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.
Molecular networking has become a key method to visualize and annotate the chemical space in non-targeted mass spectrometry data. We present Feature-Based Molecular Networking (FBMN) as an analysis method in the Global Natural Products Social Molecular Networking (GNPS) infrastructure that builds on chromatographic feature detection and alignment tools. The FBMN method brings quantitative analyses, isomeric resolution, including from ion-mobility spectrometry, into molecular networks.
In polymicrobial infections, microbes can interact with both the host immune system and one another through direct contact or the secretion of metabolites, affecting disease progression and treatment options. The thick mucus in the lungs of patients with cystic fibrosis is highly susceptible to polymicrobial infections by opportunistic pathogens, including the bacterium Pseudomonas aeruginosa and the fungus Aspergillus fumigatus. Unravelling the hidden molecular interactions within such polymicrobial communities and their metabolic exchange processes will require effective enabling technologies applied to model systems. In the present study, MALDI-TOF and MALDI-FT-ICR imaging mass spectrometry (MALDI-IMS) combined with MS/MS networking were used to provide insight into the interkingdom interaction between P. aeruginosa and A. fumigatus at the molecular level. The combination of these technologies enabled the visualization and identification of metabolites secreted by these microorganisms grown on agar. A complex molecular interplay was revealed involving suppression, increased production, and biotransformation of a range of metabolites. Of particular interest is the observation that P. aeruginosa phenazine metabolites were converted by A. fumigatus into other chemical entities with alternative properties, including enhanced toxicities and the ability to induce fungal siderophores. This work highlights the capabilities of MALDI-IMS and MS/MS network analysis to study interkingdom interactions and provides insight into the complex nature of polymicrobial metabolic exchange and biotransformations. M icrobes that colonize mammalian hosts can form polymicrobial communities, such as biofilms, where they establish commensual, mutualistic, competitive, or antagonistic interactions with one another and with the host. In microbial disease, this complex interplay can affect the outcome of antimicrobial therapy (1). Therefore, it is important to understand polymicrobial populations and their interactions at the molecular level.In persons with cystic fibrosis (CF), the lungs are lined with a viscous mucus layer susceptible to polymicrobial infections (2). Pseudomonas aeruginosa, a Gram-negative bacterial opportunistic pathogen, is the most prevalent and persistent microorganism (3) isolated from the sputum of CF lungs and leading cause of mortality in CF patients (4). Within the CF lung, P. aeruginosa exists in biofilm-like macrocolonies (5) and is refractory to antimicrobial agents and the host immune response (6). Aspergillus fumigatus, an opportunistic fungal pathogen, is the second-most persistent microbe in the CF lung, with a 10-57% prevalence rate (3), and is capable of causing allergic bronchopulmonary aspergillosis (7).Superinfection with both P. aeruginosa and A. fumigatus in CF patients leads to decreased pulmonary function compared with monoinfection with either microbe (8). Interestingly, however, in a pulmonary mouse model, mice coinfected with P. aeruginosa and A. fumigatus had a higher survival rate than mice...
The ability to correlate the production of specialized metabolites to the genetic capacity of the organism that produces such molecules has become an invaluable tool in aiding the discovery of biotechnologically applicable molecules. Here, we accomplish this task by matching molecular families with gene cluster families, making these correlations to 60 microbes at one time instead of connecting one molecule to one organism at a time, such as how it is traditionally done. We can correlate these families through the use of nanospray desorption electrospray ionization MS/MS, an ambient pressure MS technique, in conjunction with MS/MS networking and peptidogenomics. We matched the molecular families of peptide natural products produced by 42 bacilli and 18 pseudomonads through the generation of amino acid sequence tags from MS/MS data of specific clusters found in the MS/MS network. These sequence tags were then linked to biosynthetic gene clusters in publicly accessible genomes, providing us with the ability to link particular molecules with the genes that produced them. As an example of its use, this approach was applied to two unsequenced Pseudoalteromonas species, leading to the discovery of the gene cluster for a molecular family, the bromoalterochromides, in the previously sequenced strain P. piscicida JCM 20779 T . The approach itself is not limited to 60 related strains, because spectral networking can be readily adopted to look at molecular family-gene cluster families of hundreds or more diverse organisms in one single MS/MS network.MS/MS molecular networking | mass spectrometry | microbial ecology T ens of thousands of sequenced microbial genomes or rough drafts of genomes are available at this time, and this number is predicted to grow into the millions over the next decades. This wealth of sequence data has the potential to be used for the discovery of small bioactive molecules through genome mining (1-6). Genome mining is a process in which small molecules are discovered by predicting what compound will be genetically encoded based on the sequences of biosynthetic gene clusters. However, the process of mining genetically encoded small molecules is not keeping pace with the rate by which genome sequences are being obtained. In general, genome mining is still done one gene cluster at a time and requires many person-years of effort to annotate a single molecule. The time and significant expertise that current genome mining requires also make genome mining very expensive. In light of this extensive effort and cost, alternative approaches to genome mining and annotating specialized metabolites must be developed that not only take advantage of the sequenced resources available and make it efficient to perform genome mining on a more global scale but also enable the molecular analysis of unsequenced organisms. Such methods will then significantly reduce the cost of genome mining by increasing the speed with which molecules are connected to candidate genes and using resources already available. Here, we put fo...
Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.
The function of microbial interactions is to enable microorganisms to survive by establishing a homeostasis between microbial neighbors and local environments. A microorganism can respond to environmental stimuli using metabolic exchange—the transfer of molecular factors, including small molecules and proteins. Microbial interactions not only influence the survival of the microbes but also have roles in morphological and developmental processes of the organisms themselves and their neighbors. This, in turn, shapes the entire habitat of these organisms. Here we highlight our current understanding of metabolic exchange as well as the emergence of new technologies that are allowing us to eavesdrop on microbial conversations comprising dozens to hundreds of secreted metabolites that control the behavior, survival and differentiation of members of the community. The goal of the rapidly advancing field studying multifactorial metabolic exchange is to devise a microbial ‘Rosetta stone’ in order to understand the language by which microbial interactions are negotiated and, ultimately, to control the outcome of these conversations.
Cystic fibrosis (CF) lungs are filled with thick mucus that obstructs airways and facilitates chronic infections. Pseudomonas aeruginosa is a significant pathogen of this disease that produces a variety of toxic small molecules. We used molecular networking-based metabolomics to investigate the chemistry of CF sputa and assess how the microbial molecules detected reflect the microbiome and clinical culture history of the patients. Metabolites detected included xenobiotics, P. aeruginosa specialized metabolites and host sphingolipids. The clinical culture and microbiome profiles did not correspond to the detection of P. aeruginosa metabolites in the same samples. The P. aeruginosa molecules that were detected in sputum did not match those from laboratory cultures. The pseudomonas quinolone signal (PQS) was readily detectable from cultured strains, but absent from sputum, even when its precursor molecules were present. The lack of PQS production in vivo is potentially due to the chemical nature of the CF lung environment, indicating that culture-based studies of this pathogen may not explain its behavior in the lung. The most differentially abundant molecules between CF and non-CF sputum were sphingolipids, including sphingomyelins, ceramides and lactosylceramide. As these highly abundant molecules contain the inflammatory mediator ceramide, they may have a significant role in CF hyperinflammation. This study demonstrates that the chemical makeup of CF sputum is a complex milieu of microbial, host and xenobiotic molecules. Detection of a bacterium by clinical culturing and 16S rRNA gene profiling do not necessarily reflect the active production of metabolites from that bacterium in a sputum sample.
Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A lambda-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a "dehydroproline acrylamide" moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.