vaccination and identify a candidate synbiotic formulation.

Prebiotic Potential of Culinary Spices Used to Support Digestion and Bioabsorption.

Although the impact of medicinal and culinary herbs on health and disease has been studied to varying extents, scarcely little is known about the impact of these herbs on gut microbiota and how such effects might contribute to their health benefits. We applied in vitro anaerobic cultivation of human fecal microbiota followed by 16S rRNA sequencing to study the modulatory effects of 4 culinary spices: Curcuma longa (turmeric), Zingiber officinale (ginger), Piper longum (pipli or long pepper), and Piper nigrum (black pepper). All herbs analyzed possessed substantial power to modulate fecal bacterial communities to include potential prebiotic and beneficial repressive effects. We additionally analyzed the sugar composition of each herb by mass spectrometry and conducted genome reconstruction of 11 relevant sugar utilization pathways, glycosyl hydrolase gene representation, and both butyrate and propionate biosynthesis potential to facilitate our ability to functionally interpret microbiota profiles. Results indicated that sugar composition is not predictive of the taxa responding to each herb; however, glycosyl hydrolase gene representation is strongly modulated by each herb, suggesting that polysaccharide substrates present in herbs provide selective potential on gut communities. Additionally, we conclude that catabolism of herbs by gut communities primarily involves sugar fermentation at the expense of amino acid metabolism. Among the herbs analyzed, only turmeric induced changes in community composition that are predicted to increase butyrate-producing taxa. Our data suggests that substrates present in culinary spices may drive beneficial alterations in gut communities thereby altering their collective metabolism to contribute to the salubrious effects on digestive efficiency and health. These results support the potential value of further investigations in human subjects to delineate whether the metabolism of these herbs contributes to documented and yet to be discovered health benefits.

16S rRNA gene profiling and genome reconstruction reveal community metabolic interactions and prebiotic potential of medicinal herbs used in neurodegenerative disease and as nootropics.

The prebiotic potential of nervine herbal medicines has been scarcely studied. We therefore used anaerobic human fecal cultivation to investigate whether medicinal herbs commonly used as treatment in neurological health and disease in Ayurveda and other traditional systems of medicine modulate gut microbiota. Profiling of fecal cultures supplemented with either Kapikacchu, Gotu Kola, Bacopa/Brahmi, Shankhapushpi, Boswellia/Frankincense, Jatamansi, Bhringaraj, Guduchi, Ashwagandha or Shatavari by 16S rRNA sequencing revealed profound changes in diverse taxa. Principal coordinate analysis highlights that each herb drives the formation of unique microbial communities predicted to display unique metabolic potential. The relative abundance of approximately one-third of the 243 enumerated species was altered by all herbs. Additional species were impacted in an herb-specific manner. In this study, we combine genome reconstruction of sugar utilization and short chain fatty acid (SCFA) pathways encoded in the genomes of 216 profiled taxa with monosaccharide composition analysis of each medicinal herb by quantitative mass spectrometry to enhance the interpretation of resulting microbial communities and discern potential drivers of microbiota restructuring. Collectively, our results indicate that gut microbiota engage in both protein and glycan catabolism, providing amino acid and sugar substrates that are consumed by fermentative species. We identified taxa that are efficient amino acid fermenters and those capable of both amino acid and sugar fermentation. Herb-induced microbial communities are predicted to alter the relative abundance of taxa encoding SCFA (butyrate and propionate) pathways. Co-occurrence network analyses identified a large number of taxa pairs in medicinal herb cultures. Some of these pairs displayed related culture growth relationships in replicate cultures highlighting potential functional interactions among medicinal herb-induced taxa.

Comparative genomics of the KdgR regulon in Erwinia chrysanthemi 3937 and other gamma-proteobacteria.

In the plant-pathogenic enterobacterium Erwinia chrysanthemi, almost all known genes involved in pectin catabolism are controlled by the transcriptional regulator KdgR. In this study, the comparative genomics approach was used to analyse the KdgR regulon in completely sequenced genomes of eight enterobacteria, including Erw. chrysanthemi, and two Vibrio species. Application of a signal recognition procedure complemented by operon structure and protein sequence analysis allowed identification of new candidate genes of the KdgR regulon. Most of these genes were found to be controlled by the cAMP-receptor protein, a global regulator of catabolic genes. At the next step, regulation of these genes in Erw. chrysanthemi was experimentally verified using in vivo transcriptional fusions and an attempt was made to clarify the functional role of the predicted genes in pectin catabolism. Interestingly, it was found that the KdgR protein, previously known as a repressor, positively regulates expression of two new members of the regulon, phosphoenolpyruvate synthase gene ppsA and an adjacent gene, ydiA, of unknown function. Other predicted regulon members, namely chmX, dhfX, gntB, pykF, spiX, sotA, tpfX, yeeO and yjgK, were found to be subject to classical negative regulation by KdgR. Possible roles of newly identified members of the Erw. chrysanthemi KdgR regulon, chmX, dhfX, gntDBMNAC, spiX, tpfX, ydiA, yeeO, ygjV and yjgK, in pectin catabolism are discussed. Finally, complete reconstruction of the KdgR regulons in various gamma-proteobacteria yielded a metabolic map reflecting a globally conserved pathway for the catabolism of pectin and its derivatives with variability in transport and enzymic capabilities among species. In particular, possible non-orthologous substitutes of isomerase KduI and a new oligogalacturonide transporter in the Vibrio species were detected.