Due to the role, both beneficial and harmful, that fungal secondary metabolites play in society, the study of their regulation is of great importance. Genes for any one secondary metabolite are contiguously arranged in a biosynthetic gene cluster (BGC) and subject to regulation through the remodeling of chromatin. Histone modifying enzymes can place or remove post translational modifications (PTM) on histone tails which influences how tight or relaxed the chromatin is, impacting transcription of BGCs. In a recent forward genetic screen, the epigenetic reader SntB was identified as a transcriptional regulator of the sterigmatocystin BGC in A. nidulans, and regulated the related metabolite aflatoxin in A. flavus. In this study we investigate the role of SntB in the plant pathogen A. flavus by analyzing both ΔsntB and overexpression sntB genetic mutants. Deletion of sntB increased global levels of H3K9K14 acetylation and impaired several developmental processes including sclerotia formation, heterokaryon compatibility, secondary metabolite synthesis, and ability to colonize host seeds; in contrast the overexpression strain displayed fewer phenotypes. ΔsntB developmental phenotypes were linked with SntB regulation of NosA, a transcription factor regulating the A. flavus cell fusion cascade.

The maintenance of sufficient but nontoxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that, in Aspergillus fumigatus, xanthocillin and other isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay, and inductively coupled plasma mass spectrometry analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with a variety of human pathogenic fungi and bacteria established copper-dependent antimicrobial properties of xan BGC metabolites, including inhibition of laccase activity. Remediation of xanthocillin-treated Pseudomonas aeruginosa growth by copper supported the copper-chelating properties of xan BGC isocyanide products. The existence of the xan BGC in several filamentous fungi suggests a heretofore unknown role of eukaryotic natural products in copper homeostasis and mediation of interactions with competing microbes.

The maintenance of sufficient but non-toxic pools of metal micronutrients is accomplished through diverse homeostasis mechanisms in fungi. Siderophores play a well-established role for iron homeostasis; however, no copper-binding analogs have been found in fungi. Here we demonstrate that in Aspergillus fumigatus isocyanides derived from the xan biosynthetic gene cluster (BGC) bind copper, impact cellular copper content, and have significant metal-dependent antimicrobial properties. xan BGC-derived isocyanides are secreted and bind copper as visualized by a chrome azurol S (CAS) assay and inductively coupled plasma-mass spectrometry (ICP-MS) analysis of A. fumigatus intracellular copper pools demonstrated a role for xan cluster metabolites in the accumulation of copper. A. fumigatus coculture with A. nidulans, Candida albicans and a variety of pathogenic bacteria establish copper-dependent antimicrobial properties of xan BGC metabolites including inhibition of laccase activity. Similarly, inhibition of Pseudomonas aeruginosa by low concentrations of the xan isocyanide xanthocillin was copper-dependent. Other metals also reduced xanthocillin’s antimicrobial properties, but less efficiently than copper. As variations of the xan BGC exist in other filamentous fungi, we suggest that xanthocillin-like natural products represent a first example for fungal small molecules that serve to maintain copper sufficiency and mediate interactions with competing microbes.SignificanceMetal homeostasis is an integral part of metabolism for any organism. A vast array of small molecules are already known to mediate metal homeostasis in fungi and bacteria; however, unlike their bacterial counterparts, to-date there are no known fungal small molecules that function to maintain copper homeostasis. Discovery of copper binding small molecules produced by A. fumigatus gives insight into mechanisms other than the extensively studied copper transporters or metalloproteins for how fungi can regulate copper. This has important ecological implications as securing scarce nutrients is central for fitness and survival. Additionally, studying this mechanism in A. fumigatus provides a basis for investigation of copper regulation pathways in other fungi.