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Untargeted metabolomics experiments rely on spectral libraries for structure annotation, but, typically, only a small fraction of spectra can be matched. Previous in silico methods search in structure databases but cannot distinguish between correct and incorrect annotations. Here we introduce the COSMIC workflow that combines in silico structure database generation and annotation with a confidence score consisting of kernel density P value estimation and a support vector machine with enforced directionality of features. On diverse datasets, COSMIC annotates a substantial number of hits at low false discovery rates and outperforms spectral library search. To demonstrate that COSMIC can annotate structures never reported before, we annotated 12 natural bile acids. The annotation of nine structures was confirmed by manual evaluation and two structures using synthetic standards. In human samples, we annotated and manually validated 315 molecular structures currently absent from the Human Metabolome Database. Application of COSMIC to data from 17,400 metabolomics experiments led to 1,715 high-confidence structural annotations that were absent from spectral libraries. COSMIC outperforms spectral library search for metabolite annotation and annotates previously unseen structures.

Huanglongbing (HLB), associated with the psyllid-vectored phloem-limited bacterium, Candidatus Liberibacter asiaticus (C Las), is a disease threat to all citrus production worldwide. Currently, there are no sustainable curative or prophylactic treatments available. In this study, we utilized mass spectrometry (MS)-based metabolomics in combination with 3D molecular mapping to visualize complex chemistries within plant tissues to explore how these chemistries change in vivo in HLB-infected trees. We demonstrate how spatial information from molecular maps of branches and single leaves yields insight into the biology not accessible otherwise. In particular, we found evidence that flavonoid biosynthesis is disrupted in HLB-infected trees, and an increase in the polyamine, feruloylputrescine, is highly correlated with an increase in disease severity. Based on mechanistic details revealed by these molecular maps, followed by metabolic modeling, we formulated and tested the hypothesis that C Las infection either directly or indirectly converts the precursor compound, ferulic acid, to feruloylputrescine to suppress the antimicrobial effects of ferulic acid and biosynthetically downstream flavonoids. Using in vitro bioassays, we demonstrated that ferulic acid and bioflavonoids are indeed highly bactericidal to C Las, with the activity on par with a reference antibiotic, oxytetracycline, recently approved for HLB management. We propose these compounds should be evaluated as therapeutics alternatives to the antibiotics for HLB treatment. Overall, the utilized 3D metabolic mapping approach provides a promising methodological framework to identify pathogen-specific inhibitory compounds in planta for potential prophylactic or therapeutic applications.

Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes 1 – 6 ; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N -acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N -acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh- expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network. We find that bile salt hydrolase N -acyltransferase activity can form bacterial bile acid amidates that are positively correlated with the colonization of gut bacteria that assist in the regulation of the bile acid metabolic network.

Lithified layers of complex microbial mats known as microbialites are ubiquitous in the fossil record, and modern forms are increasingly identified globally. A key challenge to developing an understanding of microbialite formation and environmental role is how to investigate complex and diverse communities in situ . We selected living, layered microbialites (stromatolites) in a peritidal environment near Schoenmakerskop, Eastern Cape, South Africa to conduct a spatial survey mapping the composition and small molecule production of the microbial communities from environmental samples. Substrate core samples were collected from nine sampling stations ranging from the upper point of the freshwater inflow to the lower marine interface where tidal overtopping takes place. Substrate cores provided material for parallel analyses of microbial community diversity by 16S rRNA gene amplicon sequencing and metabolomics using LC–MS 2 . Species and metabolite diversities were correlated, and prominent specialized metabolites were targeted for preliminary characterization. A new series of cyclic hexadepsipeptides, named ibhayipeptolides, was most abundant in substrate cores of submerged microbialites. These results demonstrate the detection and identification of metabolites from mass-limited environmental samples and contribute knowledge about microbialite chemistry and biology, which facilitates future targeted studies of specialized metabolite function and biosynthesis.

Clinical testing typically relies on invasive blood draws and biopsies. Alternative methods of sample collection are continually being developed to improve patient experience; swabbing the skin is one of the least invasive sampling methods possible. To show that skin swabs in combination with untargeted mass spectrometry (metabolomics) can be used for non-invasive monitoring of an oral drug, we report the kinetics and metabolism of diphenhydramine in healthy volunteers (n = 10) over the course of 24 hours in blood and three regions of the skin. Diphenhydramine and its metabolites were observed on the skin after peak plasma levels, varying by compound and skin location, and is an illustrative example of how systemically administered molecules can be detected on the skin surface. The observation of diphenhydramine directly from the skin supports the hypothesis that both parent drug and metabolites can be qualitatively measured from a simple non-invasive swab of the skin surface. The mechanism of the drug and metabolites pathway to the skin’s surface remains unknown.

Determining the structure and phenotypic context of molecules detected in untargeted metabolomics experiments remains challenging. Here we present reverse metabolomics as a discovery strategy, whereby tandem mass spectrometry spectra acquired from newly synthesized compounds are searched for in public metabolomics datasets to uncover phenotypic associations. To demonstrate the concept, we broadly synthesized and explored multiple classes of metabolites in humans, including N -acyl amides, fatty acid esters of hydroxy fatty acids, bile acid esters and conjugated bile acids. Using repository-scale analysis 1 , 2 , we discovered that some conjugated bile acids are associated with inflammatory bowel disease (IBD). Validation using four distinct human IBD cohorts showed that cholic acids conjugated to Glu, Ile/Leu, Phe, Thr, Trp or Tyr are increased in Crohn’s disease. Several of these compounds and related structures affected pathways associated with IBD, such as interferon-γ production in CD4 + T cells 3 and agonism of the pregnane X receptor 4 . Culture of bacteria belonging to the Bifidobacterium , Clostridium and Enterococcus genera produced these bile amidates. Because searching repositories with tandem mass spectrometry spectra has only recently become possible, this reverse metabolomics approach can now be used as a general strategy to discover other molecules from human and animal ecosystems. A new discovery strategy, ‘reverse metabolomics’, facilitates high-throughput matching of mass spectrometry spectra in public untargeted metabolomics datasets, and a proof-of-concept experiment identified an association between microbial bile amidates and inflammatory bowel disease.

Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile . Bile salt hydrolases encoded by the gut microbiome shape the bile acid pool, including microbial conjugated bile acids, which impact Clostridioides difficile infection in the murine gut.

Emerging studies reveal that gut microbes can conjugate diverse amino acids to bile acids, known as microbially conjugated bile acids. However, their regulation and health effects remain unclear. Here, we analyzed early-life microbially conjugated bile acid patterns and their link to islet autoimmunity. We quantified 110 microbial bile acids in 303 stool samples collected longitudinally (3-36 months) from children who developed one or more islet autoantibodies and controls who remained autoantibody-negative. We identified distinct age-dependent trajectories of these bile acid amidates and correlated them with gut microbiome composition. We found that altered levels of ursodeoxycholic and deoxycholic acid conjugates were linked to islet autoimmunity as well as modulated monocyte activation in response to immunostimulatory lipopolysaccharide and Th17/Treg cell balance. These findings suggest that microbially conjugated bile acids influence immune development and type 1 diabetes risk.