Antibacterial insights into alternariol and its derivative alternariol monomethyl ether produced by a marine fungus.

Alternaria alternata FB1 is a marine fungus identified as a candidate for plastic degradation in our previous study. This fungus has been recently shown to produce secondary metabolites with significant antimicrobial activity against various pathogens, including methicillin-resistant Staphylococcus aureus (MRSA) and the notorious aquaculture pathogen Vibrio anguillarum. The antibacterial compounds were purified and identified as alternariol (AOH) and its derivative, alternariol monomethyl ether (AME). We found that AOH and AME primarily inhibited pathogenic bacteria (MRSA or V. anguillarum) by disordering cell division and some other key physiological and biochemical processes. We further demonstrated that AOH could effectively inhibit the unwinding activity of MRSA topoisomerases, which are closely related to cell division and are the potential action target of AOH. The antibacterial activities of AOH and AME were verified by using zebrafish as the in vivo model. Notably, AOH and AME did not significantly affect the viability of normal human liver cells at concentrations that effectively inhibited MRSA or V. anguillarum. Finally, we developed the genetic operation system of A. alternata FB1 and blocked the biosynthesis of AME by knocking out omtI (encoding an O-methyl transferase), which facilitated A. alternata FB1 to only produce AOH. The development of this system in the marine fungus will accelerate the discovery of novel natural products and further bioactivity study.IMPORTANCEMore and more scientific reports indicate that alternariol (AOH) and its derivative alternariol monomethyl ether (AME) exhibit antibacterial activities. However, limited exploration of their detailed antibacterial mechanisms has been performed. In the present study, the antibacterial mechanisms of AOH and AME produced by the marine fungus Alternaria alternata FB1 were disclosed in vitro and in vivo. Given their low toxicity on the normal human liver cell line under the concentrations exhibiting significant antibacterial activity against different pathogens, AOH and AME are proposed to be good candidates for developing promising antibiotics against methicillin-resistant Staphylococcus aureus and Vibrio anguillarum. We also succeeded in blocking the biosynthesis of AME, which facilitated us to easily obtain pure AOH. Moreover, based on our previous results, A. alternata FB1 was shown to enable polyethylene degradation.

Novel Metabolites from the Marine-Derived Fungus Peniophora sp. SCSIO41203 Show Promising In Vitro Antitumor Activity as Methuosis Inducers in PC-3 Cells.

Two new cytochalasin derivatives, peniotrinins A (1) and B (2), three new citrinin derivatives, peniotrinins C-E (4, 5, 7), and one new tetramic acid derivative, peniotrinin F (12), along with nine structurally related known compounds, were isolated from the solid culture of Peniophora sp. SCSIO41203. Their structures, including the absolute configurations of their stereogenic carbons, were fully elucidated based on spectroscopic analysis, quantum chemical calculations, and the calculated ECD. Interestingly, 1 is the first example of a rare 6/5/5/5/6/13 hexacyclic cytochalasin. We screened the above compounds for their anti-prostate cancer activity and found that compound 3 had a significant anti-prostate cancer cell proliferation effect, while compounds 1 and 2 showed weak activity at 10 µM. We then confirmed that compound 3 exerts its anti-prostate cancer effect by inducing methuosis through transmission electron microscopy and cellular immunostaining, which suggested that compound 3 might be first reported as a potential anti-prostate methuosis inducer.

Carneusones A-F, Benzophenone Derivatives from Sponge-Derived Fungus Aspergillus carneus GXIMD00543.

Six benzophenone derivatives, carneusones A-F (1-6), along with seven known compounds (7-13) were isolated from a strain of sponge-derived marine fungus Aspergillus carneus GXIMD00543. Their chemical structures were elucidated by detailed spectroscopic data and quantum chemical calculations. Compounds 5, 6, and 8 exhibited moderate anti-inflammatory activity on NO secretion using lipopolysaccharide (LPS)-induced RAW 264.7 cells with EC50 values of 34.6 ± 0.9, 20.2 ± 1.8, and 26.8 ± 1.7 µM, while 11 showed potent effect with an EC50 value of 2.9 ± 0.1 µM.

The Impact of the Culture Regime on the Metabolome and Anti-Phytopathogenic Activity of Marine Fungal Co-Cultures.

Co-cultivation, coupled with the OSMAC approach, is considered an efficient method for expanding microbial chemical diversity through the activation of cryptic biosynthetic gene clusters (BGCs). As part of our project aiming to discover new fungal metabolites for crop protection, we previously reported five polyketides, the macrolides dendrodolides E (1) and N (2), the azaphilones spiciferinone (3) and 8α-hydroxy-spiciferinone (4), and the bis-naphtho-γ-pyrone cephalochromin (5) from the solid Potato Dextrose Agar (PDA) co-culture of two marine sediment-derived fungi, Plenodomus influorescens and Pyrenochaeta nobilis. However, some of the purified metabolites could not be tested due to their minute quantities. Here we cultivated these fungi (both axenic and co-cultures) in liquid regime using three different media, Potato Dextrose Broth (PDB), Sabouraud Dextrose Broth (SDB), and Czapek-Dox Broth (CDB), with or without shaking. The aim was to determine the most ideal co-cultivation conditions to enhance the titers of the previously isolated compounds and to produce extracts with stronger anti-phytopathogenic activity as a basis for future upscaled fermentation. Comparative metabolomics by UPLC-MS/MS-based molecular networking and manual dereplication was employed for chemical profiling and compound annotations. Liquid co-cultivation in PDB under shaking led to the strongest activity against the phytopathogen Phytophthora infestans. Except for compound 1, all target compounds were detected in the co-culture in PDB. Compounds 2 and 5 were produced in lower titers, whereas the azaphilones (3 and 4) were overexpressed in PDB compared to PDA. Notably, liquid PDB co-cultures contained meroterpenoids and depside clusters that were absent in the solid PDA co-cultures. This study demonstrates the importance of culture regime in BGC regulation and chemical diversity of fungal strains in co-culture studies.

Meirols A-C: Bioactive Catecholic Compounds from the Marine-Derived Fungus Meira sp. 1210CH-42.

Three new catecholic compounds, named meirols A-C (2-4), and one known analog, argovin (1), were isolated from the marine-derived fungus Meira sp. 1210CH-42. Their structures were determined by extensive analysis of 1D, 2D NMR, and HR-ESIMS spectroscopic data. Their absolute configurations were elucidated based on ECD calculations. All the compounds exhibited strong antioxidant capabilities with EC50 values ranging from 6.01 to 7.47 µM (ascorbic acid, EC50 = 7.81 µM), as demonstrated by DPPH radical scavenging activity assays. In the α-glucosidase inhibition assay, 1 and 2 showed potent in vitro inhibitory activity with IC50 values of 184.50 and 199.70 µM, respectively (acarbose, IC50 = 301.93 µM). Although none of the isolated compounds exhibited cytotoxicity against one normal and six solid cancer cell lines, 1 exhibited moderate cytotoxicity against the NALM6 and RPMI-8402 blood cancer cell lines with GI50 values of 9.48 and 21.00 µM, respectively. Compound 2 also demonstrated weak cytotoxicity against the NALM6 blood cancer cell line with a GI50 value of 29.40 µM.

A new secondary metabolite from the marine-derived fungus Phomopsis lithocarpus FS508.

Based on the One Strain-Many Compounds (OSMAC) strategy, the secondary metabolites of Phomopsis lithocarpus FS508 were investigated. As a result, a new secondary metabolite, 4-methoxy-3-[4-(acetyloxy)-3-methyl-2-butenyl]benzoic acid (1) as well as eleven known compounds were isolated from the fermentation product of the strain FS508. Their structures were determined by NMR, IR, UV, and MS spectroscopic data analyses. All the isolated compounds were evaluated for cytotoxic and anti-inflammatory activities. Among them, compounds 3 and 9 displayed potent cytotoxicity against HepG-2 cell line, and compounds 2, 3 and 12 showed significant anti-inflammatory activities.

Isolation and Structure Elucidation of New Metabolites from the Mariana-Trench-Associated Fungus Aspergillus sp. SY2601.

Fungi are important resource for the discovery of novel bioactive natural products. This study investigated the metabolites produced by Mariana-Trench-associated fungus Aspergillus sp. SY2601 in EY liquid and rice solid media, resulting in the isolation and structure determination of 28 metabolites, including five new compounds, asperindopiperazines A-C (1-3), 5-methoxy-8,9-dihydroxy-8,9-deoxyaspyrone (21), and 12S-aspertetranone D (26). Structures of the new compounds were elucidated based on extensive NMR spectral analyses, HRESIMS data, optical rotation, ECD, and 13C NMR calculations. The new compound 12S-aspertetranone D (26) exhibited antibacterial activity against both methicillin-resistant Staphylococcus aureus and Escherichia coli with MIC values of 3.75 and 5 µg/mL, respectively.

New Phenylspirodrimanes from the Sponge-Associated Fungus Stachybotrys chartarum MUT 3308.

Two phenylspirodrimanes, never isolated before, stachybotrin J (1) and new stachybocin G (epi-stachybocin A) (2), along with the already reported stachybotrin I (3), stachybotrin H (4), stachybotrylactam (5), stachybotrylactam acetate (6), 2α-acetoxystachybotrylactam acetate (7), stachybotramide (8), chartarlactam B (9), and F1839-J (10) were isolated from the sponge-associated fungus Stachybotrys chartarum MUT 3308. Their structures were established based on extensive spectrometric (HRMS) and spectroscopic (1D and 2D NMR) analyses. Absolute configurations of the stereogenic centers of stachybotrin J (1), stachybocin G (2), and stachybotrin I (3), were determined by comparison of their experimental circular dichroism (CD) spectra with their time-dependent density functional theory (TD-DFT) circular dichroism (ECD) spectra. The putative structures of seventeen additional phenylspirodrimanes were proposed by analysis of their respective MS/MS spectra through a Feature-Based Molecular Networking approach. All the isolated compounds were evaluated for their cytotoxicity against five aggressive cancer cell lines (MP41, 786, 786R, CAL33, and CAL33RR), notably including two resistant human cancer cell lines (786R, CAL33RR), and compounds 5, 6, and 7 exhibited cytotoxicity with IC50 values in the range of 0.3-2.2 µM.

New Isocoumarins from the Marine Fungus Phaeosphaeriopsis sp. WP-26.

Five new isocoumarins, phaeosphaerins A-E (1-5), were isolated from the fermentation broth of the marine fungus Phaeosphaeriopsis sp. WP-26, along with one known isocoumarin, 6,8-dihydroxy-7-methoxy-3-methylisocoumarin (6), and two known pimarane-type diterpenes, diaportheins A (7) and B (8). Their structures were elucidated via NMR experiments, X-ray diffraction analysis, and comparison of the experimental and computed ECD curves. Compounds 1-7 displayed weak neuroprotective effects against H2O2-induced damage in SH-SY5Y cells. Moreover, compound 8 showed cytotoxicity against BEL-7402, SGC-7901, K562, A549, and HL-60 cell lines.

Potential α-Glucosidase Inhibitors from the Deep-Sea Sediment-Derived Fungus Aspergillus insulicola.

Three new phenolic compounds, epicocconigrones C-D (1-2) and flavimycin C (3), together with six known phenolic compounds: epicocconigrone A (4); 2-(10-formyl-11,13-dihydroxy-12-methoxy-14-methyl)-6,7-dihydroxy-5-methyl-4-benzofurancarboxaldehyde (5); epicoccolide B (6); eleganketal A (7); 1,3-dihydro-5-methoxy-7-methylisobenzofuran (8); and 2,3,4-trihydroxy-6-(hydroxymethyl)-5-methylbenzyl-alcohol (9), were isolated from fermentation cultures of a deep-sea sediment-derived fungus, Aspergillus insulicola. Their planar structures were elucidated based on the 1D and 2D NMR spectra and HRESIMS data. The absolute configurations of compounds 1-3 were determined by ECD calculations. Compound 3 represented a rare fully symmetrical isobenzofuran dimer. All compounds were evaluated for their α-glucosidase inhibitory activity, and compounds 1, 4-7, and 9 exhibited more potent α-glucosidase inhibitory effect with IC50 values ranging from 17.04 to 292.47 µM than positive control acarbose with IC50 value of 822.97 µM, indicating that these phenolic compounds could be promising lead compounds of new hypoglycemic drugs.

Thiolactones and Δ8,9-Pregnene Steroids from the Marine-Derived Fungus Meira sp. 1210CH-42 and Their α-Glucosidase Inhibitory Activity.

The fungal genus Meira was first reported in 2003 and has mostly been found on land. This is the first report of second metabolites from the marine-derived yeast-like fungus Meira sp. One new thiolactone (1), along with one revised thiolactone (2), two new Δ8,9-steroids (4, 5), and one known Δ8,9-steroid (3), were isolated from the Meira sp. 1210CH-42. Their structures were elucidated based on the comprehensive spectroscopic data analysis of 1D, 2D NMR, HR-ESIMS, ECD calculations, and the pyridine-induced deshielding effect. The structure of 5 was confirmed by oxidation of 4 to semisynthetic 5. In the α-glucosidase inhibition assay, compounds 2-4 showed potent in vitro inhibitory activity with IC50 values of 148.4, 279.7, and 86.0 µM, respectively. Compounds 2-4 exhibited superior activity as compared to acarbose (IC50 = 418.9 µM).

p-Terphenyl and Diphenyl Ether Derivatives from the Marine-Derived Fungus Aspergillus candidus HM5-4.

Two undescribed p-terphenyl derivatives, asperterphenylcins A-B (1-2), and two undescribed diphenyl ether derivatives, asperdiphenylcins A-B (3-4), together with three previously described p-terphenyl derivatives-4″-deoxyterprenin (5), terphenyllin (6), and 3″-hydroxyterphenyllin (7)-were obtained from the solid-rice culture of the marine-derived fungus Aspergillus candidus HM5-4, which was isolated from sponges from the South China Sea. Their structures were elucidated by HRESIMS data and NMR spectroscopic analysis. Compound 1 showed a strong inhibitory effect on Neoscytalidium dimidiatum, with an inhibition circle diameter of 31.67 ± 2.36 mm at a concentration of 10.0 µg/disc. Compounds 5 and 7 displayed cytotoxic activity against human chronic myeloid leukemia cells (K562), human liver cancer cells (BEL-7402), human gastric cancer cells (SGC-7901), human non-small cell lung cancer cells (A549) and human HeLa cervical cancer cells, with IC50 values ranging from 3.32 to 60.36 µM, respectively. Compounds 2, 6 and 7 showed potent inhibitory activity against α-glucosidase, with IC50 values of 1.26 ± 0.19, 2.16 ± 0.44 and 13.22 ± 0.55 µM, respectively.

Chemical Constituents of the Deep-sea Gammarid Shrimp-Derived Fungus Penicillium citrinum XIA-16.

One new alkaloid, (S)-2-acetamido-4-(2-(methylamino)phenyl)-4-oxobutanoic acid (1), was isolated from the deep-sea-derived Penicillium citrinum XIA-16, together with 25 known compounds including ten polyketones (2-11), eight alkaloids (12-19), six steroids (20-25), and a fatty acid (26). Their planar and relative structures were determined by an analysis of 1D and 2D nuclear magnetic resonance (NMR) as well as high resolution electrospray ionization mass spectroscopy (HR-ESI-MS) data. The absolute configuration of 1 was determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Penicitrinol B (6) significantly inhibited RSL3-induced ferroptosis (EC50 =2.0 µM) by reducing lipid peroxidation and heme oxygenase 1 (HMOX1) expression. Under the concentration of 10 µM, penicitrinol A (7) was able to inhibit cuproptosis with the cell viabilities of 68.2 % compared to the negative control (copper and elesclomol) with the cell viabilities of 14.8 %.

Exophilone, a Tetrahydrocarbazol-1-one Analogue with Anti-Pulmonary Fibrosis Activity from the Deep-Sea Fungus Exophiala oligosperma MCCC 3A01264.

A new compound, exophilone (1), together with nine known compounds (2-10), were isolated from a deep-sea-derived fungus, Exophiala oligosperma. Their chemical structures, including the absolute configuration of 1, were elucidated using nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectroscopy (HRESIMS), and electronic circular dichroism (ECD) calculation. Compounds were preliminarily screened for their ability to inhibit collagen accumulation. Compounds 1, 4, and 7 showed weaker inhibition of TGF-ß1-induced total collagen accumulation in compared with pirfenidone (73.14% inhibition rate). However, pirfenidone exhibited cytotoxicity (77.57% survival rate), while compounds 1, 4, and 7 showed low cytotoxicity against the HFL1 cell line. Particularly, exophilone (1) showed moderate collagen deposition inhibition effect (60.44% inhibition rate) and low toxicity in HFL1 cells (98.14% survival rate) at a concentration of 10 µM. A molecular docking study suggests that exophilone (1) binds to both TGF-ß1 and its receptor through hydrogen bonding interactions. Thus, exophilone (1) was identified as a promising anti-pulmonary fibrosis agent. It has the potential to be developed as a drug candidate for pulmonary fibrosis.

Paspalines C-D and Paxillines B-D: New Indole Diterpenoids from Penicillium brefeldianum WZW-F-69.

Five new indole diterpenoids named paspaline C-D (1-2) and paxilline B-D (3-5), as well as eleven known analogues (6-16), were identified from fungus Penicillium brefeldianum strain WZW-F-69, which was isolated from an abalone aquaculture base in Fujian province, China. Their structures were elucidated mainly through 1D- and 2D-NMR spectra analysis and ECD comparison. Compound 1 has a 6/5/5/6/6/8 hexacyclic ring system bearing 2,2-dimethyl-1,3-dioxocane, which is rare in natural products. Compound 2 has an unusual open F-ring structure. The cytotoxic activities against 10 cancer cell lines and antimicrobial activities against model bacteria and fungi of all compounds were assayed. No compound showed antimicrobial activity, but at a concentration of 1 µM, compounds 1 and 6 exhibited the highest inhibition rates of 71.2% and 83.4% against JeKo-1 cells and U2OS cells, respectively.

Eremophilane-Type Sesquiterpenes from a Marine-Derived Fungus Penicillium Copticola with Antitumor and Neuroprotective Activities.

Chemical examination of a marine sponge-associated Penicillium copticola fungus resulted in the isolation of ten undescribed eremophilanes, namely copteremophilanes A-J (1-10), along with two new glycosides, 5-glycopenostatin F (11) and 5-glucopenostatin I (12). Their structures were determined by extensive spectroscopic data, in association with ECD data and chemical conversions for configurational assignments. Analogs 1, 2, and 10 represent a group of uncommon skeletons of eremophilanes with an aromatic ring and a methyl migration from C-5 to C-9, and analogs 11 and 12 are characteristic of a PKS scaffold bearing a glucose unit. The incorporation of a chlorinated phenylacetic unit in 3-9 is rarely found in nature. Analog 7 showed neuroprotective effect, whereas 8 exhibited selective inhibition against human non-small cell lung cancer cells (A549). This study enriched the chemical diversity of eremophilanes and extended their bioactivities to neuroprotection.

Two New Prenylated Glycine Derivatives from the Marine-Derived Fungus Fusarium sp. TW56-10.

Two new prenylated glycine derivatives N-({4-[(3-methylbut-2-en-1-yl)oxy]phenyl}acetyl)glycine (1) and methyl N-({4-[(3-methylbut-2-en-1-yl)oxy]phenyl}acetyl)glycinate (2), along with nine known compounds (3-11) were purified from the marine-derived fungus Fusarium sp. TW56-10. Their chemical structures were determined by spectroscopic evidence, including extensive nuclear magnetic resonance (NMR), high-resolution electrospray ionization mass spectroscopy (HR-ESI-MS) data, infrared radiation (IR) and Ultraviolet spectra (UV). Compound 4 (8-O-methylfusarubin) exhibited cytotoxic activity with IC50 value of 11.45 µM for A549 cells.

Two New Cerebroside Metabolites from the Marine Fungus Hortaea werneckii.

Two new cerebroside metabolites were isolated from the fermented sponge-derived fungus extract of Hortaea werneckii. They were hortacerebroside A (1) ((2R,3E)-N-[(2S,3R,4E,8E)-1-(ß-D-glucopyranosyloxy)-3-hydroxy-9-methylhenicosa-4,8-dien-2-yl]-2-hydroxypentadec-3-enamide) and hortacerebroside B (2) ((2R)-N-[(2S,3R,4E,8E)-1-(ß-D-glucopyranosyloxy)-3-hydroxy-9-methylhenicosa-4,8-dien-2-yl]-2-hydroxypentadecanamide). Their structures were elucidated by spectroscopic analysis and by comparison of the spectroscopic data with those of related cerebroside analogs. These two compounds showed significant inhibitory effect on NO produced by lipopolysaccharide (LPS) stimulated RAW 264.7 macrophages. The IC50 values of hortacerebroside A (1) and hortacerebroside B (2) were 7 and 5 µM, respectively. These results suggested the potential application of these cerebrosides as drug leads targeting inflammatory-related disorders.

Phomaligols F-I, polyoxygenated cyclohexenone derivatives from marine-derived fungus Aspergillus flavus BB1.

By tracing the 13C NMR resonances for carbonyls and enols, four new oxidized phomaligol derivatives, phomaligols F-I (1-4), along with seven known compounds (5-11) were isolated from the culture of the fungus Aspergillus flavus BB1 isolated from the marine shellfish Meretrix meretrix collected on Hailing Island, Yangjiang, China. The chemical structures and the absolute configurations of the new compounds were elucidated by MS, NMR, ECD, optical rotation, and 13C NMR calculations. Compounds 1 and 2 represent the first examples of phomaligol derivatives that contain an unusual bicyclic skeleton. All isolated compounds were tested for their cytotoxic activity. Among them, sporogen-AO 1 (8) showed potent inhibitory activity against the cancer cell lines A549, H1299, SK-BR-3, and HCT116 with IC50 values of 0.13, 0.78, 1.19, and 1.32 µM, respectively. Phomaligol G (2) displayed cytotoxic activity against the A549 and H1299 cell lines with IC50 values of 46.86 and 51.87 µM respectively. Additionally, phomaligol H (3) demonstrated cytotoxic activity against the A549 cell line with an IC50 value of 65.53 µM. Mechanistic studies of compound 8 showed that it induced apoptosis of HCT116 cells in a dose-dependent manner.

Molecular analysis of a fungal disease in the habitat-forming brown macroalga Phyllospora comosa (Fucales) along a latitudinal gradient.

Infectious diseases affecting habitat-forming species can have significant impacts on population dynamics and alter the structure and functioning of marine ecosystems. Recently, a fungal infection was described as the causative agent of necrotic lesions on the stipe of the forest-forming macroalga Phyllospora comosa, a disease named "stipe rot" (SR). Here, we developed a quantitative PCR (qPCR) method for rapid detection and quantification of this pathogen, which was applied to evaluate the level of SR infection in eight P. comosa populations spanning the entire latitudinal distribution of this species along southeastern Australia. We also investigated the relationship between the abundance and prevalence of Stipe Rot Fungus (SRF) and potential host chemical defenses as well as its relationship with morphological and ecophysiological traits of P. comosa. qPCR estimates of SRF abundance reflected the levels of infection estimated by visual assessment, with higher numbers of SRF copies being observed in individuals showing high or intermediate levels of visual symptoms of SR. Concordance of conventional PCR and visual assessments was 92 and 94%, respectively, compared to qPCR detection. SRF prevalence was positively related to fucoxanthin content and herbivory, but not significant related to other traits measured (phlorotannin content, total length, thallus diameter, stipe width, number of branches, frond width, fouling, bleaching, gender, and photosynthetic efficiency). These results provide confidence for previous reports of this disease based upon visual assessments only, contribute to the development of monitoring and conservation strategies for safeguarding P. comosa forests, and generate insights into potential factors influencing host-pathogen interactions in this system.