Patent Application
20230043198
The microbiota metabolome results from a complex web of interactions between multiple microbial species and strains, environmental inputs (e.g., diet), and host factors. Advanced metabolomic, metagenomic and functional genomic approaches have revealed that the human microbiota can produce tens of thousands of unique small molecules (Donia and Fischbach, 2015; Milshteyn et al., 2018; Nicholson et al., 2012). However, the sheer magnitude and complexity of the gut microbiota metabolome can create significant challenges for dissecting how intra- and inter-species microbial chemistries affect host physiology (Donia and Fischbach, 2015; Fischbach, 2018). Understanding of the potential effects of the microbiota metabolome on the human host remains in its infancy.
MAOIs were the first FDA approved antidepressants (Ramachandraih, 2011); however, they have fallen out of favor due to potentially fatal dietary and drug-drug interactions (Fiedorowicz, 2004). Nonetheless, MAOIs remain an important treatment option for patients with refractory depression and other psychiatric disorders (Fiedorowicz, 2004), as well as Parkinson's disease (Riederer and Laux, 2011).
Histamine is generated via decarboxylation of L-His (Tannase, 1985). Histamine is involved in inflammatory responses and can regulate gut physiological function.
In one aspect is provided a method for determining if a test compound modulates an activity of a first protein in vivo, comprising
(a) contacting said compound with a cell comprising (i) a first nucleic acid molecule which encodes a first fusion protein comprising a first protein, a cleavage site for a protease, and a protein which activates transcription of a reporter gene in said cell, (ii) a second nucleic acid molecule which encodes a second fusion protein comprising a second protein which interacts with the first protein upon activation of the first protein and a protease or a fragment thereof capable of cleaving the protease cleavage site within the first fusion protein, and (iii) a third nucleic acid molecule which comprises a reporter gene, wherein said reporter gene is a barcode sequence operably linked to an element responsive to the protein which activates its transcription, and
(b) determining the level of transcription of said barcode sequence.
In some embodiments, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are clonally expressed to enable linkage of a specific receptor to an individual barcode. In some embodiments, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are clonally expressed through co-transfection. In some embodiments, the first nucleic acid molecule, the second nucleic acid molecule and the third nucleic acid molecule are clonally expressed through stable expression. In some embodiments, the barcode sequence comprises from 4 to 50 bases.
In some embodiments, the method further comprises
(c) concluding that the test compound activates the first protein if the level of transcription of said barcode sequence is increased in the presence of the test compound as compared to a control where no test compound is present.
In some embodiments, the method further comprises
(c) concluding that the test compound activates the first protein if the level of transcription of said barcode sequence is increased in the presence of the test compound as compared to an untreated cell.
In some embodiments, the method further comprises
(c) concluding that the test compound activates the first protein if the level of transcription of said barcode sequence is increased in the presence of the test compound as compared to a cell treated with a single dose of an agonist of the first protein in combination with an antagonist of the first protein.
In some embodiments, the first protein is a transmembrane protein. In some embodiments, the first protein is a G-protein coupled receptor (GPCR). In some embodiments, the GPCR is a non-olfactory GPCR. In some embodiments, the GPCR is an orphan GPCR. In some embodiments, the GPCR is a human GPCR.
In various embodiments, the protein which activates transcription of the reporter gene in said cell is tTA, a cas9 fusion protein, gal4/VP16, the estrogen receptor, the androgen receptor, the mineralocorticoid receptor, or the glucocorticoid receptor. In some embodiments, the cas9 fusion protein is cas9-vp64.
In various embodiments, the protein which activates transcription of the reporter gene in said cell is tTA. In some embodiments, the second protein which interacts with the first protein upon activation of the first protein is β-arrestin, a G-protein receptor kinase (GRK), or G-alpha. In some embodiments, the second protein is β-arrestin. In some embodiments, the protease is a Tobacco Etch Virus nuclear inclusion A protease (TEV protease). In some embodiments, the cell is a non-adherent mammalian cell. In some embodiments, the cell is Expi 293 T cell, Jurkat, Hela T4, raji, ramos, cho-s, or thp1 cells.
In various embodiments, the level of transcription of said barcode sequence is determined by sequencing of cDNA. In some embodiments, the cDNA is produced by reverse transcription of mRNA isolated from the cell using a polydT primer. In some embodiments, the test compound is a metabolite produced by a bacterial taxon contained within a microbiota of a subject. In some embodiments, the test compound is a metabolite produced by a bacterial strain contained within a microbiota of a subject. In some embodiments, the microbiota is a gastrointestinal (GI) microbiota. In some embodiments, the bacterial strain is clonally arrayed and cultured in vitro. In some embodiments, the method further comprises identifying the bacterial strain. In some embodiments, the bacterial strain is identified using 16S rRNA gene sequencing or whole genome sequencing. In some embodiments, the subject is human.
In some embodiments, the method is conducted in a high-throughput format, comprising:
(i) transfecting or transducing a plurality of cells separated into individual wells of a multi-well plate with the first, second and third nucleic acid molecules so that each transfected or transduced cell has a specific combination of the first protein and the barcode sequence;
(ii) mixing the transfected cells;
(iii) rearraying mixed cells into individual wells of a multi-well plate;
(iv) exposing the rearrayed cell mixtures to one or more test compounds;
(v) sequencing the barcodes, and
(vi) determining which barcode sequences are increased in the presence of the test compound(s) as compared to a control where no test compound(s) is present.
In some embodiments, the method is conducted in a high-throughput format, comprising:
(i) transfecting or transducing a plurality of cells that stably express the second nucleic acid molecule (Barr-TEV) with the first and third nucleic acid molecules (the receptor and barcode);
(ii) mixing the transfected cells;
(iii) rearraying mixed cells into individual wells of a multi-well plate;
(iv) exposing the rearrayed cell mixtures to one or more test compounds;
(v) sequencing the barcodes, and
(vi) determining which barcode sequences are increased in the presence of the test compound(s) as compared to a control where no test compound(s) is present.
In some embodiments, the first nucleic acid molecule encodes a GPCR, the second nucleic acid molecule encodes Barr-TEV, and the third nucleic acid molecule comprises a barcode.
In another aspect is provided a method for high-throughput screening of microbiota metabolites capable of modulating the activity of a plurality of G-protein coupled receptors (GPCRs), the method comprising:
a) providing a plurality of non-adherent mammalian cells, wherein each cell comprises (i) a first nucleic acid molecule encoding a first fusion protein comprising a GPCR linked to the transcription factor tTA via a cleavage site for Tobacco Etch Virus nuclear inclusion A protease (TEV protease), (ii) a second nucleic acid molecule encoding a second fusion protein comprising 3-arrestin and TEV protease configured to cleave the TEV protease site on the first fusion protein, and (iii) a third nucleic acid molecule comprising a barcode sequence operably linked to a promoter specifically activated by the tTA transcription factor, wherein each barcode sequence is thus specifically linked to an individual GPCR;
b) contacting the plurality of cells with one or more microbiota metabolites or one or more compounds;
c) sequencing the barcodes; and
d) determining which barcode sequence transcription levels are increased or decreased in the presence of the metabolites as compared to a control where no metabolites are present.
In some embodiments, the GPCR is a non-olfactory GPCR. In some embodiments, the GPCR is an orphan GPCR. In some embodiments, the GPCR is a human GPCR. In some embodiments, the cell is Expi 293 T cell. In some embodiments, the level of transcription of said barcode sequence is determined by sequencing of cDNA. In some embodiments, the cDNA is produced by reverse transcription of RNA isolated from the cell. In some embodiments, the microbiota is a gastrointestinal (GI) microbiota. In some embodiments, the method further comprises identifying a bacterial strain producing the specific metabolite which activates the specific GPCR. In some embodiments, the bacterial strain is identified using 16S rRNA sequencing.
In another aspect is provided a method of preventing or treating monoamine oxidase inhibitor (MAOI)-induced toxicity in a subject, the method comprising administering an antibiotic effective to target a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene. In yet another aspect is provided a method of preventing or treating MAOI-induced toxicity in a subject comprising administering an antibiotic effective to target Morganella spp.
In another aspect is provided a method of treating a disease or condition caused by decreased MAO activity in a subject, the method comprising administering an antibiotic effective to target an organism comprising a nucleic acid sequence encoding a phenethylamine production gene.
In some embodiments, the organism is a bacterial strain. In some embodiments of the above aspects, the bacterial strain produces phenethylamine. In some embodiments, the bacterial strain secretes phenethylamine. In some embodiments, the disease is Brunner syndrome. In some embodiments, the condition is autism or anti-social behavior. In some embodiments, the subject expresses a MAOA-L variant. In some embodiments, the antibiotic is effective to target Morganella morganii. In some embodiments, the antibiotic is cefepime, piperacillin, tazobactam, ceftazidime, cefotaxime, ceftibuten, meropenem, doripenem, ertapenem, a fluoroquinolone, or an aminoglycoside.
In another aspect is provided a method of treating depression in a subject comprising administering a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene. In some embodiments, the method further comprises administering an anti-depressant to the subject. In some embodiments, the anti-depressant is an MAOI. In some embodiments, the bacterial strain produces phenethylamine.
In another aspect is provided a method for evaluating potential toxicity of a monoamine oxidase inhibitor (MAOI) in a subject, the method comprising
a) obtaining a gastrointestinal microbiota sample from the subject, and
b) assaying the sample for the presence of a bacterial taxon or bacterial strain comprising a phenethylamine production gene.
In some embodiments, the amount of a phenethylamine producing enzyme exceeds a defined fraction of the enzymes produced by the microbiota. In some embodiments, the gastrointestinal microbiota sample is a fecal sample.
In another aspect is provided a method for evaluating potential efficacy of an MAOI in a subject, the method comprising
a) taking a fecal sample or a sample from the gastrointestinal tract of the subject, and
b) assaying for the presence of a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene.
In some embodiments, the method further comprises assaying for the amount of the bacterial strain present in the gastrointestinal tract. In some embodiments, the method further comprises treating the subject with an MAOI. In some embodiments, the method further comprises adjusting or determining the dosage of the MAOI based on the presence and/or amount of the bacterial strain present. In some embodiments, the bacterial strain is a bacterium of Morganella spp. In some embodiments, the bacterial strain is Morganella morganii.
In another aspect is provided a method of preventing or treating histamine-induced gastrointestinal disease in a subject, the method comprising administering one or more antibiotics effective to target a bacterial strain comprising a nucleic acid sequence encoding a histamine production gene. In some embodiments, the histamine production gene is a histidine decarboxylase. In some embodiments, the abundance of the histidine decarboxylase is higher in a patient with Crohn's disease as compared to a subject without inflammatory bowel disease. In some embodiments, the abundance of the histidine decarboxylase is higher in a patient with ulcerative colitis as compared to a subject without inflammatory bowel disease. In some embodiments, the bacterial strain is L. reuteri or a bacterium of Morganella spp. In some embodiments, the gastrointestinal disease is diarrhea.
In another aspect is provided a method of preventing or treating an allergy in a subject, the method comprising administering one or more antibiotics effective to target a bacterial strain comprising a nucleic acid sequence encoding a histamine production gene. In some embodiments, the bacterial strain is L. reuteri or a bacterium of Morganella spp.
In another aspect is provided a method of preventing or treating asthma in a subject, the method comprising administering one or more antibiotics effective to target a bacterial strain comprising a nucleic acid sequence encoding a histamine production gene. In some embodiments, the bacterial strain is L. reuteri or a bacterium of Morganella spp. In some embodiments, the antibiotic is cefepime, piperacillin, tazobactam, ceftazidime, cefotaxime, ceftibuten, meropenem, doripenem, ertapenem, a fluoroquinolone, or an aminoglycoside. In some embodiments, the method further comprises administering a histidine decarboxylase inhibitor. In some embodiments, the histidine decarboxylase inhibitor is rugosin D, rugosin A methyl ester, tellimagrandin II, rugosin A, pinocembrin, α-fluoromethylhistidine, brocresine, lecanoric acid, 2-hydroxy-5-carbomethoxybenzyloxyamine, and aminooxy analogs of histamine.
In another aspect is provided a method for evaluating potential effectiveness of an antibiotic to treat a gastrointestinal condition or disease, allergy or asthma in a subject, the method comprising
a) taking a fecal sample or a sample from the gastrointestinal tract of the subject, and
b) assaying for the presence of a bacterial strain comprising a nucleic acid sequence encoding a histamine production gene.
In some embodiments, the method further comprises assaying for the amount of the bacterial strain present in the gastrointestinal tract. In some embodiments, the method further comprises treating the subject with one or more antibiotics. In some embodiments, the antibiotic is cefepime, piperacillin, tazobactam, ceftazidime, cefotaxime, ceftibuten, meropenem, doripenem, ertapenem, a fluoroquinolone, or an aminoglycoside. In some embodiments, the method further comprises adjusting or determining the dosage of the antibiotic based on the presence and/or amount of the bacterial strain present. In some embodiments, the bacterial strain is a bacterium of Morganella spp. In some embodiments, the bacterial strain is Morganella morganii.
In another aspect is provided a method of preventing or treating a disease or condition resulting from production of phenethylamine, the method comprising administering one or more antibiotics effective to target a bacterial strain producing L-phenylalanine. In yet another aspect is provided a method of preventing or treating phenylketonuria (PKU) in a subject, the method comprising administering one or more antibiotics effective to target a bacterial strain producing L-phenylalanine.
In another aspect is provided a method of preventing or treating a disease or condition resulting from production of phenethylamine, the method comprising administering a Shikimate pathway inhibitor or an antagonist of aromatic L-amino acid decarboxylase. In yet another aspect is provided a method of preventing or treating phenylketonuria (PKU) in a subject, the method comprising administering a Shikimate pathway inhibitor or an antagonist of aromatic L-amino acid decarboxylase.
In some embodiments, the antagonist is carbidopa, benserazide, methyldopa, 3′,4′,5,7-Tetrahydroxy-8-methoxyisoflavone (DFMD), 3-hydroxybenzylhydrazine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), or α-difluoromethyl DOPA. In some embodiments, the bacterial strain is B. thetaiotaomicron. In some embodiments, the bacterial strain is a strain C34 of B. thetaiotaomicron.
In some embodiments, the antibiotic is ampicillin, clavulanate, tazobactam, a cephamycin, ticarcillin, piperacillin, a cephalosporin, a carbapenem, clindamycin, lincomycin, chloramphenicol, a nitroimidazole, a fluoroquinolone.
In some embodiments, the antibiotic comprises (a) a combination of ampicillin and sulbactam, (b) a combination of ticarcillin and clavulanate, or (c) a combination of piperacillin and tazobactam.
In various embodiments of the above, the method further comprises administering a probiotic composition. In some embodiments, the probiotic composition is administered after antibiotic administration.
In some embodiments, the administration of the antibiotic and the administration of the probiotic composition are repeated in a cycle.
In another aspect is provided a kit for evaluating potential toxicity of an MAOI in a subject, the kit comprising a nucleic acid, antibody, or other reagent capable of binding specifically to a nucleotide or protein expressed by a bacterial strain, wherein the bacterial strain comprises a nucleic acid sequence encoding a phenethylamine production gene. In some embodiments, the bacterial strain is a bacterium of the Morganella spp. In some embodiments, the bacterial strain is Morganella morganii.
In another aspect is provided a kit for evaluating potential effectiveness of an antibiotic to treat a gastrointestinal condition or disease, allergy or asthma in a subject, the kit comprising a nucleic acid, antibody, or other reagent capable of binding specifically to a nucleotide or protein expressed by a bacterial strain, wherein the bacterial strain comprises a nucleic acid sequence encoding a histamine production gene. In some embodiments, the bacterial strain is L. reuteri or a bacterium of Morganella spp.
In one aspect is provided a method allowing for parallel screening of multiple GPCRs (e.g., all 300 or more conventional GPCRs) in a single tube. Exemplary embodiments of this method are referred to herein as PRESTO-Salsa, in which instead of a luciferase reporter, reporter plasmids that encode unique nucleic acid barcodes are used.
Without wishing to be bound by theory, trillions of bacteria that constitutively colonize human intestines (gut microbiota) produce tens of thousands of unique small molecules that can potentially affect nearly all aspects of human physiology, from regulating immunity in the gut to shaping mood and behavior. However, this complexity can make it challenging to identify biologically relevant microbiota metabolites hidden in a sea of other chemicals. In other words, given the presence of tens of thousands of unique, unannotated metabolites in the microbiota metabolome, it can be difficult to decide which features should be prioritized for in-depth examination and characterization.
The inventors propose a revolutionary new approach to these problems. Without wishing to be bound by theory, the inventors describe host sensing of microbiota metabolites as a lens to illuminate the ‘dark matter’ that constitutes the majority of the bioactive microbiota metabolome. The inventors hypothesize that the hundreds of G-protein coupled receptors (GPCRs) encoded in the human genome can be used to identify novel bioactive microbiota metabolites from complex mixtures.
A new approach is described herein to investigate the bioactive microbiota metabolome where we used the sensing of microbiota metabolites by host GPCRs as a lens to illuminate bioactive metabolites produced by individual gut microbes. The examples described herein describe how the approach revealed a plethora of novel microbiota metabolite-GPCR interactions of potential physiological importance. For example, the inventors uncovered a diet-microbe-host axis that influences intestinal motility through the microbial production of histamine and a microbe-microbe-host axis that results in the production of the potent trace amine phenethylamine. Both of these axes can have profound effects on local and systemic host physiology. The functional profiling-based approach to understanding the contribution of the microbiota to human physiology described herein may be broadly applicable to understanding and illuminating diverse features of the bioactive microbiota metabolome.
An existing low-throughput GPCR-screening technology (PRESTO-Tango) was used by the inventors to identify metabolite mixtures from individual human gut bacteria that activated GPCRs involved in carcinogenesis, mood regulation, and immunity, as well as ‘orphan’ GPCRs whose natural ligands have evaded discovery for decades. However, because of inherent technical limitations, PRESTO-Tango can only be applied to a relatively small number of metabolite mixtures that are readily available in large quantities—e.g., metabolites produced by bacterial strains that can be cultured in vitro. PRESTO-Tango can thus only capture a small proportion of the overall bioactive microbiota metabolome, which normally results from complex in vivo interactions between multiple microbes, dietary compounds, and the host itself.
The inventors have leveraged recent developments in next-generation sequencing and nucleic acid barcode analyses to develop PRESTO-Salsa.
In one aspect a method is provided for high-throughput screening of microbiota metabolites capable of activating a plurality of G-protein coupled receptors (GPCRs). A plurality of non-adherent mammalian cells is provided. Each cell comprises (i) a first nucleic acid molecule encoding a first fusion protein comprising a GPCR linked to the transcription factor tTA via a cleavage site for Tobacco Etch Virus nuclear inclusion A protease (TEV protease), (ii) a second nucleic acid molecule encoding a second fusion protein comprising β-arrestin and TEV protease configured to cleave the TEV protease site on the first fusion protein, and (iii) a third nucleic acid molecule comprising a barcode sequence operably linked to a promoter specifically activated by the tTA transcription factor, wherein each barcode sequence is specific for an individual GPCR. The plurality of cells is contacted with a plurality of microbiota metabolites. The barcodes are sequenced. A determination is made as to which barcode sequences are increased in the presence of the metabolites as compared to a control where no metabolites are present.
Transcription of a barcode can indicate the activation of a GPCR. Without wishing to be bound by theory, when a GPCR is activated, the conformation of the GPCR changes such that a fusion protein comprising β-arrestin and TEV protease is recruited. The TEV protease then cleaves the protease site linking the tTA transcription factor and the GPCR so as to release the tTA transcription factor, which in turn drives transcription of a barcode operatively linked to the promoter.
A variety of GPCRs can be used. The GPCR may be a non-olfactory GPCR, an orphan GPCR, or a human GPCR. In preferred embodiments, the GPCR is one of the 314 conventional GPCRs described herein.
The microbiota metabolites may come from any number of samples, such as a fecal sample, or a sample from the gastrointestinal tract. The fecal sample or the sample from the gastrointestinal tract may be cultured and/or exposed to nutrients so as to enhance production of a particular microbiota metabolite. For example, additional L-Phe and L-His can be supplied to increase sensitivity of detection of M. morganii, which can directly convert L-Phe and L-His into phenethylamine and histamine, respectively. The sample may also be fractionated based on chemical properties, for example by reversed phase HPLC.
Without wishing to be bound by theory, the use of barcodes can greatly improve sensitivity of detection and allow for high-throughput screening. A larger number of cells expressing a wider variety of unique GPCR-barcode pairs can be used. Highly sensitive quantitative methods can be used to assay for the expression of all barcodes, e.g., as described in Example 1.
As shown in
By pooling cells that express unique GPCR-barcode pairs and then quantifying the expression of specific barcodes after stimulation, activation or inhibition of hundreds of GPCRs in a single tube can be simultaneously screened for. See,
The method can further comprise identifying a bacterial strain producing the specific metabolite which activates the specific GPCR. Bacterial strains can be classified by 16S rRNA sequencing, for example, or by whole genome sequencing.
Individual bacterial strains can be isolated and cultured from a particular sample of interest, with metabolites from each cultured strain further tested in the various PRESTO-Salsa embodiments described herein. Alternatively, a bacterial strain can be added to a microbiota sample to assess the metabolites produced by the bacterial strain in the context of a given microbiome. The microbiota sample can then be cultured to allow the added bacterial strain to produce additional metabolites. A differential analysis of the barcodes produced by the PRESTO-Salsa method can be undertaken.
It was found that dozens of human gut bacteria from diverse phyla, families, species, and strains produced small molecules that activated various GPCRs, including both well-characterized GPCRs and orphan GPCRs. Patterns of metabolite production were observed that were largely predictable based on phylogeny, as well as strain-specific differences within a given species. Future studies will be necessary to determine when and why specific pathways are conserved or not in distinct species and strains. Metabolites resulting from core metabolic processes essential to a given microbe might be highly conserved, while metabolites involved in competitive processes may show considerable strain variation that could depend on the niche that is occupied and the need to compete with other bacterial species, non-bacterial microbes, or with the host. Regardless of the initial impetus for microbial metabolite expression, the data described herein support the concept that human-associated microbes exhibit impressive regulatory capabilities and are likely one of the richest sources of small molecules that impact human biology.
The high throughput of PRESTO-Salsa can allow for ability to easily examine biological or technical replicates, as well as perform dose response curves, for all samples. Finally, PRESTO-Salsa may be resistant to well-known off-target effects of certain chemicals on luciferase stability and thus may also reduce false positives.
PRESTO-Salsa provides a major technical advantage in that hundreds of receptors may be screened simultaneously in a single tube using small sample input volumes. PRESTO-Salsa is modular and flexible, and thus can be expanded to include other receptor families. Also, PRESTO-Salsa can easily be modified to simultaneously monitor multiple signaling outputs downstream of the same receptors (e.g., G-protein based signaling versus B-arrestin based signaling by GPCRs) to allow for identification of biased agonists and antagonists. PRESTO-Salsa enables screening of precious low-volume samples against hundreds of sensors while a similar sample volume used in PRESTO-Tango would only have been sufficient to examine the effects on one or two receptors. PRESTO-Salsa can provide for imminent scalability and automation. A major economic advantage of PRESTO-Salsa is that PRESTO-Salsa can enable simultaneous screening of hundreds of receptors in a single tube using low input volumes; prior technologies would require multiple plates of cells and a large input sample volume to accomplish this same goal. The use of next-gen sequencing as the final readout in PRESTO-Salsa is considerably cheaper than the prior Luciferase based screening (especially as next-gen sequencing continues to decline in cost). The cost of screening using PRESTO-Salsa may be lower than that of established assays by at least an order of magnitude.
The microbiota metabolome results from a complex web of interactions between multiple microbial species and strains, environmental inputs (e.g., diet), and host factors. Using a reductionist approach, the inventors discovered two bacterial isolates that traffic in the same small molecule: a unique strain of B. theta that is a prolific producer of L-Phe and M. morganii, which efficiently converts L-Phe into phenethylamine. The approaches described herein can reveal metabolic exchanges that may be missed when examining endpoint microbiota metabolomes produced by complex mixtures of microorganisms (e.g., complete gut microbial communities).
In various embodiments of the methods described herein, any GPCR agonists can be further tested in vivo, for example to examine the possibility that production of GPCR agonists by specific microbes would shape host physiology in vivo. The data in the examples show that histamine production by M. morganii or L. reuteri promotes increased colon motility and that feeding with exogenous histidine further increases microbial production of histamine and colonic motility. M. morganii monocolonized mice fed with histidine exhibited elevated levels of systemic histamine, indicating that microbiota-derived histamine may also shape systemic immune responses.
The inventors' studies also uncovered a specific Bacteroides strain that uniquely produces high levels of the essential amino acid L-Phe and revealed that L-Phe could activate the orphan GPCRs GPR56 and GPR97. GPR56 is highly expressed in the small intestine and human pancreatic islets (Amisten et al., 2013; Duner et al., 2016), and L-Phe concentrations in the jejunum can reach concentrations up to 2 mM after a meal (Adibi, 1973). Thus, GPR56 may act as a nutrient sensor to regulate digestion and satiety.
Although L-Phe levels in the serum usually are well below the levels necessary to activate GPR56/97, patients with phenylketonuria (PKU) who cannot degrade L-Phe exhibit serum concentrations of L-Phe higher than 1 mM (Williams, 2008). Thus, activation of GPR56 and/or GPR97 may contribute to some of the symptoms of PKU. Notably, GPR56 is highly expressed in neural progenitor cells, oligodendrocytes, astrocytes and microglia (Giera et al., 2018; Haitina et al., 2008), and GPR56/AGRG1 deficiency can cause severe neurodevelopmental diseases such as bilateral frontoparietal polymicrogyria (BFPP) (Sotnikova et al., 2004).
Without wishing to be bound by theory, dietary amino-acid availability can be important in the production of biogenic amines that can shape host physiology. The studies herein can highlight other members of the microbiota as an alternative source of substrates that are often thought of as primarily derived from diet (e.g., essential amino acids). Microbial-produced amino acids may potentially supplement or even replace dietary amino acids in microbial biotransformations. Microbe-derived L-Phe may be used as a substrate for biotransformation by M. morganii using a simplified and highly-artificial diet that lacks L-Phe. However, bacterial L-Phe may also be important under physiological conditions. For example, dietary amino acids are largely absorbed in the small intestine (Adibi, 1973); thus, colonic microbes such as M. morganii have relatively limited access to dietary amino acids as compared to small intestinal organisms. Also, low-protein diets naturally decrease microbial access to dietary amino acids, and fasting may reduce intestinal amino acid availability even further (Pezeshki et al., 2016). Thus, microbial production of amino acids in the colon may play a critical role in the production of various bioactive microbiota metabolites under a variety of physiologically relevant conditions.
Also provided are various therapeutic and diagnostic methods.
In one aspect is provided a method of preventing or treating monoamine oxidase inhibitor (MAOI)-induced toxicity in a subject. An antibiotic effective to target a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene is administered.
In a related aspect is provided a method of preventing or treating monoamine oxidase inhibitor (MAOI)-induced toxicity in a subject. A small molecule antagonist of a bacterial phenethylamine synthesis enzyme is administered.
In various embodiments, the method of preventing or treating MAOI-induced toxicity in a subject comprising administering an antibiotic effective to target Morganella spp.
Although M. morganii was previously reported to produce dopamine, the inventors found that all isolates of M. morganii primarily produced the potent trace amine phenethylamine rather than dopamine or tyramine. The inventors also found that treatment of M. morganii monocolonized mice with an MAOI led to systemic accumulation of phenethylamine and mortality. Phenethylamine is a potent neuroactive chemical that, unlike dopamine and tyramine, can readily cross the blood-brain barrier (Oldendorf, 1971). The effects of phenethylamine are thought to be mediated primarily through activation of the trace amine-associated receptors and subsequent release of norepinephrine and dopamine (Borowsky et al., 2001; Bunzow, 2001; Sotnikova et al., 2004). However, the data here suggests that phenethylamine also can act as a selective agonist for DRD2-4. It will be fascinating to dissect the cellular and molecular mechanisms by which microbiota-derived phenethylamine can influence host biology both locally and systemically.
Without wishing to be bound by theory, the inventors' findings suggest that interindividual variability in microbial production of phenethylamine may explain some of the variable effects of MAOIs on depression; this possibility is particularly intriguing given the reported beneficial effects of phenethylamine on mood and the ability of phenethylamine to cross the blood-brain barrier (Irsfeld et al., 2013). In addition, biogenic amine production by gut microbes may explain some of the side effects of MAOIs.
One of the most prominent adverse events associated with MAOIs is ‘tyramine poisoning’, which typically results from ingestion of food products containing high levels of tyramine, such as certain cheeses (Fiedorowicz, 2004). The data herein indicates that specific gut microbes also act as a source of biogenic amines that may have similar effects on host physiology and that it is possible that pharmacological inhibitors of biogenic amine receptors that are meant to act at specific sites (e.g., in the brain) may also impinge upon natural host-microbiota interactions.
Also provided is a method of treating a disease or condition caused by decreased MAO activity in a subject. An antibiotic effective to target a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene is administered.
In a related aspect is provided a method of treating a disease or condition caused by decreased MAO activity in a subject. A small molecule antagonist of a bacterial phenethylamine synthesis enzyme is administered.
The bacterial strain may produce phenethylamine, such as M. morganii. The bacterial strain may secrete phenethylamine. Various diseases or conditions caused by decreased MAO activity can be treated, including Brunner syndrome, autism or anti-social behavior. The subject may express a MAOA-L variant.
Various antibiotics effective to target Morganella morganii may be used, such as cefepime, piperacillin, tazobactam, ceftazidime, cefotaxime, ceftibuten, meropenem, doripenem, ertapenem, a fluoroquinolone, or an aminoglycoside.
Also provided is a method of treating depression in a subject comprising administering a bacterial strain comprising a nucleic acid sequence encoding a phenethylamine production gene. An anti-depressant, such as an MAOI, can also be administered to the subject.
Also provided is a method for evaluating potential toxicity of a monoamine oxidase inhibitor (MAOI) in a subject. A gastrointestinal microbiota sample is obtained from the subject. The sample is assayed for the presence of a bacterial strain comprising a phenethylamine production gene, such as Morganella morganii. The levels of the bacterial strain can also be quantitatively assayed so as to further assess the risk of potential MAOI toxicity. The presence of, and/or levels of, the bacterial strain can be used to assess the risk of MAOI toxicity. From this information, a different anti-depressant or therapeutic besides a MAOI may be administered. Alternatively, the dosage levels of MAOI may be adjusted or reduced.
Also provided is a method for evaluating method for evaluating potential efficacy of an MAOI in a subject. The method comprises taking a fecal sample or a sample from the gastrointestinal tract of the subject, and assaying for the presence of a bacterial strain (e.g., Morganella morganii) comprising a nucleic acid sequence encoding a phenethylamine production gene. A quantitative assay for the amount of the bacterial strain may also be conducted. The subject may then be treated with an MAOI. The dosage or type of MAOI can be adjusted based on the presence of, and/or amount of, the bacterial strain.
Also provided is a method for method of preventing or treating histamine-induced gastrointestinal disease in a subject. Notably, the inventors discovered that histidine decarboxylases, including M. morganii histidine decarboxylase, are enriched in patients with Crohn's disease as compared to healthy controls or UC patients (see
An exemplary disease is histamine intolerance, which can mimic the symptoms of food allergy. Another is diarrhea. Histamine-induced gastrointestinal diseases can be caused by, or exacerbated by, interference with the activity of the enzymes DAO and HNMT. Without wishing to be bound by theory, the presence of, or an excess amount of, L. reuteri or a bacterium of Morganella spp. can cause or exacerbate intestinal disease. These can directly convert L-His into histamine.
Another exemplary disease is inflammatory bowel disease. The abundance of Morganella spp. encoding histidine decarboxylase is increased in Crohn's disease versus healthy controls and ulcerative colitis (
Also provided is a method of preventing or treating an allergy, or asthma, in a subject. One or more antibiotics effective to target a bacterial strain comprising a nucleic acid sequence encoding a histamine production gene is administered. The bacterial strain can be L. reuteri or a bacterium of Morganella spp. Without wishing to be bound by theory, the presence of either or both strains can increase overall histamine levels in the subject, which would induce or exacerbate allergies or asthma. Exemplary antibiotics include cefepime, piperacillin, tazobactam, ceftazidime, cefotaxime, ceftibuten, meropenem, doripenem, ertapenem, a fluoroquinolone, or an aminoglycoside.
The antibiotic may be selected for based on the outcome of a method for evaluating potential effectiveness of an antibiotic to treat a gastrointestinal condition or disease, allergy or asthma in a subject. The method comprises a) taking a fecal sample or a sample from the gastrointestinal tract of the subject, and b) assaying for the presence of a bacterial strain (e.g., bacterium of Morganella spp.) comprising a nucleic acid sequence encoding a histamine production gene. The method can comprise assaying for the amount of the bacterial strain present in the gastrointestinal tract. The subject may be treated with the antibiotic. In some embodiments, an adjusting or determining the dosage of the antibiotic is undertaken based on the presence and/or amount of the bacterial strain present.
Also provided is a method of preventing or treating a disease or condition resulting from production of phenethylamine, the method comprising administering one or more antibiotics effective to target a bacterial strain producing L-phenylalanine. In a related aspect is provided a method of preventing or treating a disease or condition resulting from production of phenethylamine, the method comprising administering a small molecule antagonist of a phenethylamine synthesis enzyme. The small molecule antagonist may be a Shikimate pathway inhibitor. The small molecule antagonist may be an antagonist of aromatic L-amino acid decarboxylase (AADC or AAAD), such as carbidopa, benserazide, methyldopa, 3′,4′,5,7-Tetrahydroxy-8-methoxyisoflavone (DFMD), 3-hydroxybenzylhydrazine, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), and α-difluoromethyl DOPA.
Further provided is a method of preventing or treating phenylketonuria (PKU) in a subject, the method comprising administering one or more antibiotics effective to target a bacterial strain producing L-phenylalanine. Also provided is a related method of preventing or treating phenylketonuria (PKU) in a subject, the method comprising administering a small molecule targeting the bacterial phenylalanine synthesis enzymes of bacterial strains secreting L-phenylalanine for the treatment or prevention of diseases resulting from overproduction of L-phenylalanine.
Exemplary bacterial strains include B. thetaiotaomicron (e.g., strain C34). The method may comprise administration of an antibiotic effective to target the bacterial strain, such as ampicillin, clavulanate, tazobactam, a cephamycin, ticarcillin, piperacillin, a cephalosporin, a carbapenem, clindamycin, lincomycin, chloramphenicol, a nitroimidazole, a fluoroquinolone. Alternatively, a combination of antibiotics may be administered, such as (a) a combination of ampicillin and sulbactam, (b) a combination of ticarcillin and clavulanate, or (c) a combination of piperacillin and tazobactam.
In various embodiments of the above methods involving administration of an antibiotic, a probiotic composition may be administered. The probiotic composition may be administered after a course of antibiotics is administered, for example. Without wishing to be bound by theory, the probiotic composition may be effective to colonize the gut with different bacteria and prevent the targeted bacteria from quickly reestablishing itself. In additional embodiments, the antibiotic can be administered in a cyclic manner with the probiotic composition. The antibiotic could even be administered concurrently with the probiotic composition.
Also provided is a kit for evaluating potential toxicity of an MAOI in a subject. The kit can comprise a nucleic acid, antibody, or other reagent capable of binding specifically to a nucleotide or protein expressed by a bacterial strain, where the bacterial strain comprises a nucleic acid sequence encoding a phenethylamine production gene.
Also provided is a kit for evaluating potential effectiveness of an antibiotic to treat a gastrointestinal condition or disease, allergy or asthma in a subject. The kit comprises a nucleic acid, antibody, or other reagent capable of binding specifically to a nucleotide or protein expressed by a bacterial strain, where the bacterial strain comprises a nucleic acid sequence encoding a histamine production gene (such as one detected in Crohn's disease patients according to data shown in
The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
The following table shows the reagents used throughout the following examples:
B. fragilis
B. ovatus
B. thetaiotaomicron
B. uniformis
M. morganii
To facilitate pooling of GPCR-barcode pairs, an Expi 293T cell line was generated that stably expresses the Barr-TEV fusion protein. These cells can be efficiently co-transfected with a plasmid that encodes a given GPCR-tTA fusion and a reporter plasmid that encodes a defined barcode downstream of a tet-responsive element. To enable concurrent screening of all GPCRs, the stable Expi 293T cell line was co-transfected with all 314 defined GPCR-barcode reporter plasmid pairs in individual wells in 96 well plates. After about 24 hours, all cells were mixed together in a single tube before the cellular mixtures were redistributed into approximately four 96 well plates. Importantly, Expi 293T cells are non-adherent, which facilitates pooling and redistribution of cells.
After redistribution, each well was stimulated with a given metabolite mixture before harvesting RNA, synthesizing cDNA and then sequencing barcodes via Illumina amplicon sequencing. Relative expression levels of a given barcode were compared to control samples so as to represent the relative activation of a given GPCR by that metabolite or metabolite mixture. To assess the sensitivity and specificity of PRESTO-Salsa, the inventors first established this system using well-known GPCR ligands or microbial metabolite mixtures that contain known GPCR agonists, as shown in
These data demonstrate the specificity and sensitivity of the PRESTO-Salsa technology. Importantly, the sequencing-based readout used for PRESTO-Salsa was considerably more sensitive than PRESTO-Tango given the relative sensitivity of sequencing versus luciferase expression.
144 unique human gut bacteria spanning five phyla, nine classes, eleven orders, and twenty families were isolated from 11 inflammatory bowel disease patients via high-throughput anaerobic culturomics and massively barcoded 16S rRNA gene sequencing. All strains were cultured in gut microbiota medium (Goodman et al., 2011) or Gifu broth at 37° C. under anaerobic conditions and the identities of all strains were confirmed by 16S rRNA gene sequencing. The isolated human gut bacteria are listed in Table 1. Each isolate was grown in monoculture in a medium specialized for the cultivation of human gut microbes (gut microbiota medium; GMM).
Supernatants from individual bacterial monocultures were screened against the near-complete non-olfactory GPCRome (314 conventional GPCRs) using the high-throughput assay Parallel Receptor-ome Expression and Screening via Transcriptional Output-Tango (PRESTO-Tango).
The PRESTO-Tango assay was conducted as follows. HTLA cells, a HEK293 cell line that stably expresses β-arrestin-TEV and tTA-Luciferase (a kind gift from Gilad Barnea, Brown University), were plated in 96-well tissue culture plates (Eppendorf) in DMEM containing 10% FBS and 1% Penicillin/Streptomycin. One day after plating (after reaching approximately 90% confluence) Tango plasmids were transfected into HTLA cells using polyethylenimine (Polysciences). 16-24 hours after transfection, medium was replaced with 180 μl fresh DMEM containing 1% Penicillin/Streptomycin and 10 mM HEPES and 20 μl bacterial medium alone or bacterial supernatant. For PRESTO-Tango screening, all commensals were cultured in gut microbiota medium or Gifu broth for 2 days in an anaerobic chamber (Coy). Commensal supernatants were sterilized by high-speed centrifugation followed by sterile filtration (0.22 μm). For in vitro studies, M. morganii was cultured in minimal medium (MM), or MM with 10 mM L-Phe, 2.5 mM L-Tyr, 10 mM L-DOPA, or 10 mM L-His for 24 hours. Bacterial supernatants were analyzed by LC-MS.
16-24 hours after stimulation, 50 ul per well of Bright-Glo solution (Promega) diluted 20-fold with PBS containing 20 mM HEPES was added into each well. After 20 min incubation at room temperature, luminescence was quantified using a Spectramax i3x (Molecular Devices). Activation fold for each sample was calculated by dividing relative luminescence units (RLU) for each condition by RLUs from media alone controls.
GPCR activation by metabolomes from 144 bacterial strains isolated from the human gut microbiota as measured by PRESTO-Tango. Data is displayed as a heatmap (
Bacteroides
fragilis
Streptococcus
luteciae
Clostridium
perfringens
Fusobacterium
Streptococcus
Bacteroides
fragilis
Megasphaera
Clostridium
perfringens
Bacteroides
fragilis
Bifidobacterium
Bacteroides
fragilis
Morganella
Bacteroides
Bacteroides
ovatus
Bifidobacterium
Bifidobacterium
Bacteroides
fragilis
Fusobacterium
Bifidobacterium
adolescentis
Bifidobacterium
adolescentis
Morganella
Sutterella
Bacteroides
Bacteroides
Bacteroides
fragilis
Bifidobacterium
adolescentis
Lactobacillus
Pediococcus
Pediococcus
Bacteroides
fragilis
Bifidobacterium
Bifidobacterium
Morganella
Streptococcus
Clostridium
perfringens
Bifidobacterium
Erysipelotrichaceae
Collinsella
aerofaciens
Collinsella
aerofaciens
Clostridium
perfringens
Streptococcus
Bacteroides
ovatus
Bacteroides
Bacteroides
fragilis
Bacteroides
Bacteroides
fragilis
Parabacteroides
distasonis
Clostridium
perfringens
Morganella
Peptoniphilus
Morganella
Peptoniphilus
Collinsella
aerofaciens
gnavus
gnavus
Bacteroides
Bacteroides
uniformis
Bacteroides
ovatus
Collinsella
stercoris
Collinsella
stercoris
Collinsella
stercoris
Lactobacillus
reuteri
Lactobacillus
reuteri
Bacteroides
uniformis
Bacteroides
Nevskia
Lactobacillus
reuteri
Lactobacillus
reuteri
Nevskia
Acidaminococcus
Bacteroides
uniformis
Bacteroides
uniformis
Bifidobacterium
Acidaminococcus
Acidaminococcus
Bacteroides
Acidaminococcus
Acidaminococcus
Bifidobacterium
Bifidobacterium
Bifidobacterium
Bifidobacterium
Bacteroides
uniformis
Bacteroides
ovatus
Megasphaera
gnavus
Pediococcus
Pediococcus
Parabacteroides
distasonis
Eggerthela
lenta
Blautia
producta
Faecalibacterium
prausnitzii
Clostridium
perfringens
Streptococcus
Eggerthela
lenta
torques
Bacteroides
uniformis
Allobaculum
Parabacteroides
Collinsella
stercoris
Peptoniphilus
Bacteroides
fragilis
Oscillospira
Morganella
Eggerthela
lenta
Morganella
Oscillospira
Oscillospira
Mogibacterium
Enhydrobacter
The culture collection can allow for examination of the effects of phylogenetically diverse taxa while also providing insights into potential strain-specific differences between members of the same species.
An analysis of the above cultures indicated that human gut microbes produce compounds that activate both well-characterized and orphan GPCRs. PRESTO-Tango screening revealed a diverse array of hits, including bacterial-derived metabolite mixtures that activated well-characterized GPCRs as well as mixtures that activated orphan receptors. According to the data in
Specific patterns of GPCR activation emerged based on gross phylogeny. Tables 2 and 3 show the degree of receptor activation for each bacteria according to PRESTO-Tango analysis in each of GMM and Gifu media, respectively. For example, most strains belonging to the phyla Bacteroidetes and Proteobacteria potently activated the succinate receptor (Sucr1), while strains belonging to the phyla Firmicutes, Fusobacteria and Actinobacteria largely failed to activate this receptor (Tables 2-3). However, many activation patterns did not correlate with phylogeny, including multiple examples of bacterial strains that exhibited unique GPCR agonist activities despite being assigned to the same species (Tables 2-3).
Ruminococcus
Ruminococcus
Ruminococcus
Ruminococcus
gnavus-1#
gnavus-2#
gnavus-3#
torques
Faecalibacterium
Oscillospira
Oscillospira
Lactobacillus
Lactobacillus
prausnitzii
reuteri-1#
reuteri-2#
Lactacillus
Pediococcus
Pediococcus
Pediococcus
Streptococcus
Streptococcus
Streptococcus
Streptococcus
luteciae
Acidamino-
Acidamino-
Acidamino-
Pepto-
Pepto-
coccus
coccus
coccus
Megasphaera
Megasphaera
niphilus
niphilus
Bacteroids
fragilis-1#
Bacteroids
Bacteroids
Bacteroids
Bacteroids
Bacteroids
Bacteroids
Bacteroids
Bacteroids
fragilis-2#
fragilis-3#
fragilis-4#
fragilis-5#
fragilis-6#
fragilis-7#
fragilis-8#
fragilis-9#
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
ovatus-1#
ovatus-2#
ovatus-3#
uniformis-1#
uniformis-2#
uniformis-3#
uniformis-4#
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Parabacteroides
distasonis-1#
Fusobacterium
Fusobacterium
Collinsella
Collinsella
Collinsella
Collinsella
Collinsella
aerofaciens-1#
aerofaciens-2#
aerofaciens-3#
stercoris-1#
stercoris-2#
Bifidobac-
Bifidobac-
Bifidobac-
Bifidobac-
Bifidobac-
Bifidobac-
Bifidobac-
Eggerthela
terium
terium
terium
terium
terium
terium
terium
lenta
Bifidobacterium
Bifidobacterium
Bifidobacterium
Morganella
Morganella
Morganella
Morganella
Morganella
adolescentis-1#
adolescentis-2#
adolescentis-3#
Morganella
Morganella
morganii-
morganii-
Morganella
Ruminococcus
Ruminococcus
Ruminococcus
Blautia
gnavus-1#
gnavus-2#
torques
producta
Faecalibacterium
Oscillospira
Oscillospira
Oscillospira
Lactobacillus
Lactobacillus
Lactobacillus
Lactobacillus
prausnitzii
reuteri-1#
reuteri-2#
reuteri-3#
Pediococcus
Pediococcus
Pediococcus
Streptococcus
Streptococcus
Streptococcus
Streptococcus
luteciae
Acidamino-
Acidamino-
Acidamino-
Acidamino-
coccus
coccus
coccus
coccus
Megasphaera
Megasphaera
Peptoniphilus
Peptoniphilus
Peptoniphilus
Bacteroids
Bacteroids
Bacteroids
Bacteroides
Bacteroides
Bacteroides
Bacteroides
fragilis-1#
fragilis-2#
fragilis-3#
ovatus-1#
ovatus-2#
ovatus-3#
ovatus-4#
B.
B.
B.
B.
B.
B.
Bacteroides
Bacteroides
ovatus-5#
thetao-1#
thetao-2#
eggerthii
Caccae
copri
uniformis-1#
uniformis-2#
Bacteroides
Bacteroides
Bacteroides
Bacteroides
Parabacteroides
Parabacteroides
Fusobacterium
Fusobacterium
uniformis-3#
distasonis-1#
distasonis-2#
Bifidobac-
Bifidobac-
Collinsella
Collinsella
Collinsella
Collinsella
Collinsella
Collinsella
terium
terium
aerofaciens-1#
aerofaciens-2#
aerofaciens-3#
stercoris-1#
stercoris-2#
stercoris-3#
Bifidobacterium
Bifidobacterium
Bifidobacterium
Morganella
Morganella
Morganella
Morganella
Morganella
adolescentis-1#
adolescentis-2#
adolescentis-3#
Morganella
Besides the succinate receptor, the next most prevalent class of GPCRs activated by gut commensals was the aminergic receptors, which are expressed in diverse tissues and cell types and regulate a wide variety of core physiological processes ranging from neurotransmission to immunity. See,
In contrast, two strains of L. reuteri that were found to activate HRHs, while two distinct strains of L. reuteri failed to activate HRHs despite displaying similar growth kinetics. See,
M. morganii was previously reported to produce dopamine as measured by high-performance liquid chromatography (HPLC) (Ozogul, 2004). However, it was observed that all M. morganii supernatants potently activated DRD2-4, but not DRD1 and DRD5, as shown in
The inventors then examined the ability of all possible upstream and downstream metabolites in the mammalian dopamine pathway to activate DRD1-5 via PRESTO-Tango (
Previous reports have also suggested that M. morganii produces histamine (Ozogul, 2004). M. morganii strains indeed secreted significant amounts of histamine, as confirmed by ELISA (
Similarly, two strains of L. reuteri and two strains from the Enterobacteriaceae family that activated histamine receptors also secreted histamine (
In mammals, phenethylamine, dopamine, and tyramine are produced via the decarboxylation of L-Phe, L-DOPA, and L-Tyr, respectively, by the aromatic L-amino acid decarboxylase (AADC;
However, supplementation with L-Phe or L-His led to the production of high levels of phenethylamine or histamine, and activation of DRD2-4 or HRH2-4, as shown in
Histamine is generated via decarboxylation of L-His (Tannase, 1985). Eight strains of M. morganii and two strains of L. reuteri generated histamine in vitro. Supplementation with L-His significantly increased histamine production by these strains. In contrast, two distinct strains of L. reuteri failed to produce histamine regardless of supplementation with L-His, according to the results shown in
To test whether M. morganii and L. reuteri can also produce histamine in vivo, germ-free mice were colonized with two distinct mock communities containing (i) 9 or 10 diverse human gut microbes or (ii) M. morganii, with or without supplementation of 1% L-His in the drinking water ad libitum to approximate the effect of an L-His-rich diet, e.g., a meat-heavy diet. 6-12 weeks-old germ-free wild-type C57Bl/6 mice of both sexes were used in all experiments. The germ-free C57Bl/6 mice were colonized via oral gavage with 200 μl of individual bacterial cultures or mixed bacterial consortia. Mock communities A and B consisted of the following taxa: Community A: Streptococcus spp; C. perfringens; B. fragilis; Erisipelotrichaceae spp; C. aerofaciens; Bacteroides UC; B. producta; Allobaculum spp and Oscillospira spp; and Community B: Bacteroides spp; P. distasonis; Peptoniphilus spp; B. ovatus; Clostridiales UC/UC; Lachnospiraceae UC/UC; C. stercoris; B. uniformis and Parabacteroides spp. All gnotobiotic mice were maintained in Techniplast P Isocages and manipulated aseptically for the duration of the experiment.
The data is shown in
The location of M. morganii was next determined in vivo. Modified Niven's agar was used to enumerate M. morganii CFUs in gnotobiotic mice colonized with two mock communities of 9 or 10 diverse human gut microbes plus M. morganii (Mavromatis, 2002). The inventors found that M. morganii primarily inhabits the cecum and colon, and is nearly absent in the small intestine. See,
Oral gavage with histamine has been reported to increase colon motility in rodents (Kim et al., 2011; Tyagi et al., 2009). The inventors thus hypothesized that gut microbe-derived histamine might also increase intestinal motility. The inventors assayed intestinal motility in mice colonized with two mock communities containing phylogenetically diverse human gut microbes or with M. morganii with or without administration of 1% L-His in the water. Intestinal motility was assayed by measuring fecal output as follows. Individual mice were housed in an empty container (¼ gallon) for 1 hour after which time the fecal pellets were counted and weighed. For mice fed with L-His, mice were given water containing 1% L-His ad libitum for 2 weeks before fecal output measurements.
Colonization with M. morganii led to a significant increase in fecal output, which was further increased upon supplementation with L-His. See,
While abundant histamine production by M. morganii was detected both in vitro and in vivo, only low levels of phenethylamine were detected in the colons of M. morganii colonized mice (
Increased phenethylamine was observed in the colons of M. morganii colonized mice by triple quadrupole MS (
Unlike many other irreversible MAOIs, phenelzine is still used clinically for the treatment of major depressive disorder, as well as a variety of other psychological disorders including panic, social anxiety, and post-traumatic stress disorders (Fiedorowicz, 2004). The inventors found that mice colonized with M. morganii became lethargic within days after treatment with MAOI, and more than half of all mice colonized with M. morganii died before the seventh day of treatment. See,
Morbidity and mortality after MAOI treatment correlated with elevated levels of phenethylamine in the colon, serum and brains of M. morganii monocolonized mice treated with phenelzine, as measured by QQQ MS. The results are shown in
Specific bacterial supernatants were observed to activate select orphan GPCRs (
Since there is no known endogenous small molecule ligand for GPR56/AGRG1 (Purcell, 2018), the inventors next attempted to identify the specific metabolite produced by B. theta C34 that activated GPR56/AGRG1. B. theta C34 supernatants were lyophilized, extracted with methanol and subjected to fractionation by reversed-phase HPLC. All resulting fractions were analyzed for activity via GPR56 PRESTO-Tango, with fraction 11 was identified as the active fraction. See,
Metabolomic assays were performed as follows. NMR spectra were taken using an Agilent 600 MHz NMR system with a cryoprobe. High-resolution MS and tandem MS (MS/MS) data were obtained using an Agilent iFunnel 6550 ESI-HRMS-QTOF (Electron Spray Ionization-High Resolution Mass Spectrometry-Quadrupole Time-of-Flight) instrument on Phenomenex Kinetex 5 μm C18 100 Å (4.6×250 mm) columns. The Agilent 1260 Infinity system with a Phenomenex Kinetex 5 μm C18 100 Å column (4.6×250 mm) or an Agilent Poroshell 120 EC-C18 2.7 μm (3.0×50 mm) column and a photodiode array (PDA) detector was used for routine sample analysis. An Agilent Prepstar HPLC system with an Agilent Polaris C18-A 5 μm (21.2×250 mm) columns were used for sample fractionation and purification.
High resolution mass spectrometry, NMR and coinjection analyses of F11 showed that the essential amino acid phenylalanine (Phe) is the primary constituent of F11 (
The inventors next tested whether pure L-Phe or structurally related amino acids could activate GPR56/AGRG1 using GPR56-Tango. B. thetaiotaomicron strain C11 was grown in 10 mL of gut microbiota medium under anaerobic conditions at 37° C. for 24 hr. Supernatant was harvested, lyophilized and extracted with 2 mL methanol. The crude extract was then dried and fractionated using a preparative C18 HPLC system. The gradient used was 10-50% acetonitrile in water (with 0.01% trifluoroacetic acid) for 30 min, then 100% for 5 min. The fractions, which were collected every minute, were dried, resuspended in PBS buffer, and tested for bioactivity using PRESTO-Tango. The active fraction was characterized using ESI-HRMS-QTOF and NMR analyses. Stereochemistry was confirmed by advanced Marfey's analysis (
L-Phe and, to a lesser extent, L-Tyr stereoselectively activated GPR56/AGRG1, while L-Trp and L-His, D-Phe, D-Trp, D-His and D-Tyr showed no activity. See,
GPR56/AGRG1 is a member of the adhesion GPCR family. Adhesion GPCRs characteristically possess large extracellular domains that mediate interactions with a variety of protein ligands, such as components of the extracellular matrix (Purcell, 2018). The inventors assayed whether the extracellular domain of GPR56/AGRG1 was also required for activation by the small molecule L-Phe by constructing a truncation mutant of GPR56/AGRG1. Although this mutant is expressed normally (Kishore et al., 2016), it failed to respond to L-Phe. See,
The inventors next examined whether other orphan GPCRs might also respond to L-Phe. PRESTO-Tango screening was performed on all adhesion and orphan GPCRs stimulated with L-Phe. It was found that GPR97/AGRG3 was also activated by this compound, as shown in
The above reductionist studies revealed that B. theta C34 produces large amounts of L-Phe while M. morganii can process L-Phe into the trace amine phenethylamine. The inventors then performed an assay to address whether these two bacteria might participate in an active metabolic exchange in vivo. The first step in investigating this hypothesis was to determine whether B. theta C34 can directly synthesize L-Phe. Using a defined minimal medium lacking L-Phe (SACC), the inventors observed that B. theta C34 could directly synthesize significant amounts of L-Phe in vitro (
The inventors next examined whether M. morganii would directly process B. theta C34-derived L-Phe into phenethylamine. B. theta C34 was cultured in SACC medium and then transferred B. theta supernatant to a culture of M. morganii. B. theta C34-derived L-Phe was efficiently converted into phenethylamine by M. morganii, as shown in
These mice were then treated with phenelzine to reveal the potential production of phenethylamine. Mice colonized with M. morganii alone and fed an L-Phe deficient diet remained healthy and produced minimal phenethylamine (as measured by DRD2 activation by cecal and colonic extracts) despite MAOI treatment (
Salsa reporter cells (a stable cell line expressing a fusion protein of B-arrestin and TEV protease) were seeded into poly-D-lysine pretreated 96-well plate with 1004, DMEM+10% FBS+1% Pen/Strep. When cell density reached 90%, the cells were transfected with 100 ng of plasmid encoding each GPCR and 100 ng of a unique Salsa reporter plasmid in each well (1 well per GPCR and 314 wells total). Six hours after transfection, the cell medium was discarded followed by addition of 50 μl trypsin and 10 min incubation at 37 degrees Celsius. After digestion, an additional 504, of cell medium was added, and cells were pipetted 15 times to separate cell clusters into single cells. All transfected cells were then pooled into one 15 mL tube to generate a mixed cell library and centrifuged at 3,000 rpm for 5 min.
The supernatant was discarded, and cells were resuspended in the same volume of fresh cell medium. The cell pellet was pipetted 15 times to resuspend the cells, and then cells were reseeded at 100 μL/well into poly-D-lysine pretreated 96-well plates. Twelve hours after reseeding, the cell medium was discarded and replaced with 1004, of fresh DMEM+1% Pen/Strep, followed by stimulation with serial dilutions of the indicated GPCR ligands. Nine hours after ligand stimulation, mRNA was extracted from each well and used to prepare a amplicon DNA library for next-generation sequencing. Barcode reads after ligand stimulation divided by barcode reads without ligand stimulation were calculated to produce the fold activation for each GPCR and for each sample.
The data is shown in
The present application describes a number of examples and embodiments of the invention. Nevertheless, it must be borne in mind that various modifications of the described examples and embodiments can be developed, while not departing from the scope and the essence of the invention in principle. With this in mind, other embodiments are included in the scope of the items listed below. At that, all the numerical ranges described herein include all the sub ranges contained therein, as well as any individual values within the scope of these ranges. All publications, patents and patent applications mentioned in this description are hereby incorporated by reference.
This application claims priority to U.S. Provisional Application No. 62/777,480, filed Dec. 10, 2018, which application is herein incorporated by reference in its entirety.
This invention was made with government support under grant AI123477 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/065226 | 12/9/2019 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62777480 | Dec 2018 | US |
