Targeted isolation of induced and bioactive metabolites from fungal co-cultures

Abstract

The prevalence of Fusarium spp. as causative agent of onychomycoses is rising and Fusarium spp. as well as other non-dermatophyte fungi appear to be insensitive to systemic standard treatment [1]. Hence, new antifungal agents active against Fusarium spp. are needed. In a large screening [2], different plant and human pathogenic fungi were co-cultured with Fusarium spp. in Petri dishes. Few fungi were able to keep Fusarium at bay, among them the Basidiomycete Hohenbuehelia reniformis. An induction of red pigments was visually observed in the co-culture experiments but was not particularly highlighted among the metabolites revealed by UHPLC-TOFMS-based chemometrics. Nevertheless, using a specific UHPLC-UV analysis, it could be demonstrated that several pigments were upregulated by Fusarium sp. upon co-culture. On the other hand, MS-based chemometrics revealed upregulation of metabolites produced by H. reniformis. Extracts of the mycelium of fungi grown in Petri dishes contain mainly saccharides as confirmed by NMR of the total extract. Prefractionation with the resin HP20SS was successfully applied to separate secondary metabolites from the saccharides. For the co-culture of H. reniformis and Fusarium sp., an enriched – and sugar-free – prefraction showed activity against human pathogenic Fusarium sp. Preliminary identification of this fraction's metabolites was done by dereplication of the high resolution MS data. These compounds are only present in small amounts (< 1% of total extract), thus co-cultures were produced on solid media at a large scale. The analytical strategy for the isolation of Fusarium pigments and anti-Fusarium constituents from fungal co-cultures on solid media are presented.