Host-microbe co-metabolism via MCAD generates circulating metabolites including hippuric acid.

The human gut microbiota produces dozens of small molecules that circulate in blood, accumulate to comparable levels as pharmaceutical drugs, and influence host physiology. Despite the importance of these metabolites to human health and disease, the origin of most microbially-produced molecules and their fate in the host remains largely unknown. Here, we uncover a host-microbe co-metabolic pathway for generation of hippuric acid, one of the most abundant organic acids in mammalian urine. Combining stable isotope tracing with bacterial and host genetics, we demonstrate reduction of phenylalanine to phenylpropionic acid by gut bacteria; the host re-oxidizes phenylpropionic acid involving medium-chain acyl-CoA dehydrogenase (MCAD). Generation of germ-free male and female MCAD-/- mice enabled gnotobiotic colonization combined with untargeted metabolomics to identify additional microbial metabolites processed by MCAD in host circulation. Our findings uncover a host-microbe pathway for the abundant, non-toxic phenylalanine metabolite hippurate and identify ß-oxidation via MCAD as a novel mechanism by which mammals metabolize microbiota-derived metabolites.

Oxidative ornithine metabolism supports non-inflammatory C. difficile colonization.

The enteric pathogen Clostridioides difficile (Cd) is responsible for a toxin-mediated infection that causes more than 200,000 recorded hospitalizations and 13,000 deaths in the United States every year1. However, Cd can colonize the gut in the absence of disease symptoms. Prevalence of asymptomatic colonization by toxigenic Cd in healthy populations is high; asymptomatic carriers are at increased risk of infection compared to noncolonized individuals and may be a reservoir for transmission of Cd infection2,3. Elucidating the molecular mechanisms by which Cd persists in the absence of disease is necessary for understanding pathogenesis and developing refined therapeutic strategies. Here, we show with gut microbiome metatranscriptomic analysis that mice recalcitrant to Cd infection and inflammation exhibit increased community-wide expression of arginine and ornithine metabolic pathways. To query Cd metabolism specifically, we leverage RNA sequencing in gnotobiotic mice infected with two wild-type strains (630 and R20291) and isogenic toxin-deficient mutants of these strains to differentiate inflammation-dependent versus -independent transcriptional states. A single operon encoding oxidative ornithine degradation is consistently upregulated across non-toxigenic Cd strains. Combining untargeted and targeted metabolomics with bacterial and host genetics, we demonstrate that both diet- and host-derived sources of ornithine provide a competitive advantage to Cd, suggesting a mechanism for Cd persistence within a non-inflammatory, healthy gut.

A metabolomics pipeline for the mechanistic interrogation of the gut microbiome.

Gut microorganisms modulate host phenotypes and are associated with numerous health effects in humans, ranging from host responses to cancer immunotherapy to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microorganisms has hindered efforts to define mechanistic connections between individual microbial strains and host phenotypes. One key way in which the gut microbiome influences host physiology is through the production of small molecules1-3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect the products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites in diverse sample types. We report the metabolic profiles of 178 gut microorganism strains using our library of 833 metabolites. Using this metabolomics resource, we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover a previously undescribed type of metabolism in Bacteroides, and reveal candidate biochemical pathways using comparative genomics. Microbiota-dependent metabolites can be detected in diverse biological fluids from gnotobiotic and conventionally colonized mice and traced back to the corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microorganisms and interactions between microorganisms and their host.

Enantiomers of 4-aminopentanoic acid act as false GABAergic neurotransmitters and impact mouse behavior.

Imbalance in the metabolic pathway linking excitatory and inhibitory neurotransmission has been implicated in multiple psychiatric and neurologic disorders. Recently, we described enantiomer-specific effects of 2-methylglutamate, which is not decarboxylated to the corresponding methyl analogue of gamma-aminobutyric acid (GABA): 4-aminopentanoic acid (4APA). Here, we tested the hypothesis that 4APA also has enantiomer-specific actions in brain. Mouse cerebral synaptosome uptake (nmol/mg protein over 30 min) of (R)-4APA or (S)-4APA was time and temperature dependent; however, the R enantiomer had greater uptake, reduction of endogenous GABA concentration, and release following membrane depolarization than did the S enantiomer. (S)-4APA exhibited some weak agonist (GABAA α4ß3δ, GABAA α5ß2γ2, and GABAB B1/B2) and antagonist (GABAA α6ß2γ2) activity while (R)-4APA showed weak agonist activity only with GABAA α5ß2γ2. Both 4APA enantiomers (100 mg/kg IP) were detected in mouse brain 10 min after injection, and by 1 hr had reached concentrations that were stable over 6 hr; both enantiomers were cleared rapidly from mouse serum over 6 hr. Two-month-old mice had no mortality following 100-900 mg/kg IP of each 4APA enantiomer but did have similar dose-dependent reduction in distance moved in a novel cage. Neither enantiomer at 30 or 100 mg/kg impacted outcomes in 23 measures of well-being, activity chamber, or withdrawal from hot plate. Our results suggest that enantiomers of 4APA are active in mouse brain, and that (R)-4APA may act as a novel false neurotransmitter of GABA. Future work will focus on disease models and on possible applications as neuroimaging agents.

Enantiomers of 2-methylglutamate and 2-methylglutamine selectively impact mouse brain metabolism and behavior.

Imbalance of excitatory and inhibitory neurotransmission is implicated in a wide range of psychiatric and neurologic disorders. Here we tested the hypothesis that insertion of a methyl group on the stereogenic alpha carbon of L-Glu or L-Gln would impact the γ-aminobutyric acid (GABA) shunt and the glutamate-glutamine cycle. (S)-2-methylglutamate, or (S)-2MeGlu, was efficiently transported into brain and synaptosomes where it was released by membrane depolarization in a manner equivalent to endogenous L-Glu. (R)-2MeGlu was transported less efficiently into brain and synaptosomes but was not released by membrane depolarization. Each enantiomer of 2MeGlu had limited activity across a panel of over 30 glutamate and GABA receptors. While neither enantiomer of 2MeGlu was metabolized along the GABA shunt, (S)-2MeGlu was selectively converted to (S)-2-methylglutamine, or (S)-2MeGln, which was subsequently slowly hydrolyzed back to (S)-2MeGlu in brain. rac-2MeGln was also transported into brain, with similar efficiency as (S)-2MeGlu. A battery of behavioral tests in young adult wild type mice showed safety with up to single 900 mg/kg dose of (R)-2MeGlu, (S)-2MeGlu, or rac-2MeGln, suppressed locomotor activity with single ≥ 100 mg/kg dose of (R)-2MeGlu or (S)-2MeGlu. No effect on anxiety or hippocampus-dependent learning was evident. Enantiomers of 2MeGlu and 2MeGln show promise as potential pharmacologic agents and imaging probes for cells that produce or transport L-Gln.

Enhancing the Antiviral Efficacy of RNA-Dependent RNA Polymerase Inhibition by Combination with Modulators of Pyrimidine Metabolism.

Genome-wide analysis of the mode of action of GSK983, a potent antiviral agent, led to the identification of dihydroorotate dehydrogenase as its target along with the discovery that genetic knockdown of pyrimidine salvage sensitized cells to GSK983. Because GSK983 is an ineffective antiviral in the presence of physiological uridine concentrations, we explored combining GSK983 with pyrimidine salvage inhibitors. We synthesized and evaluated analogs of cyclopentenyl uracil (CPU), an inhibitor of uridine salvage. We found that CPU was converted into its triphosphate in cells. When combined with GSK983, CPU resulted in large drops in cellular UTP and CTP pools. Consequently, CPU-GSK983 suppressed dengue virus replication in the presence of physiological concentrations of uridine. In addition, the CPU-GSK983 combination markedly enhanced the effect of RNA-dependent RNA polymerase (RdRp) inhibition on viral infection. Our findings highlight a new host-targeting strategy for potentiating the antiviral activity of RdRp inhibitors.

A Plasma Protein Network Regulates PM20D1 and N-Acyl Amino Acid Bioactivity.

N-acyl amino acids are a family of cold-inducible circulating lipids that stimulate thermogenesis. Their biosynthesis is mediated by a secreted enzyme called PM20D1. The extracellular mechanisms that regulate PM20D1 or N-acyl amino acid activity in the complex environment of blood plasma remains unknown. Using quantitative proteomics, here we show that PM20D1 circulates in tight association with both low- and high-density lipoproteins. Lipoprotein particles are powerful co-activators of PM20D1 activity in vitro and N-acyl amino acid biosynthesis in vivo. We also identify serum albumin as a physiologic N-acyl amino acid carrier, which spatially segregates N-acyl amino acids away from their sites of production, confers resistance to hydrolytic degradation, and establishes an equilibrium between thermogenic "free" versus inactive "bound" fractions. These data establish lipoprotein particles as principal extracellular sites of N-acyl amino acid biosynthesis and identify a lipoprotein-albumin network that regulates the activity of a circulating thermogenic lipid family.

Cooperative enzymatic control of N-acyl amino acids by PM20D1 and FAAH.

The N-acyl amino acids are a family of bioactive lipids with pleiotropic physiologic functions, including in energy homeostasis. Their endogenous levels are regulated by an extracellular mammalian N-acyl amino acid synthase/hydrolase called PM20D1 (peptidase M20 domain containing 1). Using an activity-guided biochemical approach, we report the molecular identification of fatty acid amide hydrolase (FAAH) as a second intracellular N-acyl amino acid synthase/hydrolase. In vitro, FAAH exhibits a more restricted substrate scope compared to PM20D1. In mice, genetic ablation or selective pharmacological inhibition of FAAH bidirectionally dysregulates intracellular, but not circulating, N-acyl amino acids. Dual blockade of both PM20D1 and FAAH reveals a dramatic and non-additive biochemical engagement of these two enzymatic pathways. These data establish FAAH as a second intracellular pathway for N-acyl amino acid metabolism and underscore enzymatic division of labor as an enabling strategy for the regulation of a structurally diverse bioactive lipid family.

Complete Reconstitution and Deorphanization of the 3 MDa Nocardiosis-Associated Polyketide Synthase.

Several Nocardia strains associated with nocardiosis, a potentially life-threatening disease, house a nonamodular assembly line polyketide synthase (PKS) that presumably synthesizes an unknown polyketide. Here, we report the discovery and structure elucidation of the NOCAP (nocardiosis-associated polyketide) aglycone by first fully reconstituting the NOCAP synthase in vitro from purified protein components followed by heterologous expression in E. coli and spectroscopic analysis of the purified products. The NOCAP aglycone has an unprecedented structure comprised of a substituted resorcylaldehyde headgroup linked to a 15-carbon tail that harbors two conjugated all-trans trienes separated by a stereogenic hydroxyl group. This report is the first example of reconstituting a trans-acyltransferase assembly line PKS in vitro and of using these approaches to "deorphanize" a complete assembly line PKS identified via genomic sequencing. With the NOCAP aglycone in hand, the stage is set for understanding how this PKS and associated tailoring enzymes confer an advantage to their native hosts during human Nocardia infections.

A Metabolic Pathway for Activation of Dietary Glucosinolates by a Human Gut Symbiont.

Consumption of glucosinolates, pro-drug-like metabolites abundant in Brassica vegetables, has been associated with decreased risk of certain cancers. Gut microbiota have the ability to metabolize glucosinolates, generating chemopreventive isothiocyanates. Here, we identify a genetic and biochemical basis for activation of glucosinolates to isothiocyanates by Bacteroides thetaiotaomicron, a prominent gut commensal species. Using a genome-wide transposon insertion screen, we identified an operon required for glucosinolate metabolism in B. thetaiotaomicron. Expression of BT2159-BT2156 in a non-metabolizing relative, Bacteroides fragilis, resulted in gain of glucosinolate metabolism. We show that isothiocyanate formation requires the action of BT2158 and either BT2156 or BT2157 in vitro. Monocolonization of mice with mutant BtΔ2157 showed reduced isothiocyanate production in the gastrointestinal tract. These data provide insight into the mechanisms by which a common gut bacterium processes an important dietary nutrient.

A Pathogen-Responsive Gene Cluster for Highly Modified Fatty Acids in Tomato.

In response to biotic stress, plants produce suites of highly modified fatty acids that bear unusual chemical functionalities. Despite their chemical complexity and proposed roles in pathogen defense, little is known about the biosynthesis of decorated fatty acids in plants. Falcarindiol is a prototypical acetylenic lipid present in carrot, tomato, and celery that inhibits growth of fungi and human cancer cell lines. Using a combination of untargeted metabolomics and RNA sequencing, we discovered a biosynthetic gene cluster in tomato (Solanum lycopersicum) required for falcarindiol production. By reconstituting initial biosynthetic steps in a heterologous host and generating transgenic pathway mutants in tomato, we demonstrate a direct role of the cluster in falcarindiol biosynthesis and resistance to fungal and bacterial pathogens in tomato leaves. This work reveals a mechanism by which plants sculpt their lipid pool in response to pathogens and provides critical insight into the complex biochemistry of alkynyl lipid production.

Depletion of microbiome-derived molecules in the host using Clostridium genetics.

The gut microbiota produce hundreds of molecules that are present at high concentrations in the host circulation. Unraveling the contribution of each molecule to host biology remains difficult. We developed a system for constructing clean deletions in Clostridium spp., the source of many molecules from the gut microbiome. By applying this method to the model commensal organism Clostridium sporogenes, we knocked out genes for 10 C. sporogenes-derived molecules that accumulate in host tissues. In mice colonized by a C. sporogenes for which the production of branched short-chain fatty acids was knocked out, we discovered that these microbial products have immunoglobulin A-modulatory activity.

Family-wide Annotation of Enzymatic Pathways by Parallel In Vivo Metabolomics.

Enzymes catalyze fundamental biochemical reactions that control cellular and organismal homeostasis. Here we present an approach for de novo biochemical pathway discovery across entire mammalian enzyme families using parallel viral transduction in mice and untargeted liquid chromatography-mass spectrometry. Applying this method to the M20 peptidases uncovers both known pathways of amino acid metabolism as well as a previously unknown CNDP2-regulated pathway for threonyl dipeptide catabolism. Ablation of CNDP2 in mice elevates threonyl dipeptides across multiple tissues, establishing the physiologic relevance of our biochemical assignments. Taken together, these data underscore the utility of parallel in vivo metabolomics for the family-wide discovery of enzymatic pathways.

CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity.

Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.

Tunable Enzymatic Synthesis of the Immunomodulator Lipid IVA To Enable Structure-Activity Analysis.

The Lipid A family of glycolipids, found in the outer membranes of all Gram-negative bacteria, exhibits considerable structural diversity in both lipid and glycan moieties. The lack of facile methods to prepare analogues of these natural products represents a major roadblock in understanding the relationship between their structure and immunomodulatory activities. Here we present a modular, cell-free multienzymatic platform to access these structure-activity relationships. By individually purifying 19 Escherichia coli proteins and reconstituting them in vitro in the presence of acetyl-CoA, UDP- N-acetylglucosamine, NADPH, and ATP, we have developed a system capable of synthesizing Lipid IVA, the first bioactive intermediate in the Lipid A pathway. Our reconstituted multienzyme system revealed considerable promiscuity for orthologs with distinct substrate specificity, as illustrated by swapping enzymes from distantly related cyanobacterial and Pseudomonas species. Analysis of the agonistic and antagonistic activities of the resulting products against the THP-1 human monocytic cell line revealed hitherto unrecognized trends, while opening the door to harnessing the potent biological activities of these complex glycolipid natural products.

Thioesterase-Catalyzed Aminoacylation and Thiolation of Polyketides in Fungi.

Fungal highly reducing polyketide synthases (HRPKSs) biosynthesize polyketides using a single set of domains iteratively. Product release is a critical step in HRPKS function to ensure timely termination and enzyme turnover. Nearly all of the HRPKSs characterized to date employ a separate thioesterase (TE) or acyltransferase enzyme for product release. In this study, we characterized two fungal HRPKSs that have fused C-terminal TE domains, a new domain architecture for fungal HRPKSs. We showed that both HRPKS-TEs synthesize aminoacylated polyketides in an ATP-independent fashion. The KU42 TE domain selects cysteine and homocysteine and catalyzes transthioesterification using the side-chain thiol group as the nucleophile. In contrast, the KU43 TE domain selects leucine methyl ester and performs a direct amidation of the polyketide, a reaction typically catalyzed by nonribosomal peptide synthetase (NRPS) domains. The characterization of these HRPKS-TE enzymes showcases the functional diversity of HRPKS enzymes and provides potential TE domains as biocatalytic tools to diversify HRPKS structures.

N-hydroxy-pipecolic acid is a mobile metabolite that induces systemic disease resistance in Arabidopsis.

Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N-hydroxy-pipecolic acid (N-OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N-OH-Pip from pipecolic acid in planta, and exogenously applied N-OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N-OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N-OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance.

HEx: A heterologous expression platform for the discovery of fungal natural products.

For decades, fungi have been a source of U.S. Food and Drug Administration-approved natural products such as penicillin, cyclosporine, and the statins. Recent breakthroughs in DNA sequencing suggest that millions of fungal species exist on Earth, with each genome encoding pathways capable of generating as many as dozens of natural products. However, the majority of encoded molecules are difficult or impossible to access because the organisms are uncultivable or the genes are transcriptionally silent. To overcome this bottleneck in natural product discovery, we developed the HEx (Heterologous EXpression) synthetic biology platform for rapid, scalable expression of fungal biosynthetic genes and their encoded metabolites in Saccharomyces cerevisiae. We applied this platform to 41 fungal biosynthetic gene clusters from diverse fungal species from around the world, 22 of which produced detectable compounds. These included novel compounds with unexpected biosynthetic origins, particularly from poorly studied species. This result establishes the HEx platform for rapid discovery of natural products from any fungal species, even those that are uncultivable, and opens the door to discovery of the next generation of natural products.

D2O Labeling to Measure Active Biosynthesis of Natural Products in Medicinal Plants.

Plant natural products have served as a prominent source of medicines throughout human history, and are still used today as clinically-approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow-growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically-relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids, and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short time scales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes.

Genetic Mapping and Biochemical Basis of Yellow Feather Pigmentation in Budgerigars.

Parrot feathers contain red, orange, and yellow polyene pigments called psittacofulvins. Budgerigars are parrots that have been extensively bred for plumage traits during the last century, but the underlying genes are unknown. Here we use genome-wide association mapping and gene-expression analysis to map the Mendelian blue locus, which abolishes yellow pigmentation in the budgerigar. We find that the blue trait maps to a single amino acid substitution (R644W) in an uncharacterized polyketide synthase (MuPKS). When we expressed MuPKS heterologously in yeast, yellow pigments accumulated. Mass spectrometry confirmed that these yellow pigments match those found in feathers. The R644W substitution abolished MuPKS activity. Furthermore, gene-expression data from feathers of different bird species suggest that parrots acquired their colors through regulatory changes that drive high expression of MuPKS in feather epithelia. Our data also help formulate biochemical models that may explain natural color variation in parrots. VIDEO ABSTRACT.