Identification of the pigment and its role in UV resistance in Paecilomyces variotii, a Chernobyl isolate, using genetic manipulation strategies.

Fungi produce secondary metabolites that are not directly involved in their growth, but often contribute to their adaptation to extreme environmental stimuli and enable their survival. Conidial pigment or melanin is one of the secondary metabolites produced naturally by a polyketide synthesis (PKS) gene cluster in several filamentous fungi and is known to protect these fungi from extreme radiation conditions. Several pigmented or melanized fungi have been shown to grow under extreme radiation conditions at the Chernobyl nuclear accident site. Some of these fungi, including Paecilomyces variotii, were observed to grow towards the source of radiation. Therefore, in this study, we wanted to identify if the pigment produced by P. variotii, contributes to providing protection against radiation condition. We first identified the PKS gene responsible for synthesis of pigment in P. variotii and confirmed its role in providing protection against UV irradiation through CRISPR-Cas9 mediated gene deletion. This is the first report that describes the use of CRISPR methodology to create gene deletions in P. variotii. Further, we showed that the pigment produced by this fungus, was not inhibited by DHN-melanin pathway inhibitors, indicating that the fungus does not produce melanin. We then identified the pigment synthesized by the PKS gene of P. variotii, as a naptho-pyrone Ywa1, by heterologously expressing the gene in Aspergillus nidulans. The results obtained will further aid in understanding the mechanistic basis of radiation resistance.

Efficient identification of fungal antimicrobial principles by tandem MS and NMR database.

The continuous re-isolation of the known and non-applicable compounds that is time-consuming and wasting resources is still a critical problem in the discovery of bioactive entities from natural resources. To efficiently address the problem, high performance liquid chromatography-diode array detector-microfractionation (HPLC-DAD-microfractionation) guided by disk agar diffusion assay was developed, and the active compounds were further identified using the tandem mass spectrometry (MS/MS)-based molecular networking. Of 150 fungal strains screened, the methanolic extracts of Phoma herbarum PPM7487, Cryptosporiopsis ericae PPM7405, and Albifimbria verrucaria PPM945 exhibited potent antimicrobial activity against Candida albicans SC5314 and Cryptococcus neoformans H99 in the preliminary agar diffusion assay. The concept of OSMAC (one strain many compounds) was employed in the fungal cultures in order to enrich the diversity of the 2nd metabolites in this study. HPLC coupled with off-line bioactivity-directed profiling of the extracts enabled a precise localization of the compounds responsible for the conspicuous antimicrobial activity. The purified active compounds were identified based mainly on MS/MS database, and further supported by 13C nuclear magnetic resonance (NMR) spectral data compared to the literatures. In addition to nineteen known compounds, a new trichothecene derivative 1, namely trichoverrin D, was isolated and identified through this protocol. The antifungal activities of all the pure isolates were evaluated, and the structure activity relationships were also inferred. This report has demonstrated the combination of HPLC microfractination and MS/MS coupled by NMR spectral dereplication for speeding up the antimicrobial natural products discovery process.

Phomaketide A Inhibits Lymphangiogenesis in Human Lymphatic Endothelial Cells.

Lymphangiogenesis is an important biological process associated with cancer metastasis. The development of new drugs that block lymphangiogenesis represents a promising therapeutic strategy. Marine fungus-derived compound phomaketide A, isolated from the fermented broth of Phoma sp. NTOU4195, has been reported to exhibit anti-angiogenic and anti-inflammatory effects. However, its anti-lymphangiogenic activity has not been clarified to date. In this study, we showed that phomaketide A inhibited cell growth, migration, and tube formation of lymphatic endothelial cells (LECs) without an evidence of cytotoxicity. Mechanistic investigations revealed that phomaketide A reduced LECs-induced lymphangiogenesis via vascular endothelial growth factor receptor-3 (VEGFR-3), protein kinase Cδ (PKCδ), and endothelial nitric oxide synthase (eNOS) signalings. Furthermore, human proteome array analysis indicated that phomaketide A significantly enhanced the protein levels of various protease inhibitors, including cystatin A, serpin B6, tissue factor pathway inhibitor (TFPI), and tissue inhibitor matrix metalloproteinase 1 (TIMP-1). Importantly, phomaketide A impeded tumor growth and lymphangiogenesis by decreasing the expression of LYVE-1, a specific marker for lymphatic vessels, in tumor xenograft animal model. These results suggest that phomaketide A may impair lymphangiogenesis by suppressing VEGFR-3, PKCδ, and eNOS signaling cascades, while simultaneously activating protease inhibitors in human LECs. We document for the first time that phomaketide A inhibits lymphangiogenesis both in vitro and in vivo, which suggests that this natural product could potentially treat cancer metastasis.

Angiogenesis Inhibitors and Anti-Inflammatory Agents from Phoma sp. NTOU4195.

Seven new polyketides, phomaketides A-E (1-5) and pseurotins A3 (6) and G (7), along with the known compounds FR-111142, pseurotins A, A1, A2, D, and F2, 14-norpseurotin A, α-carbonylcarbene, tyrosol, cyclo(-l-Pro-l-Leu), and cyclo(-l-Pro-l-Phe), were purified from the fermentation broth and mycelium of the endophytic fungal strain Phoma sp. NTOU4195 isolated from the marine red alga Pterocladiella capillacea. The structures were established through interpretation of spectroscopic data. The antiangiogenic and anti-inflammatory effects of 1-7 and related analogues were evaluated using human endothelial progenitor cells (EPCs) and lipopolysaccharide (LPS)-activated murine macrophage RAW264.7 cells, respectively. Of the compounds tested, compound 1 exhibited the most potent antiangiogenic activity by suppressing the tube formation of EPCs with an IC50 of 8.1 µM, and compound 3 showed the most selective inhibitory activity of LPS-induced NO production in RAW264.7 macrophages with an IC50 value of 8.8 µM.

Terpenoids from the Fermented Broths of Coprinellus radians.

One new sesquiterpenoid, namely coprinol (1), along with guanacastanes J (2), E (3) and N (4), were isolated from the ethyl acetate extracts of the fermented broths of the fungal strain Coprinellus radians ≠1168. Their structures were elucidated on the basis of spectroscopic data analysis. The growth inhibitory activities against A549 of 1-4 were evaluated, and only 4 exhibited moderate growth inhibitory activity with a GI50 value of 18.2 µM compared with fluorouracil (GI50 = 3.6 µM). All the compounds were also subjected to antifungal assay against Candida albicans ATCC 18804, C. albicans SC-5314, Cryptococcus neoformans ATCC 13690 and Saccharomyces cerevisiae ATCC 2345; all showed mild antifungal activity with MIC values of 128.0 µg/mL for 1-3 and 64.0 µg/mL for 4 in comparison with amphotericin B (MIC = 0.25 µg/mL) and ketoconazole (MIC = 1.0 µg/mL).

New Furanone and Sesquiterpene from the Pericarp of Calocedrus formosana.

One new γ-lactone, namely calolactone (1), together with one new drimane-type sesquiterpene, namely caloterpene (2), were isolated from the pericarp of Calocedrus formosana Florin. Their structures were elucidated by spectroscopic and mass spectrometric analysis.

Aliphatic phenolic ethers from Trichobotrys effusa.

Four novel aliphatic phenolic ethers, namely, trichoethers A-D (1-4), possessing a unique C11-O-C10 skeleton, together with coriloxin, zythiostromic acid A, radicicol, and 3,5-dihydroxytoluene were isolated from the ethyl acetate extracts of the fermented broths of Trichobotrys effusa YMJ1179. The structures of all the compounds were determined based on spectroscopic data analysis. The configurations of 1-4 were established by J values and NOESY and compared with published data. Compounds 1-4 and radicicol exhibited growth-inhibitory activities against the A549 non-small-cell lung cancer cell line with GI50 values of 25.61, 19.32, 16.19, 24.31, and 1.43 µM, respectively, in comparison with 5-fluorouracil (GI50 = 4.55 µM).