Isolation, structure, and biological activity of Phaeofungin, a cyclic lipodepsipeptide from a Phaeosphaeria sp. Using the Genome-Wide Candida albicans Fitness Test.

Phaeofungin (1), a new cyclic depsipeptide isolated from Phaeosphaeria sp., was discovered by application of reverse genetics technology, using the Candida albicans fitness test (CaFT). Phaeofungin is comprised of seven amino acids and a ß,γ-dihydroxy-γ-methylhexadecanoic acid arranged in a 25-membered cyclic depsipeptide. Five of the amino acids were assigned with d-configurations. The structure was elucidated by 2D-NMR and HRMS-MS analysis of the natural product and its hydrolyzed linear peptide. The absolute configuration of the amino acids was determined by Marfey's method by complete and partial hydrolysis of 1. The CaFT profile of the phaeofungin-containing extract overlapped with that of phomafungin (3), another structurally different cyclic lipodepsipeptide isolated from a Phoma sp. using the same approach. Comparative biological characterization further demonstrated that these two fungal lipodepsipeptides are functionally distinct. While phomafungin was potentiated by cyclosporin A (an inhibitor of the calcineurin pathway), phaeofungin was synergized with aureobasidin A (2) (an inhibitor of the sphingolipid biosynthesis) and to some extent caspofungin (an inhibitor of glucan synthase). Furthermore, phaeofungin caused ATP release in wild-type C. albicans strains but phomafungin did not. It showed modest antifungal activity against C. albicans (MIC 16-32 µg/mL) and better activity against Aspergillus fumigatus (MIC 8-16 µg/mL) and Trichophyton mentagrophytes (MIC 4 µg/mL). The linear peptide was inactive, suggesting that the macrocyclic depsipeptide ring is essential for target engagement and antifungal activity.

Distribution of the antifungal agents sordarins across filamentous fungi.

Sordarins are a class of natural antifungal agents which act by specifically inhibiting fungal protein synthesis through their interaction with the elongation factor 2, EF2. A number of natural sordarins produced by diverse fungi of different classes have been reported in the literature. We have run an exhaustive search of sordarin-producing fungi using two different approaches consecutively, the first one being a differential sensitivity screen using a sordarin-resistant mutant yeast strain run in parallel with a wild type strain, and the second one an empiric screen against Candida albicans followed by early detection of sordarins by LC-MS analysis. Using these two strategies we have detected as many as 22 new strains producing a number of different sordarin analogues, either known (sordarin, xylarin, zofimarin) or novel (isozofimarin and 4'-O-demethyl sordarin). Sordarin and xylarin were the most frequently found compounds in the class. The producing strains were subjected to sequencing of the ITS region to determine their phylogenetic affinities. All the strains were shown to belong to the Xylariales, being distributed across three families in this order, the Xylariaceae, the Amphisphaeriaceae, and the Diatrypaceae. Despite being screened in large numbers, we did not find sordarin production in any other fungal group, including those orders where sordarin producing fungi are known to exist (i.e., Sordariales, Eurotiales, and Microascales), suggesting that the production of sordarin is a trait more frequently associated to members of the Xylariales than to any other fungal order.

Four illudane sesquiterpenes from Coprinopsis episcopalis.

Four new illudane derivatives with antibiotic and cytotoxic properties, illudins I (1), I(2) (2), J (3) and J(2) (4), have been isolated from the fungus Coprinopsis episcopalis (syn. Coprinus episcopalis). These sesquiterpenes are stereoisomers, and their relative structures have been determined taking into consideration 2D NMR data.

Novel illudins from Coprinopsis episcopalis (syn. Coprinus episcopalis), and the distribution of illudin-like compounds among filamentous fungi.

The illudins are a family of fungal sesquiterpenes that have been studied as anti-tumor agents, and they also have antibacterial activity. Over a four-year period, 25 304 fungal isolates (approximately 97% ascomycetes and 3% basidiomycetes), were screened for antibacterial activity against methicillin-resistant Staphylococcus aureus. Illudin-like compounds with antibacterial and cytotoxic activity against tumor cell lines were observed in 10 basidiomycete strains. The isolates were recovered from different types of substrata using indirect methods and only formed sterile mycelia in pure culture. The isolates were genetically related but not identical, based on PCR-based fingerprinting techniques. DNA sequencing of the ITS1-5.8 S-ITS2 region of the strains revealed that nine had identical sequences, indicating that they were conspecific. The sequence of the remaining isolate was 96.34% similar, suggesting that it was a closely related species. The D1-D2 region of the 25 S rRNA gene of the two strain types was also sequenced. Both sequences were 99.39% similar, and Coprinopsis gonophylla (syn. Coprinus gonophyllus) was the closest match for both. Strains were grown in pure culture on a rice-based medium that allowed the development of basidiomata from one culture of the main strain type, which was identified as C. episcopalis, a close relative of C. gonophyllus. Both species (or strain types) produced different types of illudin-like compounds. Three novel illudins (I, I2 and J2) were found to be produced by the cultures identified as C. episcopalis, while only illudinic acid was produced by the other Coprinopsis sp. The taxonomical relationships of the Coprinops is species identified in this study with other illudin producers previously reported in the literature are discussed.