Xerophytic Lichens from Gypsiferous Outcrops of Arid Areas of Andalusia as a Source of Anti-Phytopathogenic Depsides.

In a survey to evaluate the potential of lichens associated with gypsum areas as sources of new antifungal metabolites, six species of lichens were collected in the gypsum outcrops of the Sorbas Desert (Diploschistes ocellatus and Seirophora lacunosa) and the Tabernas Desert (Cladonia foliacea, Acarospora placodiformis, Squamarina lentigera and Xanthoparmelia pokornyi) in southern Spain. Raw lichen acetone extracts were tested against a panel of seven phytopathogenic fungi, including Botrytis cinerea, Colletotrichum acutatum, Fusarium oxysporum f.sp cubense TR4, Fusarium ploriferaum, Magnaporthe grisea, Verticillium dahliae and Zymoseptoria tritici. Active extracts of Cladonia foliacea, Xanthoparmelia pokornyi and Squamarina lentigera were analyzed by HPLC-MS/MS and Molecular Networking to identify possible metabolites responsible for the antifungal activity. A total of ten depside-like metabolites were identified by MS/MS dereplication and NMR experiments, of which one was a new derivative of fumaroprotocetraric acid. The compounds without previously described biological activity were purified and tested against the panel of fungal phytopathogens. Herein, the antifungal activity against fungal phytopathogens of 4'-O-methylpaludosic acid, divaricatic acid and stenosporic acid is reported for the first time. Stenosporic and divaricatic acids displayed a broad antifungal spectrum against seven relevant fungal phytopathogens in a micromolar range, including the extremely resistant fungus F. oxysporum f. sp. cubense Tropical Race 4 (TR4). 4'-O-methylpaludosic acid exhibited specific antifungal activity against the wheat pathogen Z. tritici, with an IC50 of 38.87 µg/mL (87.1 µM) in the absorbance-based assay and 24.88 µg/mL (55.52 µM) in the fluorescence-based assay.

MS/MS Molecular Networking Unveils the Chemical Diversity of Biscembranoid Derivatives, Neutrophilic Inflammatory Mediators from the Cultured Soft Coral Sarcophyton trocheliophorum.

Biscembranoids are the distinctive tetraterpenoids owing a 14/6/14 membered tricyclic scaffold that have been mainly discovered in the soft corals, especially the genera Sarcophyton, Lobophytum and Sinularia. Recent findings have demonstrated the great anti-inflammatory potential of biscembranoid analogues in human neutrophils, motivating more chemical and biological explorations targeting these marine-derived natural products. In the current study, the chemical diversity of biscembranoids derived from the cultured-type Sarcophyton trocheliophorum von Marenzeller was illustrated through MS/MS molecular networking (MN) profiling approach. Based on the MN patterns, the prioritization of unknown biscembranoid derivatives was putatively analyzed. As a result, the biscembrane targeting isolation afforded two new metabolites, sarcotrochelides A (1) and B (2), along with six known analogues (3-8). Their structures and relative configurations were determined by spectroscopic methods. In vitro neutrophil inflammatory inhibition was further investigated for all isolates based on reduced superoxide anion (O2•-) generation detections. Compounds 5-8 showed significant dose-dependently inhibitory effects, suggesting the cruciality of 6,7-dihydrooxepin-2(5H)-one moiety and saturated γ-lactone ring in their reactive oxygen species (ROS)-dependent anti-inflammatory properties.

Molecular networking assisted discovery and combinatorial biosynthesis of new antimicrobial pleuromutilins.

Pleuromutilins, the unique fungal metabolites possessing 5/6/8 tricyclic skeleton, are potent antibacterial leading compounds for the development of new antibiotics. We applied the MS/MS molecular networking technique and the combinatorial biosynthesis approach to discover new pleuromutilin analogues. Ten pleuromutilin derivatives including seven new compounds (1-7) were obtained from the solid culture of Omphalina mutila. The gene cluster for the biosynthesis of pleuromutilins in the mushroom of O. mutila was identified and further expressed in yeast. Nine pleuromutilin-type diterpenes including three new "unnatural" pleuromutilins (16-18) were generated in a GGPP-engineered Saccharomyces cerevisiae. The antimicrobial bioassays indicated that compounds 3, 9, 10, 15, and 17 exhibited potent inhibition against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE). Several pleuromutilins were found to show immunomodulatory activities by promoting the cell viability, enhancing the ROS and NO production, or increasing the levels of proinflammatory cytokines IL-6 and TNF-α in the macrophage RAW 264.7. The structure-activity relationship for pleuromutilins was analyzed.

Assessing the Diversity and Biomedical Potential of Microbes Associated With the Neptune's Cup Sponge, Cliona patera.

Marine sponges are known to host a complex microbial consortium that is essential to the health and resilience of these benthic invertebrates. These sponge-associated microbes are also an important source of therapeutic agents. The Neptune's Cup sponge, Cliona patera, once believed to be extinct, was rediscovered off the southern coast of Singapore in 2011. The chance discovery of this sponge presented an opportunity to characterize the prokaryotic community of C. patera. Sponge tissue samples were collected from the inner cup, outer cup and stem of C. patera for 16S rRNA amplicon sequencing. C. patera hosted 5,222 distinct OTUs, spanning 26 bacterial phyla, and 74 bacterial classes. The bacterial phylum Proteobacteria, particularly classes Gammaproteobacteria and Alphaproteobacteria, dominated the sponge microbiome. Interestingly, the prokaryotic community structure differed significantly between the cup and stem of C. patera, suggesting that within C. patera there are distinct microenvironments. Moreover, the cup of C. patera had lower diversity and evenness as compared to the stem. Quorum sensing inhibitory (QSI) activities of selected sponge-associated marine bacteria were evaluated and their organic extracts profiled using the MS-based molecular networking platform. Of the 110 distinct marine bacterial strains isolated from sponge samples using culture-dependent methods, about 30% showed quorum sensing inhibitory activity. Preliminary identification of selected QSI active bacterial strains revealed that they belong mostly to classes Alphaproteobacteria and Bacilli. Annotation of the MS/MS molecular networkings of these QSI active organic extracts revealed diverse classes of natural products, including aromatic polyketides, siderophores, pyrrolidine derivatives, indole alkaloids, diketopiperazines, and pyrone derivatives. Moreover, potential novel compounds were detected in several strains as revealed by unique molecular families present in the molecular networks. Further research is required to determine the temporal stability of the microbiome of the host sponge, as well as mining of associated bacteria for novel QS inhibitors.

Genome Mining, Microbial Interactions, and Molecular Networking Reveals New Dibromoalterochromides from Strains of Pseudoalteromonas of Coiba National Park-Panama.

The marine bacterial genus Pseudoalteromonas is known for their ability to produce antimicrobial compounds. The metabolite-producing capacity of Pseudoalteromonas has been associated with strain pigmentation; however, the genomic basis of their antimicrobial capacity remains to be explained. In this study, we sequenced the whole genome of six Pseudoalteromonas strains (three pigmented and three non-pigmented), with the purpose of identifying biosynthetic gene clusters (BGCs) associated to compounds we detected via microbial interactions along through MS-based molecular networking. The genomes were assembled and annotated using the SPAdes and RAST pipelines and mined for the identification of gene clusters involved in secondary metabolism using the antiSMASH database. Nineteen BGCs were detected for each non-pigmented strain, while more than thirty BGCs were found for two of the pigmented strains. Among these, the groups of genes of nonribosomal peptide synthetases (NRPS) that code for bromoalterochromides stand out the most. Our results show that all strains possess BGCs for the production of secondary metabolites, and a considerable number of distinct polyketide synthases (PKS) and NRPS clusters are present in pigmented strains. Furthermore, the molecular networking analyses revealed two new molecules produced during microbial interactions: the dibromoalterochromides D/D' (11-12).

Combined MS/MS-NMR Annotation Guided Discovery of Iris lactea var. chinensis Seed as a Source of Viral Neuraminidase Inhibitory Polyphenols.

In this study, the chemical diversity of polyphenols in Iris lactea var. chinensis seeds was identified by combined MS/MS-NMR analysis. Based on the annotated chemical profile, the isolation of stilbene oligomers was conducted, and consequently, stilbene oligomers (1-10) were characterized. Of these, compounds 1 and 2 are previously undescribed stilbene dimer glycoside (1) and tetramer glycoside (2), respectively. Besides, to evaluate this plant seed as a rich source of stilbene oligomers, we quantified three stilbene oligomers of I. lactea var. chinensis seeds. The contents of three major stilbene oligomers-trans-ε-viniferin (3), vitisin A (6), and vitisin B (9)-in I. lactea var. chinensis seeds were quantified as 2.32 (3), 4.95 (6), and 1.64 (9) mg/g dry weight (DW). All the isolated compounds were tested for their inhibitory activities against influenza neuraminidase. Compound 10 was found to be active with the half maximal inhibitory concentration (IC50) values at 4.76 µM. Taken together, it is concluded that I. lactea var. chinensis seed is a valuable source of stilbene oligomers with a human health benefit.

SweetNET: A Bioinformatics Workflow for Glycopeptide MS/MS Spectral Analysis.

Glycoproteomics has rapidly become an independent analytical platform bridging the fields of glycomics and proteomics to address site-specific protein glycosylation and its impact in biology. Current glycopeptide characterization relies on time-consuming manual interpretations and demands high levels of personal expertise. Efficient data interpretation constitutes one of the major challenges to be overcome before true high-throughput glycopeptide analysis can be achieved. The development of new glyco-related bioinformatics tools is thus of crucial importance to fulfill this goal. Here we present SweetNET: a data-oriented bioinformatics workflow for efficient analysis of hundreds of thousands of glycopeptide MS/MS-spectra. We have analyzed MS data sets from two separate glycopeptide enrichment protocols targeting sialylated glycopeptides and chondroitin sulfate linkage region glycopeptides, respectively. Molecular networking was performed to organize the glycopeptide MS/MS data based on spectral similarities. The combination of spectral clustering, oxonium ion intensity profiles, and precursor ion m/z shift distributions provided typical signatures for the initial assignment of different N-, O- and CS-glycopeptide classes and their respective glycoforms. These signatures were further used to guide database searches leading to the identification and validation of a large number of glycopeptide variants including novel deoxyhexose (fucose) modifications in the linkage region of chondroitin sulfate proteoglycans.

MS/MS networking guided analysis of molecule and gene cluster families.

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.