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.

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.

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.

Pentadecaibins I-V: 15-Residue Peptaibols Produced by a Marine-Derived Trichoderma sp. of the Harzianum Clade.

In the course of investigations on peptaibol chemodiversity from marine-derived Trichoderma spp., five new 15-residue peptaibols named pentadecaibins I-V (1-5) were isolated from the solid culture of the strain Trichoderma sp. MMS1255 belonging to the T. harzianum species complex. Phylogenetic analyses allowed precise positioning of the strain close to T. lentiforme lineage inside the Harzianum clade. Peptaibol sequences were elucidated on the basis of their MS/MS fragmentation and extensive 2D NMR experiments. Amino acid configurations were determined by Marfey's analyses. The pentadecaibins are based on the sequences Ac-Aib1-Gly2-Ala3-Leu4-Aib/Iva5-Gln6-Aib/Iva7-Val/Leu8-Aib9-Ala10-Aib11-Aib12-Aib13-Gln14-Pheol15. Characteristic of the pentadecaibin sequences is the lack of the Aib-Pro motif commonly present in peptaibols produced by Trichoderma spp. Genome sequencing of Trichoderma sp. MMS1255 allowed the detection of a 15-module NRPS-encoding gene closely associated with pentadecaibin biosynthesis. Pentadecaibins were assessed for their potential antiproliferative and antimicrobial activities.

Steroids from Marine-Derived Fungi: Evaluation of Antiproliferative and Antimicrobial Activities of Eburicol.

The most common sterol in fungi is ergosterol, which has frequently been investigated in human pathogenic fungal strains. This sterol, and others isolated from fungal strains, has also demonstrated cytotoxicity against cancer cell lines and antimicrobial activities. Marine fungi can produce high amounts of bioactive compounds. So, a screening was performed to study sterol composition using GC/MS in 19 marine fungal strains and ergosterol was always the major one. One strain, Clonostachys rosea MMS1090, was selected due to its high amount of eburicol and a one strain many compounds approach was performed on seven culture media to optimize its production. After purification and structural identification by NMR, eburicol was assessed against four cancer cell lines, MCF-7, MDA-MB-231, NSCLC-N6-L16 and A549, and seven human pathogenic bacteria Staphylococcus aureus, Bacillus sp., Bacillus cereus, Listeria ivanovii, Escherichia coli, Citrobacter freundii and Salmonella spp. The most significant activity was cytotoxicity against MCF-7 cells (2 µM). This is the first report of such an accumulation of eburicol in the marine fungal strain C. rosea confirming its potential in the production of bioactive lipids.

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.

Hyporientalin A, an anti-Candida peptaibol from a marine Trichoderma orientale.

A Trichoderma orientale strain LSBA1 was isolated from the Mediterranean marine sponge Cymbaxinella damicornis. The crude extract of T. orientale mycelium showed inhibitory activity against growth of Gram-positive and Gram-negative bacteria as well as clinical isolates of Candida albicans. Purification of the anti-Candida component was performed using a combination of open silica gel-60 column and reverse phase high performance liquid chromatography. The active compound called hyporientalin A has been identified as a peptaibol analogue of longibrachin-A-II using mass spectrometry. It exhibited fungicidal activity against clinical isolates of C. albicans with minimal inhibitory concentrations (MICs) ranging from 2.49 to 19.66 µM, comparable to that of the antifungal agent amphotericin B. Our data support the use of hyporientalin A as a promising new and efficient antifungal drug in the treatment of candidiasis while controlling toxicity.

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.

The Marine-Derived Fungus Clonostachys rosea, Source of a Rare Conjugated 4-Me-6E,8E-hexadecadienoic Acid Reducing Viability of MCF-7 Breast Cancer Cells and Gene Expression of Lipogenic Enzymes.

A marine-derived strain of Clonostachys rosea isolated from sediments of the river Loire estuary (France) was investigated for its high lipid production. The fungal strain was grown on six different culture media to explore lipid production changes. An original branched conjugated fatty acid, mainly present in triglycerides and mostly produced when grown on DCA (23% of total fatty acid composition). It was identified as 4-Me-6E,8E-hexadecadienoic on the basis of spectroscopic analyses. This fatty acid reduced viability of MCF-7 breast cancer cells in a dose dependent manner (up to 63%) at physiological free fatty acid human plasma concentration (100 µM). Reduction of gene expression of two lipogenic enzymes, the acetyl CoA carboxylase (ACC) and the fatty acid synthase (FAS) was evaluated to explore the mechanisms of action of 4-Me-6E,8E-16:2 acid. At 50 µM, 50% and 35% of mRNA gene expression inhibition were observed for ACC and FAS, respectively.

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.

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.

Unprecedented 17-residue peptaibiotics produced by marine-derived Trichoderma atroviride.

In the course of investigations on marine-derived toxigenic fungi, five strains of Trichoderma atroviride were studied for their production of peptaibiotics. While these five strains were found to produce classical 19-residue peptaibols, three of them exhibited unusual peptidic sodium-adduct [M + 2 Na](2+) ion peaks at m/z between 824 and 854. The sequencing of these peptides led to two series of unprecedented 17-residue peptaibiotics based on the model Ac-XXX-Ala-Ala-XXX-XXX-Gln-Aib-Aib-Aib-Ala/Ser-Lxx-Aib-Pro-XXX-Aib-Lxx-[C(129) ]. The C-terminus of these new peptides was common to all of them, and its elemental formula C5 H9 N2 O2 was established by HR-MS. It could correspond to the cyclized form of N(δ) -hydroxyornithine which has already been observed at the C-terminus of various peptidic siderophores. The comparison of the sequences of 17- and 19-residue peptides showed similarities for positions 1-16. This observation seems to indicate a common biosynthesis pathway. Both new 17-residue peptaibiotics and 19-residue peptaibols exhibited weak in vitro cytotoxicities against KB cells.

Heterologous Expression and Biochemical Characterization of a New Chloroperoxidase Isolated from the Deep-Sea Hydrothermal Vent Black Yeast Hortaea werneckii UBOCC-A-208029.

The initiation of this study relies on a targeted genome-mining approach to highlight the presence of a putative vanadium-dependent haloperoxidase-encoding gene in the deep-sea hydrothermal vent fungus Hortaea werneckii UBOCC-A-208029. To date, only three fungal vanadium-dependent haloperoxidases have been described, one from the terrestrial species Curvularia inaequalis, one from the fungal plant pathogen Botrytis cinerea, and one from a marine derived isolate identified as Alternaria didymospora. In this study, we describe a new vanadium chloroperoxidase from the black yeast H. werneckii, successfully cloned and overexpressed in a bacterial host, which possesses higher affinity for bromide (Km = 26 µM) than chloride (Km = 237 mM). The enzyme was biochemically characterized, and we have evaluated its potential for biocatalysis by determining its stability and tolerance in organic solvents. We also describe its potential three-dimensional structure by building a model using the AlphaFold 2 artificial intelligence tool. This model shows some conservation of the 3D structure of the active site compared to the vanadium chloroperoxidase from C. inaequalis but it also highlights some differences in the active site entrance and the volume of the active site pocket, underlining its originality.

Two classes of new peptaibols are synthesized by a single non-ribosomal peptide synthetase of Trichoderma virens.

Peptaibols are a group of small peptides having a high α-aminoisobutyric acid (Aib) content and produced by filamentous fungi, especially by the members of the genus Trichoderma (anamorph Hypocrea). These antibiotics are economically important for their anti-microbial and anti-cancer properties as well as ability to induce systemic resistance in plants against microbial invasion. In this study we present sequences of two classes (11-residue and 14-residue) of peptaibols produced by the biocontrol fungus Trichoderma virens. Of the 35 11-residue peptaibols sequenced, 18 are hitherto not described, and all the 53 14-residue sequences described by us here are new. We have also identified a peptaibol synthetase (non-ribosomal peptide synthetase, NRPS) with 14 complete modules in the genome of this fungus and disruption of this single gene (designated as tex2) resulted in the loss of both the classes of peptaibols. We, thus present here an unprecedented case where a single NRPS encodes for two classes of peptaibols. The new peptaibols identified here could have applications as therapeutic agents for the management of human and plant health.

Targeting bioactive compounds in natural extracts - Development of a comprehensive workflow combining chemical and biological data.

In natural product drug discovery, several strategies have emerged to highlight specifically bioactive compound(s) within complex mixtures (fractions or crude extracts) using metabolomics tools. In this area, a great deal of interest has raised among the scientific community on strategies to link chemical profiles and associated biological data, leading to the new field called "biochemometrics". This article falls into this emerging research by proposing a complete workflow, which was divided into three major steps. The first one consists in the fractionation of the same extract using four different chromatographic stationary phases and appropriated elution conditions to obtain five fractions for each column. The second step corresponds to the acquisition of chemical profiles using HPLC-HRMS analysis, and the biological evaluation of each fraction. The last step evaluates the links between the relative abundances of molecules present in fractions (peak area) and the global bioactivity level observed for each fraction. To this purpose, an original bioinformatics script (encoded with R Studio software) using the combination of four statistical models (Spearman, F-PCA, PLS, PLS-DA) was here developed leading to the generation of a "Super list" of potential bioactive compounds together with a predictive score. This strategy was validated by its application on a marine-derived Penicillium chrysogenum extract exhibiting antiproliferative activity on breast cancer cells (MCF-7 cells). After the three steps of the workflow, one main compound was highlighted as responsible for the bioactivity and identified as ergosterol. Its antiproliferative activity was confirmed with an IC50 of 0.10 µM on MCF-7 cells. The script efficiency was further demonstrated by comparing the results obtained with a different recently described approach based on NMR profiling and by virtually modifying the data to evaluate the computational tool behaviour. This approach represents a new and efficient tool to tackle some of the bottlenecks in natural product drug discovery programs.

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.

New Trichobrachins, 11-residue peptaibols from a marine strain of Trichoderma longibrachiatum.

A marine strain of Trichoderma longibrachiatum isolated from blue mussels (Mytilus edulis) was investigated for short peptaibol production. Various 11-residue peptaibols, obtained as microheterogenous mixtures after a chromatographic fractionation, were identified by positive mass spectrometry fragmentation (ESI-IT-MS(n), CID-MS(n) and GC/EI-MS). Thirty sequences were identified, which is the largest number of analogous sequences so far observed at once. Twenty-one sequences were new, and nine others corresponded to peptaibols already described. These peptaibols belonged to the same peptidic family based on the model Ac-Aib-xxx-xxx-xxx-Aib-Pro-xxx-xxx-Aib-Pro-xxol. They were named trichobrachin A when the residue in position 2 was an Asn, and trichobrachin C when it was a Gln. Major chromatographic sub-fractions, corresponding to purified peptaibols, were assayed for their cytotoxic activity. Trichobrachin A-IX and trichobrachin C exhibited the highest activities. There was an exponential relation between their relative hydrophobicity and their cytotoxicity on KB cells.

Determination of peptaibol trace amounts in marine sediments by liquid chromatography/electrospray ionization-ion trap-mass spectrometry.

Extraction followed by reverse phase liquid chromatography (LC)/electrospray ionization-ion trap-mass spectrometry (ESI-IT-MS) analysis has been successfully developed for the determination of peptaibols, fungal toxic metabolites, in marine sediments. Spiking experiments showed that the mean recovery of target compounds exceeded 85% at a spiking level of 10 ng/g of sediment (wet weight). Detection and quantification limits were 250 and 830 pg/g of sediment, respectively. The method developed constituted the first sensitive assay for quantification of peptaibol trace amounts in a natural environment. A concentration of 5 ng/g in sediment samples collected from Fier d'Ars was found.

Toxicity assessment of peptaibols and contaminated sediments on Crassostrea gigas embryos.

Peptaibols are known membrane-modifying peptides that were recently detected in marine sediments and mussels collected from a shellfish farming area (Fier d'Ars, Atlantic coast, France). In this investigation, embryotoxicity bioassays with oysters (Crassostrea gigas) were performed to assess acute toxicity of alamethicin and different groups of peptaibols produced by a Trichoderma longibrachiatum strain isolated from marine environment. C. gigas embryos appeared very sensitive to all the metabolites examined with higher toxic effects for long-sequence peptides (EC50 ranging from 10 to 64 nM). D-shaped larvae with mantle abnormality were particularly noticed when peptaibol concentrations increased. Disturbances of embryogenesis were also observed following exposure to organic and aqueous extract of sediments from Fier d'Ars (EC50=42.4 and 6.6 g L(-1) dry weight, respectively). Although peptaibol concentrations measured in these sediments could explain only a part of the toxic effects observed, this study suggests that these mycotoxins can induce larval abnormalities in a population of exposed animals at environmentally realistic concentrations. Their detection in coastal areas devoted to bivalve culture should be taken into account.