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

Abstract

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.

Transcription Factor Repurposing Offers Insights into Evolution of Biosynthetic Gene Cluster Regulation

“…S1b ). The P. expansum BGC contains the two essential enzymes required for xanthocillin synthesis, XanB, an isocyanide synthase-dioxygenase, and XanG, a cytochrome P450 ( 20 , 26 ). The P. expansum cluster lacks orthologs of AfXanE and AfXanF that modify xan BGC products through methylation ( 26 ).…”

“…The P. expansum BGC contains the two essential enzymes required for xanthocillin synthesis, XanB, an isocyanide synthase-dioxygenase, and XanG, a cytochrome P450 ( 20 , 26 ). The P. expansum cluster lacks orthologs of AfXanE and AfXanF that modify xan BGC products through methylation ( 26 ). The National Center for Biotechnology Information (NCBI) incorrectly annotated PeXanC as 337 amino acids, which we determined to be 316 amino acids by multiple sequence alignment ( Fig.…”

“…3b ). This motif was found in all the promoters of A. fumigatus xan genes ( xanA , xanB , xanE , xanF , xanG ) regulated by AfXanC but not in the promoters of xanC itself or xanD , which was not regulated by XanC ( 20 ) and did not contribute to product formation ( 26 ).…”

“…Both TWW31.1 and TWW32.1 were grown in GMM, and their gross colony phenotype, AfxanB gene expression, and xan BGC product titer were compared to those of the A. fumigatus xan BGC wild-type (WT) strain (TFYL81.5) ( 26 , 38 ) and the previously characterized ΔAfxanC (TNLR9.1) and OE:: AfxanC (TNLR1.2) strains ( 20 , 26 ). Consistent with the previous data ( 20 ), the OE:: AfxanC strain showed a decreased colony diameter and accumulation of a yellow pigment at the bottom of the plate in comparison to the control and ΔAfxanC strains ( Fig.…”

“…No metabolites were observed in the WT control and ΔAfxanC strains ( Fig. 3g ), and of the six quantifiable xan BGC products produced by the OE:: AfxanC strain [BU-4705, xanthocillin X monomethyl ether, fumiformamide, N , N -((1 Z ,3 Z )-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide, melanocin E, and melanocin F] ( 20 , 26 ), only a small amount of N , N -((1 Z ,3 Z )-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide was detected in extracts of the binding site mutant. In contrast, two metabolites [ N , N -((1 Z ,3 Z )-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide and melanocin F] were produced by the binding site control at the same level ( P = 0.1620, P = 0.7921), and the other four metabolites were reduced by approximately 50% ( P < 0.0001) compared to the OE:: AfxanC strain ( Fig.…”