Reverse metabolomics for the discovery of chemical structures from humans.

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 analysis1,2, 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 cells3 and agonism of the pregnane X receptor4. 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.

Commensal bacteria promote type I interferon signaling to maintain immune tolerance in mice.

Type I interferons (IFNs) exert a broad range of biological effects important in coordinating immune responses, which have classically been studied in the context of pathogen clearance. Yet, whether immunomodulatory bacteria operate through IFN pathways to support intestinal immune tolerance remains elusive. Here, we reveal that the commensal bacterium, Bacteroides fragilis, utilizes canonical antiviral pathways to modulate intestinal dendritic cells (DCs) and regulatory T cell (Treg) responses. Specifically, IFN signaling is required for commensal-induced tolerance as IFNAR1-deficient DCs display blunted IL-10 and IL-27 production in response to B. fragilis. We further establish that IFN-driven IL-27 in DCs is critical in shaping the ensuing Foxp3+ Treg via IL-27Rα signaling. Consistent with these findings, single-cell RNA sequencing of gut Tregs demonstrated that colonization with B. fragilis promotes a distinct IFN gene signature in Foxp3+ Tregs during intestinal inflammation. Altogether, our findings demonstrate a critical role of commensal-mediated immune tolerance via tonic type I IFN signaling.

The Host-Microbiome Response to Hyperbaric Oxygen Therapy in Ulcerative Colitis Patients.

BACKGROUND & AIMS: Hyperbaric oxygen therapy (HBOT) is a promising treatment for moderate-to-severe ulcerative colitis. However, our current understanding of the host and microbial response to HBOT remains unclear. This study examined the molecular mechanisms underpinning HBOT using a multi-omic strategy. METHODS: Pre- and post-intervention mucosal biopsies, tissue, and fecal samples were collected from HBOT phase 2 clinical trials. Biopsies and fecal samples were subjected to shotgun metaproteomics, metabolomics, 16s rRNA sequencing, and metagenomics. Tissue was subjected to bulk RNA sequencing and digital spatial profiling (DSP) for single-cell RNA and protein analysis, and immunohistochemistry was performed. Fecal samples were also used for colonization experiments in IL10-/- germ-free UC mouse models. RESULTS: Proteomics identified negative associations between HBOT response and neutrophil azurophilic granule abundance. DSP identified an HBOT-specific reduction of neutrophil STAT3, which was confirmed by immunohistochemistry. HBOT decreased microbial diversity with a proportional increase in Firmicutes and a secondary bile acid lithocholic acid. A major source of the reduction in diversity was the loss of mucus-adherent taxa, resulting in increased MUC2 levels post-HBOT. Targeted database searching revealed strain-level associations between Akkermansia muciniphila and HBOT response status. Colonization of IL10-/- with stool obtained from HBOT responders resulted in lower colitis activity compared with non-responders, with no differences in STAT3 expression, suggesting complementary but independent host and microbial responses. CONCLUSIONS: HBOT reduces host neutrophil STAT3 and azurophilic granule activity in UC patients and changes in microbial composition and metabolism in ways that improve colitis activity. Intestinal microbiota, especially strain level variations in A muciniphila, may contribute to HBOT non-response.

Multi-omics analyses of the ulcerative colitis gut microbiome link Bacteroides vulgatus proteases with disease severity.

Ulcerative colitis (UC) is driven by disruptions in host-microbiota homoeostasis, but current treatments exclusively target host inflammatory pathways. To understand how host-microbiota interactions become disrupted in UC, we collected and analysed six faecal- or serum-based omic datasets (metaproteomic, metabolomic, metagenomic, metapeptidomic and amplicon sequencing profiles of faecal samples and proteomic profiles of serum samples) from 40 UC patients at a single inflammatory bowel disease centre, as well as various clinical, endoscopic and histologic measures of disease activity. A validation cohort of 210 samples (73 UC, 117 Crohn's disease, 20 healthy controls) was collected and analysed separately and independently. Data integration across both cohorts showed that a subset of the clinically active UC patients had an overabundance of proteases that originated from the bacterium Bacteroides vulgatus. To test whether B. vulgatus proteases contribute to UC disease activity, we first profiled B. vulgatus proteases found in patients and bacterial cultures. Use of a broad-spectrum protease inhibitor improved B. vulgatus-induced barrier dysfunction in vitro, and prevented colitis in B. vulgatus monocolonized, IL10-deficient mice. Furthermore, transplantation of faeces from UC patients with a high abundance of B. vulgatus proteases into germfree mice induced colitis dependent on protease activity. These results, stemming from a multi-omics approach, improve understanding of functional microbiota alterations that drive UC and provide a resource for identifying other pathways that could be inhibited as a strategy to treat this disease.

Urinary Exosomes Identify Inflammatory Pathways in Vancomycin Associated Acute Kidney Injury.

BACKGROUND: Vancomycin is commonly used as a first line therapy for gram positive organisms such as methicillin resistant Staphylococcusaureus. Vancomycin-induced acute kidney injury (V-AKI) has been reported in up to 43% of patients, especially in those with higher targeted trough concentrations. The precise mechanism of injury in humans remains elusive, with recent evidence directed towards proximal tubule cell apoptosis. In this study, we investigated the protein contents of urinary exosomes in patients with V-AKI to further elucidate biomarkers of mechanisms of injury and potential responses. METHODS: Urine samples from patients with V-AKI who were enrolled in the DIRECT study and matched healthy controls from the UAB-UCSD O'Brien Center Biorepository were included in the analysis. Exosomes were extracted using solvent exclusion principle and polyethylene glycol induced precipitation. Protein identity and quantification was determined by label-free liquid chromatography mass spectrometry (LC/MS). The mean peak serum creatinine was 3.7 ± 1.4 mg/dL and time to kidney injury was 4.0 ± 3.0 days. At discharge, 90% of patients demonstrated partial recovery; 33% experienced full recovery by day 28. Proteomic analyses on five V-AKI and 7 control samples revealed 2009 proteins in all samples and 251 proteins significantly associated with V-AKI (Pi-score > 1). The top discriminatory proteins were complement C3, complement C4, galectin-3-binding protein, fibrinogen, alpha-2 macroglobulin, immunoglobulin heavy constant mu and serotransferrin. CONCLUSION: Urinary exosomes reveal up-regulation of inflammatory proteins after nephrotoxic injury in V-AKI. Further studies are necessary in a large patient sample to confirm these findings for elucidation of pathophysiologic mechanisms and validation of potential injury biomarkers.

Kinetic profiling of metabolic specialists demonstrates stability and consistency of in vivo enzyme turnover numbers.

Enzyme turnover numbers (kcats) are essential for a quantitative understanding of cells. Because kcats are traditionally measured in low-throughput assays, they can be inconsistent, labor-intensive to obtain, and can miss in vivo effects. We use a data-driven approach to estimate in vivo kcats using metabolic specialist Escherichia coli strains that resulted from gene knockouts in central metabolism followed by metabolic optimization via laboratory evolution. By combining absolute proteomics with fluxomics data, we find that in vivo kcats are robust against genetic perturbations, suggesting that metabolic adaptation to gene loss is mostly achieved through other mechanisms, like gene-regulatory changes. Combining machine learning and genome-scale metabolic models, we show that the obtained in vivo kcats predict unseen proteomics data with much higher precision than in vitro kcats. The results demonstrate that in vivo kcats can solve the problem of inconsistent and low-coverage parameterizations of genome-scale cellular models.

Development of muscle atrophy and loss of function in a Gulf-War illness model: underlying mechanisms.

Gulf War illness (GWI) afflicts military personnel who served during the Persian Gulf War and is notable for cognitive deficits, depression, muscle pain, weakness, intolerance to exercise, and fatigue. Suspect causal agents include the chemicals pyridostigmine (PB), permetrim (PM) and N,N-diethyl-m-toluamide (DEET) used as protectants against insects and nerve gases. No pre-clinical studies have explored the effects on skeletal muscle (SkM). Young male rats were provided PB, PM and DEET at equivalent human doses and physical restraint (to induce stress) for 3 weeks followed a 3-week recovery. GWI gastrocnemius weight was ~ 35% lower versus controls, which correlated with decreases in myofiber area, limb strength, and treadmill time/distance. In GWI rats, SkM fiber type relative abundance changed towards slow type I. Muscle wasting pathway proteins were upregulated while those that promote growth decreased as did mitochondrial endpoints and muscle ATP levels. Proteomic analysis of SkM also documented unique alterations in mitochondrial and metabolic pathways. Thus, exposure to GWI chemicals/stress adversely impacts key metabolic pathways leading to muscle atrophy and loss of function. These changes may account for GWI Veterans symptoms.

Mortality Risk Profiling of Staphylococcus aureus Bacteremia by Multi-omic Serum Analysis Reveals Early Predictive and Pathogenic Signatures.

Staphylococcus aureus bacteremia (SaB) causes significant disease in humans, carrying mortality rates of ∼25%. The ability to rapidly predict SaB patient responses and guide personalized treatment regimens could reduce mortality. Here, we present a resource of SaB prognostic biomarkers. Integrating proteomic and metabolomic techniques enabled the identification of >10,000 features from >200 serum samples collected upon clinical presentation. We interrogated the complexity of serum using multiple computational strategies, which provided a comprehensive view of the early host response to infection. Our biomarkers exceed the predictive capabilities of those previously reported, particularly when used in combination. Last, we validated the biological contribution of mortality-associated pathways using a murine model of SaB. Our findings represent a starting point for the development of a prognostic test for identifying high-risk patients at a time early enough to trigger intensive monitoring and interventions.

EGFR is required for Wnt9a-Fzd9b signalling specificity in haematopoietic stem cells.

Wnt signalling drives many processes in development, homeostasis and disease; however, the role and mechanism of individual ligand-receptor (Wnt-Frizzled (Fzd)) interactions in specific biological processes remain poorly understood. Wnt9a is specifically required for the amplification of blood progenitor cells during development. Using genetic studies in zebrafish and human embryonic stem cells, paired with in vitro cell biology and biochemistry, we determined that Wnt9a signals specifically through Fzd9b to elicit ß-catenin-dependent Wnt signalling that regulates haematopoietic stem and progenitor cell emergence. We demonstrate that the epidermal growth factor receptor (EGFR) is required as a cofactor for Wnt9a-Fzd9b signalling. EGFR-mediated phosphorylation of one tyrosine residue on the Fzd9b intracellular tail in response to Wnt9a promotes internalization of the Wnt9a-Fzd9b-LRP signalosome and subsequent signal transduction. These findings provide mechanistic insights for specific Wnt-Fzd signals, which will be crucial for specific therapeutic targeting and regenerative medicine.