Dual-purpose isocyanides produced by Aspergillus fumigatus contribute to cellular copper sufficiency and exhibit antimicrobial activity

Abstract

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 xanthocillins 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.