Mass Spectrometry Based Molecular 3D-Cartography of Plant Metabolites

Plants play an essential part in global carbon fixing through photosynthesis and are the primary food and energy source for humans. Understanding them thoroughly is therefore of highest interest for humanity. Advances in DNA and RNA sequencing and in protein and metabolite analysis allow the systematic description of plant composition at the molecular level. With imaging mass spectrometry, we can now add a spatial level, typically in the micrometer-to-centimeter range, to their compositions, essential for a detailed molecular understanding. Here we present an LC-MS based approach for 3D plant imaging, which is scalable and allows the analysis of entire plants. We applied this approach in a case study to pepper and tomato plants. Together with MS/MS spectra library matching and spectral networking, this non-targeted workflow provides the highest sensitivity and selectivity for the molecular annotations and imaging of plants, laying the foundation for studies of plant metabolism and plant-environment interactions

Prioritizing Natural Product Diversity in a Collection of 146 Bacterial Strains Based on Growth and Extraction Protocols

In order to expedite the rapid and efficient discovery and isolation of novel specialized metabolites, while minimizing the waste of resources on rediscovery of known compounds, it is crucial to develop efficient approaches for strain prioritization, rapid dereplication, and the assessment of favored cultivation and extraction conditions. Herein we interrogated bacterial strains by systematically evaluating cultivation and extraction parameters with LC-MS/MS analysis and subsequent dereplication through the Global Natural Product Social Molecular Networking (GNPS) platform. The developed method is fast, requiring minimal time and sample material, and is compatible with high-throughput extract analysis, thereby streamlining strain prioritization and evaluation of culturing parameters. With this approach, we analyzed 146 marine Salinispora and Streptomyces strains that were grown and extracted using multiple different protocols. In total, 603 samples were analyzed, generating approximately 1.8 million mass spectra. We constructed a comprehensive molecular network and identified 15 molecular families of diverse natural products and their analogues. The size and breadth of this network shows statistically supported trends in molecular diversity when comparing growth and extraction conditions. The network provides an extensive survey of the biosynthetic capacity of the strain collection and a method to compare strains based on the variety and novelty of their metabolites. This approach allows us to quickly identify patterns in metabolite production that can be linked to taxonomy, culture conditions, and extraction methods, as well as informing the most valuable growth and extraction conditions

Molecular and Microbial Microenvironments in Chronically Diseased Lungs Associated with Cystic Fibrosis.

To visualize the personalized distributions of pathogens and chemical environments, including microbial metabolites, pharmaceuticals, and their metabolic products, within and between human lungs afflicted with cystic fibrosis (CF), we generated three-dimensional (3D) microbiome and metabolome maps of six explanted lungs from three cystic fibrosis patients. These 3D spatial maps revealed that the chemical environments differ between patients and within the lungs of each patient. Although the microbial ecosystems of the patients were defined by the dominant pathogen, their chemical diversity was not. Additionally, the chemical diversity between locales in the lungs of the same individual sometimes exceeded interindividual variation. Thus, the chemistry and microbiome of the explanted lungs appear to be not only personalized but also regiospecific. Previously undescribed analogs of microbial quinolones and antibiotic metabolites were also detected. Furthermore, mapping the chemical and microbial distributions allowed visualization of microbial community interactions, such as increased production of quorum sensing quinolones in locations where Pseudomonas was in contact with Staphylococcus and Granulicatella, consistent with in vitro observations of bacteria isolated from these patients. Visualization of microbe-metabolite associations within a host organ in early-stage CF disease in animal models will help elucidate the complex interplay between the presence of a given microbial structure, antibiotics, metabolism of antibiotics, microbial virulence factors, and host responses.IMPORTANCE Microbial infections are now recognized to be polymicrobial and personalized in nature. Comprehensive analysis and understanding of the factors underlying the polymicrobial and personalized nature of infections remain limited, especially in the context of the host. By visualizing microbiomes and metabolomes of diseased human lungs, we reveal how different the chemical environments are between hosts that are dominated by the same pathogen and how community interactions shape the chemical environment or vice versa. We highlight that three-dimensional organ mapping methods represent hypothesis-building tools that allow us to design mechanistic studies aimed at addressing microbial responses to other microbes, the host, and pharmaceutical drugs

Creating a 3D microbial and chemical snapshot of a human habitat

Kapono CA, Morton JT, Bouslimani A, et al. Creating a 3D microbial and chemical snapshot of a human habitat. Scientific Reports. 2018;8(1): 3669

Lugdunomycin, an Angucycline-Derived Molecule with Unprecedented Chemical Architecture

The angucyclines form the largest family of polycyclic aromatic polyketides, and have been studied extensively. Herein, we report the discovery of lugdunomycin, an angucycline-derived polyketide, produced by Streptomyces species QL37. Lugdunomycin has unique structural characteristics, including a heptacyclic ring system, a spiroatom, two all-carbon stereocenters, and a benzaza-[4,3,3]propellane motif. Considering the structural novelty, we propose that lugdunomycin represents a novel subclass of aromatic polyketides. Metabolomics, combined with MS-based molecular networking analysis of Streptomyces sp. QL37, elucidated 24 other rearranged and non-rearranged angucyclines, 11 of which were previously undescribed. A biosynthetic route for the lugdunomycin and limamycins is also proposed. This work demonstrates that revisiting well-known compound families and their producer strains still is a promising approach for drug discovery

Formation of Amyloid-Like Fibrils by Y-Box Binding Protein 1 (YB-1) Is Mediated by Its Cold Shock Domain and Modulated by Disordered Terminal Domains

YB-1, a multifunctional DNA- and RNA-binding nucleocytoplasmic protein, is involved in the majority of DNA- and mRNA-dependent events in the cell. It consists of three structurally different domains: its central cold shock domain has the structure of a β-barrel, while the flanking domains are predicted to be intrinsically disordered. Recently, we showed that YB-1 is capable of forming elongated fibrils under high ionic strength conditions. Here we report that it is the cold shock domain that is responsible for formation of YB-1 fibrils, while the terminal domains differentially modulate this process depending on salt conditions. We demonstrate that YB-1 fibrils have amyloid-like features, including affinity for specific dyes and a typical X-ray diffraction pattern, and that in contrast to most of amyloids, they disassemble under nearly physiological conditions

Global chemical effects of the microbiome include new bile-acid conjugations

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis

Open Access Repository-Scale Propagated Nearest Neighbor Suspect Spectral Library for Untargeted Metabolomics

Abstract Despite the increasing availability of tandem mass spectrometry (MS/MS) community spectral libraries for untargeted metabolomics over the past decade, the majority of acquired MS/MS spectra remain uninterpreted. To further aid in interpreting unannotated spectra, we created a nearest neighbor suspect spectral library, consisting of 87,916 annotated MS/MS spectra derived from hundreds of millions of public MS/MS spectra. Annotations were propagated based on structural relationships to reference molecules using MS/MS-based spectrum alignment. We demonstrate the broad relevance of the nearest neighbor suspect spectral library through representative examples of propagation-based annotation of acylcarnitines, bacterial and plant natural products, and drug metabolism. Our results also highlight how the library can help to better understand an Alzheimer’s brain phenotype. The nearest neighbor suspect spectral library is openly available through the GNPS platform to help investigators hypothesize candidate structures for unknown MS/MS spectra in untargeted metabolomics data

American Gut: an Open Platform for Citizen Science Microbiome Research

McDonald D, Hyde E, Debelius JW, et al. American Gut: an Open Platform for Citizen Science Microbiome Research. mSystems. 2018;3(3):e00031-18

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry techniques are well-suited to high-throughput characterization of natural products, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social molecular networking (GNPS, http://gnps.ucsd.edu), an open-access knowledge base for community wide organization and sharing of raw, processed or identified tandem mass (MS/MS) spectrometry data. In GNPS crowdsourced curation of freely available community-wide reference MS libraries will underpin improved annotations. Data-driven social-networking should facilitate identification of spectra and foster collaborations. We also introduce the concept of ‘living data’ through continuous reanalysis of deposited data