Cryptic Aspergillus nidulans antimicrobials

Abstract

Secondary metabolite (SM) production by fungi is hypothesized to provide some fitness attribute for the producing organisms. However, most SM clusters are "silent" when fungi are grown in traditional laboratory settings, and it is difficult to ascertain any function or activity of these SM cluster products. Recently, the creation of a chromatin remodeling mutant in Aspergillus nidulans induced activation of several cryptic SM gene clusters. Systematic testing of nine purified metabolites from this mutant identified an emodin derivate with efficacy against both human fungal pathogens (inhibiting both spore germination and hyphal growth) and several bacteria. The ability of catalase to diminish this antimicrobial activity implicates reactive oxygen species generation, specifically, the generation of hydrogen peroxide, as the mechanism of emodin hydroxyl activity.

Fig. 1.

A. nidulans cclAΔ inhibits the growth of A. fumigatus strains on GMM agar. Both 2-OH-overproducing strains [cclAΔ and alcA(p)::mdpE] prevented hyphal intercalation of the radial growth of both A. nidulans (data not shown) and A. fumigatus wild-type strains. In contrast, the mycelia of wild-type A. nidulans and A. fumigatus strains overlapped with no signs of inhibitory activity. The cclAΔ mdpGΔ strain, which failed to produce 2-OH, showed interactions similar to wild type. All strains were grown on GMM with the exception of the confrontation between A. nidulans alcA(p)::mdpE and A. fumigatus, which was conducted on LMM with cyclopentanone.

Fig. 2.

AF293 germination and germling morphology are affected by cclAΔ but not wild-type extracts. (A) Germination of spores is inhibited by addition of organic extracts from cclAΔ strains, but not from WT strains. These effects included decreased percentages of germination and decreased conidial swelling. (B) Addition of purified 2-OH reconstitutes these effects. DMSO is the control solvent for 2-OH. (C) Quantification of the percentage of germinated spores for each treatment. Letters (a to c) indicate differences with statistical significance (P < 0.05) according to the Tukey-Kramer multiple comparison test.

Fig. 3.

Morphology abberancies in A. nidulans mutants. (A) Colony morphology varies between strains. cclAΔ leads to a decrease in colony diameter, likely independent of production of emodin derivatives, as the cclAΔ mdpGΔ strain devoid of emodin derivative production also was inhibited in diameter. However, the decreased conidiation observed in both the cclAΔ and alcA(p)::mdpE strains appeared to be associated with the increased expression of the monodictyophenone cluster. (B) Microscopic examination of hyphal morphology differs among strains but is not dependent upon production of emodin derivatives.