Exploring the complementarity of fast multipulse and multidimensional NMR methods for metabolomics: a chemical ecology case study.

This study investigates the potential and complementarity of high-throughput multipulse and multidimensional NMR methods for metabolomics. Through a chemical ecology case study, three methods are investigated, offering a continuum of methods with complementary features in terms of resolution, sensitivity and experiment time. Ultrafast 2D COSY, adiabatic INEPT and SYMAPS HSQC are shown to provide a very good classification ability, comparable to the reference 1D 1H NMR method. Moreover, a detailed analysis of discriminant buckets upon supervised statistical analysis shows that all methods are highly complementary, since they are able to highlight discriminant signals that could not be detected by 1D 1H NMR. In particular, fast 2D methods appear very efficient to discriminate signals located in highly crowded regions of the 1H spectrum. Overall, the combination of these recent methods within a single NMR metabolomics workflow allows to maximize the accessible metabolic information, and also raises exciting challenges in terms of NMR data analysis for chemical ecology.

Nature-Inspired Chemistry of Complex Alkaloids: Combining Targeted Molecular Networking Approach and Semisynthetic Strategy to Access Rare Communesins in a Marine-Derived Penicillium expansum.

Communesins are rare alkaloids isolated from fungi of the genus Penicillium. In this work, the extract of a marine-derived Penicillium expansum strain was studied using targeted molecular networking approach allowing to detect 65 communesins including 55 new ones. A fragmentation pattern for dimethylvinyl communesins was established and a script was implemented allowing to predict the structure and map all communesins in a global molecular network. A semisynthetic strategy was carried out to obtain some minor congeners from the two isolated communesins A and B. Nine communesins were then synthetised: two of them were already described as produced by the studied strain; four are new natural products which occurrence in the extracts was confirmed; three are new semi-synthetic analogues never described so far. These communesins were evaluated for their cytotoxicity on two human cancer cell lines KB and MCF-7 leading to a preliminary study of their structure-activity relationships.

Deciphering interactions between the marine dinoflagellate Prorocentrum lima and the fungus Aspergillus pseudoglaucus.

The comprehension of microbial interactions is one of the key challenges in marine microbial ecology. This study focused on exploring chemical interactions between the toxic dinoflagellate Prorocentrum lima and a filamentous fungal species, Aspergillus pseudoglaucus, which has been isolated from the microalgal culture. Such interspecies interactions are expected to occur even though they were rarely studied. Here, a co-culture system was designed in a dedicated microscale marine-like condition. This system allowed to explore microalgal-fungal physical and metabolic interactions in presence and absence of the bacterial consortium. Microscopic observation showed an unusual physical contact between the fungal mycelium and dinoflagellate cells. To delineate specialized metabolome alterations during microalgal-fungal co-culture metabolomes were monitored by high-performance liquid chromatography coupled to high-resolution mass spectrometry. In-depth multivariate statistical analysis using dedicated approaches highlighted (1) the metabolic alterations associated with microalgal-fungal co-culture, and (2) the impact of associated bacteria in microalgal metabolome response to fungal interaction. Unfortunately, only a very low number of highlighted features were fully characterized. However, an up-regulation of the dinoflagellate toxins okadaic acid and dinophysistoxin 1 was observed during co-culture in supernatants. Such results highlight the importance to consider microalgal-fungal interactions in the study of parameters regulating toxin production.

Halogenation in Fungi: What Do We Know and What Remains to Be Discovered?

In nature, living organisms produce a wide variety of specialized metabolites to perform many biological functions. Among these specialized metabolites, some carry halogen atoms on their structure, which can modify their chemical characteristics. Research into this type of molecule has focused on how organisms incorporate these atoms into specialized metabolites. Several families of enzymes have been described gathering metalloenzymes, flavoproteins, or S-adenosyl-L-methionine (SAM) enzymes that can incorporate these atoms into different types of chemical structures. However, even though the first halogenation enzyme was discovered in a fungus, this clade is still lagging behind other clades such as bacteria, where many enzymes have been discovered. This review will therefore focus on all halogenation enzymes that have been described in fungi and their associated metabolites by searching for proteins available in databases, but also by using all the available fungal genomes. In the second part of the review, the chemical diversity of halogenated molecules found in fungi will be discussed. This will allow the highlighting of halogenation mechanisms that are still unknown today, therefore, highlighting potentially new unknown halogenation enzymes.

Complementary Strategies to Unlock Biosynthesis Gene Clusters Encoding Secondary Metabolites in the Filamentous Fungus Podospora anserina.

The coprophilous ascomycete Podospora anserina is known to have a high potential to synthesize a wide array of secondary metabolites (SMs). However, to date, the characterization of SMs in this species, as in other filamentous fungal species, is far less than expected by the functional prediction through genome mining, likely due to the inactivity of most SMs biosynthesis gene clusters (BGCs) under standard conditions. In this work, our main objective was to compare the global strategies usually used to deregulate SM gene clusters in P. anserina, including the variation of culture conditions and the modification of the chromatin state either by genetic manipulation or by chemical treatment, and to show the complementarity of the approaches between them. In this way, we showed that the metabolomics-driven comparative analysis unveils the unexpected diversity of metabolic changes in P. anserina and that the integrated strategies have a mutual complementary effect on the expression of the fungal metabolome. Then, our results demonstrate that metabolite production is significantly influenced by varied cultivation states and epigenetic modifications. We believe that the strategy described in this study will facilitate the discovery of fungal metabolites of interest and will improve the ability to prioritize the production of specific fungal SMs with an optimized treatment.

Genomic diversity of Serratia proteamaculans and Serratia liquefaciens predominant in seafood products and spoilage potential analyses.

Serratia sp. cause food losses and waste due to spoilage; it is noteworthy that they represent a dominant population in seafood. The main spoilage associated species comprise S. liquefaciens, S. grimesii, S. proteamaculans and S. quinivorans, also known as S. liquefaciens-like strains. These species are difficult to discriminate since classical 16S rRNA gene-based sequences do not possess sufficient resolution. In this study, a phylogeny based on the short-length luxS gene was able to speciate 47 Serratia isolates from seafood, with S. proteamaculans being the main species from fresh salmon and tuna, cold-smoked salmon, and cooked shrimp while S. liquefaciens was only found in cold-smoked salmon. The genome of the first S. proteamaculans strain isolated from the seafood matrix (CD3406 strain) was sequenced. Pangenome analyses of S. proteamaculans and S. liquefaciens indicated high adaptation potential. Biosynthetic pathways involved in antimicrobial compounds production and in the main seafood spoilage compounds were also identified. The genetic equipment highlighted in this study contributed to gain further insights into the predominance of Serratia in seafood products and their capacity to spoil.

Untargeted Metabolomics Approach for the Discovery of Environment-Related Pyran-2-ones Chemodiversity in a Marine-Sourced Penicillium restrictum.

Very little is known about chemical interactions between fungi and their mollusc host within marine environments. Here, we investigated the metabolome of a Penicillium restrictum MMS417 strain isolated from the blue mussel Mytilus edulis collected on the Loire estuary, France. Following the OSMAC approach with the use of 14 culture media, the effect of salinity and of a mussel-derived medium on the metabolic expression were analysed using HPLC-UV/DAD-HRMS/MS. An untargeted metabolomics study was performed using principal component analysis (PCA), orthogonal projection to latent structure discriminant analysis (O-PLSDA) and molecular networking (MN). It highlighted some compounds belonging to sterols, macrolides and pyran-2-ones, which were specifically induced in marine conditions. In particular, a high chemical diversity of pyran-2-ones was found to be related to the presence of mussel extract in the culture medium. Mass spectrometry (MS)- and UV-guided purification resulted in the isolation of five new natural fungal pyran-2-one derivatives-5,6-dihydro-6S-hydroxymethyl-4-methoxy-2H-pyran-2-one (1), (6S, 1'R, 2'S)-LL-P880ß (3), 5,6-dihydro-4-methoxy-6S-(1'S, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (4), 4-methoxy-6-(1'R, 2'S-dihydroxy pent-3'(E)-enyl)-2H-pyran-2-one (6) and 4-methoxy-2H-pyran-2-one (7)-together with the known (6S, 1'S, 2'S)-LL-P880ß (2), (1'R, 2'S)-LL-P880γ (5), 5,6-dihydro-4-methoxy-2H-pyran-2-one (8), (6S, 1'S, 2'R)-LL-P880ß (9), (6S, 1'S)-pestalotin (10), 1'R-dehydropestalotin (11) and 6-pentyl-4-methoxy-2H-pyran-2-one (12) from the mussel-derived culture medium extract. The structures of 1-12 were determined by 1D- and 2D-MMR experiments as well as high-resolution tandem MS, ECD and DP4 calculations. Some of these compounds were evaluated for their cytotoxic, antibacterial, antileishmanial and in-silico PTP1B inhibitory activities. These results illustrate the utility in using host-derived media for the discovery of new natural products.

New Insight into Antimicrobial Compounds from Food and Marine-Sourced Carnobacterium Species through Phenotype and Genome Analyses.

Carnobacterium maltaromaticum and Carnobacterium divergens, isolated from food products, are lactic acid bacteria known to produce active and efficient bacteriocins. Other species, particularly those originating from marine sources, are less studied. The aim of the study is to select promising strains with antimicrobial potential by combining genomic and phenotypic approaches on large datasets comprising 12 Carnobacterium species. The biosynthetic gene cluster (BGCs) diversity of 39 publicly available Carnobacterium spp. genomes revealed 67 BGCs, distributed according to the species and ecological niches. From zero to six BGCs were predicted per strain and classified into four classes: terpene, NRPS (non-ribosomal peptide synthetase), NRPS-PKS (hybrid non-ribosomal peptide synthetase-polyketide synthase), RiPP (ribosomally synthesized and post-translationally modified peptide). In parallel, the antimicrobial activity of 260 strains from seafood products was evaluated. Among the 60% of active strains, three genomes were sequenced and submitted to a dereplication process. C. inhibens MIP2551 produced a high amountof H2O2, probably thanks to the presence of four oxidase-encoding genes. C. maltaromaticum EBP3019 and SF668 strains were highly efficient against Listeria monocytogenes. A new extracellular 16 kDa unmodified bacteriocin in the EBP3019 strain and five different bacteriocins in SF668 were highlighted. In this study, the overview of antimicrobial BGC and inhibitory activities of Carnobacterium spp. allowed the prediction of potential innovative natural products that could be relevant for biotechnological applications.

Expanding the chemical diversity through microorganisms co-culture: Current status and outlook.

Natural products (NPs) are considered as a cornerstone for the generation of bioactive leads in drug discovery programs. However, one of the major limitations of NP drug discovery program is "rediscovery" of known compounds, thereby hindering the rate of drug discovery efficiency. Therefore, in recent years, to overcome these limitations, a great deal of attention has been drawn towards understanding the role of microorganisms' co-culture in inducing novel chemical entities. Such induction could be related to activation of genes which might be silent or expressed at very low levels (below detection limit) in pure-strain cultures under normal laboratory conditions. In this review, chemical diversity of compounds isolated from microbial co-cultures, is discussed. For this purpose, chemodiversity has been represented as a chemical-structure network based on the "Tanimoto Structural Similarity Index". This highlights the huge structural diversity induced by microbial co-culture. In addition, the current trends in microbial co-culture research are highlighted. Finally, the current challenges (1 - induction monitoring, 2 - reproducibility, 3 - growth time effect and 4 - up-scaling for isolation purposes) are discussed. The information in this review will support researchers to design microbial co-culture strategies for future research efforts. In addition, guidelines for co-culture induction reporting are also provided to strengthen future reporting in this NP field.

Metabolomics-Driven Discovery of Meroterpenoids from a Mussel-Derived Penicillium ubiquetum.

Penicillium ubiquetum MMS330 isolated from the blue mussel Mytilus edulis collected on the Loire estuary in France was here investigated. As very few secondary metabolites have been documented for this species, its metabolome was studied following the OSMAC approach to enhance as many biosynthetic pathways as possible. Interestingly, HPLC-HRMS based hierarchical clustering analysis together with MS/MS molecular networking highlighted the selective overproduction of some structurally related compounds when the culture was performed on seawater CYA (Czapek Yeast extract Agar) medium. Mass-guided purification from large scale cultivation on this medium led to the isolation of nine meroterpenoids including two new analogues, 22-deoxyminiolutelide A (1) and 4-hydroxy-22-deoxyminiolutelide B (2), together with seven known compounds (3-9). The structures of 1 and 2 were elucidated on the basis of HR-ESIMS and NMR spectroscopic data analysis. Furthermore, NMR signals of 22-deoxyminiolutelide B (3) were reassigned.

Benzofuro[3,2-d]pyrimidines inspired from cercosporamide CaPkc1 inhibitor: Synthesis and evaluation of fluconazole susceptibility restoration.

In a context of growing resistance to classical antifungal therapy, the design of new drugs targeting alternative pathways is highly expected. Benzofuro[3,2-d]pyrimidine derivatives, derived from (-)-cercosporamide, were synthesized and evaluated as potential Candida albicans PKC inhibitors in the aim of restoring susceptibility to azole treatment. Co-administration assay of benzofuropyrimidinedione 23 and fluconazole highlighted a synergistic effect on inhibition of cell growth of a Candida albicans resistant strain.

Automated Detection of Natural Halogenated Compounds from LC-MS Profiles-Application to the Isolation of Bioactive Chlorinated Compounds from Marine-Derived Fungi.

A collection of culture extracts obtained from several marine-derived fungal strains collected on the French Atlantic coast was investigated by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) in order to prospect for halogenated compounds and to identify potentially new ones. To achieve a fast, automated, and efficient data analysis, a bioinformatics tool named MeHaloCoA (Marine Halogenated Compound Analysis) was developed and included into R. After extraction of all the peaks from the metabolic fingerprints and their associated mass spectra, a mathematical filter based on mass isotopic profiles allowed the selective detection of halogenated (Cl and Br) molecules. Integrating MeHaloCoA into a dereplication approach allowed the identification of known and new halogenated compounds in a competitive amount of time. Subsequent targeted purification led to the isolation of several chlorinated metabolites, including two new natural products with bioactive potential, griseophenone I and chlorogriseofulvin, from a marine-derived Penicillium canescens strain.

Time Dependency of Chemodiversity and Biosynthetic Pathways: An LC-MS Metabolomic Study of Marine-Sourced Penicillium.

This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter "time" for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry) each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry) data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome.

Synthesis and antiproliferative activity of ligerin and new fumagillin analogs against osteosarcoma.

Ligerin (1) is a natural chlorinated merosesquiterpenoid related to fumagillin (2) exhibiting a selective antiproliferative activity against osteosarcoma cell lines and an in vivo antitumor activity in a murine model. Semisynthesis of ligerin analogs was performed in order to study the effects of the C3-spiroepoxide substitution by a halogenated moiety together with the modulation of the C6 chain. Results showed that all derivatives exhibited an in vitro antiproliferative activity against osteosarcoma cell lines and that chlorohydrin compounds were equally or more active than their spiroepoxy analogs. Among semisynthetic analogs, the parent compound 1 was the best candidate for further studies since it exhibited higher or equivalent activity compared to TNP470 (3) against SaOS2 and MG63 human osteosarcoma cells with a four times weaker toxicity against HFF2 human fibroblasts. Quantitative videomicroscopy analysis was conducted and allowed a better understanding of the mechanism of its antiproliferative activity.

Cytotoxicity, fractionation and dereplication of extracts of the dinoflagellate Vulcanodinium rugosum, a producer of pinnatoxin G.

Pinnatoxin G (PnTX-G) is a marine toxin belonging to the class of cyclic imines and produced by the dinoflagellate Vulcanodinium rugosum. In spite of its strong toxicity to mice, leading to the classification of pinnatoxins into the class of "fast-acting toxins", its hazard for human health has never been demonstrated. In this study, crude extracts of V. rugosum exhibited significant cytotoxicity against Neuro2A and KB cells. IC50 values of 0.38 µg mL⁻¹ and 0.19 µg mL⁻¹ were estimated on Neuro2A cells after only 24 h of incubation and on KB cells after 72 h of incubation, respectively. In the case of Caco-2 cells 48 h after exposure, the crude extract of V. rugosum induced cell cycle arrest accompanied by a dramatic increase in double strand DNA breaks, although only 40% cytotoxicity was observed at the highest concentration tested (5 µg mL⁻¹). However, PnTX-G was not a potent cytotoxic compound as no reduction of the cell viability was observed on the different cell lines. Moreover, no effects on the cell cycle or DNA damage were observed following treatment of undifferentiated Caco-2 cells with PnTX-G. The crude extract of V. rugosum was thus partially purified using liquid-liquid partitioning and SPE clean-up. In vitro assays revealed strong activity of some fractions containing no PnTX-G. The crude extract and the most potent fraction were evaluated using full scan and tandem high resolution mass spectrometry. The dereplication revealed the presence of a major compound that could be putatively annotated as nakijiquinone A, N-carboxy-methyl-smenospongine or stachybotrin A, using the MarinLit™ database. Further investigations will be necessary to confirm the identity of the compounds responsible for the cytotoxicity and genotoxicity of the extracts of V. rugosum.

Ligerin, an antiproliferative chlorinated sesquiterpenoid from a marine-derived Penicillium strain.

A new chlorinated sesquiterpenoid analogue of fumagillin, ligerin (1), was isolated from a marine-derived strain of Penicillium, belonging to the subgenus Penicillium, along with the known compounds penicillic acid (2), orcinol, and orsellinic acid. Chemical structures were established by an interpretation of spectroscopic data including IR, UV, and HRESIMS, together with analyses of 1D and 2D NMR spectra and X-ray analysis for the determination of the absolute configuration. Ligerin (1) displayed strong inhibitory activity against an osteosarcoma cell line. This is the first report of the isolation of a fumagillin analogue from a marine-derived Penicillium strain.

Patulin and secondary metabolite production by marine-derived Penicillium strains.

Genus Penicillium represents an important fungal group regarding to its mycotoxin production. Secondary metabolomes of eight marine-derived strains belonging to subgenera Furcatum and Penicillium were investigated using dereplication by liquid chromatography (LC)-Diode Array Detector (DAD)-mass spectrometry (MS)/MS. Each strain was grown on six different culture media to enhance the number of observable metabolites. Thirty-two secondary metabolites were detected in crude extracts with twenty first observations for studied species. Patulin, a major mycotoxin, was classically detected in extracts of Penicillium expansum, and was also isolated from Penicillium antarcticum cultures, whose secondary metabolome is still to be done. These detections constituted the first descriptions of patulin in marine strains of Penicillium, highlighting the risk for shellfish and their consumers due to the presence of these fungi in shellfish farming areas. Patulin induced acute neurotoxicity on Diptera larvae, indicating the interest of this bioassay as an additional tool for detection of this major mycotoxin in crude extracts.

Enhancement of domoic acid neurotoxicity on Diptera larvae bioassay by marine fungal metabolites.

Peptaibols are small linear fungal peptides which are produced in the marine environment. They exhibit neurotoxicity by forming pores in neuronal membranes. This work describes their combine effect with domoic acid, a neurotoxic phycotoxin, on Diptera larvae. The Acute toxicity bioassay on this biological model was tested with a panel of different toxins (microbial, algal or fungal). It allowed the discrimination of neurotoxins and non-neurotoxic toxins, and an evaluation of the toxicity level (MED and ED(50)) which were correlated with published LD(50) in mice for neurotoxins tested. The highest activities on this test were found for Na(+) channel blockers tetrodotoxin (ED(50) = 0.026 mg/kg) and saxitoxin (ED(50) = 0.18 mg/kg). Domoic acid was less active with an ED(50) = 7.6 mg/kg. For synergism study, longibrachin-A-I, a 20-mer peptaibol isolated from cultures of a marine-derived strain of Trichoderma longibrachiatum Rifai was chosen. Bioassay results confirmed its neuroactivity. Its level of toxicity (ED(50) = 270 mg/kg) was lower than those of phycotoxins tested but higher than mycotoxin ones. Injected together, longibrachin-A-I and domoic acid exhibited an increase of their activities. With doses of longibrachin-A-I below its Minimal Effective Dose (MED), the synergism factor which expresses the enhancement of domoic acid toxicity could reach 34.5. Both domoic acid and longibrachin-A-I are acting on ion channels and pores in neuronal membranes which contribute to the intake of Ca(2+) into cells.

Structural investigation and elucidation of new communesins from a marine-derived Penicillium expansum Link by liquid chromatography/electrospray ionization mass spectrometry.

Penicillium expansum is a ubiquitous species for which there are only few reports for chemical investigation in marine environments. Among the numerous secondary metabolites produced by this species, communesins represent a new class of cytotoxic and insecticidal indole alkaloids. In this study, we investigated a marine P. expansum strain exhibiting neuroactivity on a Diptera larvae bioassay. Bio-guided purification led to the isolation and the identification of communesin B as the main active compound by HRMS and 1H and 13C NMR. Liquid chromatography analyses with detection by electrospray ionization coupled with tandem mass spectrometry (LC/ESI-MS/MS) and high-resolution tandem mass spectrometry (LC/HRMS/MS) allowed the identification and characterization of four other known communesins (A, D, E and F) in the crude extract. A fragmentation model for dimethyl epoxide communesins was proposed after detailed interpretation of their MS/MS spectra. Further analyses of the extract using the modelled fragmentations led to the detection of seven new communesins found as minor compounds. Chemical structural elucidation of these new derivatives is discussed based on their fragmentation characteristics.

Effects of seawater on growth and gliotoxin excretion of marine strains of Aspergillus fumigatus Fres.

In order to enhance the knowledge of the putative toxinic risk linked to mycotoxin excretion in shellfish farming areas, the influence of seawater salinity was studied on 2 marine-derived Aspergillus fumigatus strains. This fungal species produces gliotoxin, an epipolythiodioxopiperazine immunosuppressive mycotoxin that can be accumulated in the meat of cultured blue mussel (Mytilus edulis), and could be responsible for disease when ingested. Two marine strains were grown in vitro both on a non-saline and a saline culture media and were compared with 13 terrestrial strains to observe the effects of seawater on fungal growth and gliotoxin excretion in the exudate produced. Daily measurement of the colony areas showed that the seawater salinity significantly reduced the rate of growth of all the strains. Marine and terrestrial strains appeared to be almost similar as regards the appearance, growth and gliotoxin excretion, but the marine strains exudation seemed to be less influenced by seawater salinity than the terrestrial strains. Seawater salinity, however, enhanced exudation and gliotoxin excretion by A. fumigatus, and thus seems to be an aggravating factor for the toxicity of this species in the marine environment.