Dual-purpose isocyanides produced byAspergillus fumigatuscontribute 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 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.

Copper homeostasis and cuproptosis in health and disease

“…Given that Cu has also been shown to destabilize Fe-S proteins in bacteria and yeast, it is reasonable to speculate that a Cu-containing complex might be developed to trigger cuproptosis in bacteria as an antimicrobial agent. [261][262][263] Cuproptosis as a putative therapeutic target for Wilson's disease As mentioned above, mutations in the ATP7B gene, which encodes the Cu-transporting P-type ATPase ATP7B, are associated with Wilson's disease, a life-threatening disorder in which patients present with progressive Cu accumulation in several tissues, particularly the liver, brain, and cornea. Importantly, Cu deposition in the liver and brain can cause cellular damage and severe hepatic and neurological symptoms.…”

Siderophores: a potential role as a diagnostic for invasive fungal disease

“…First, some host cells control microbial growth through nutrient limitation. In addition to iron and zinc sequestration [56], restricted access to other metal ions such as Cu 2+ and Mn 2+ can reduce microbial fitness and growth [57]. Most intracellular bacteria are auxotrophic for purines, pyridines and amino acids, which, coupled with the known metabolic restrictions of immune cells, indicates a link between host cell metabolism and growth limitation of intracellular pathogens [58].…”

In Living Color: Pigment-Based Microbial Ecology At the Mineral–Air Interface

“…In the same way, fungal melanin is an excellent metal-binding compound, and its metal scavenging activity allows the bioabsorption of essential metals from rocks and other mineral niches (Oh et al 2021 ), conferring a competitive advantage by enhancing the mineral use. The yellow fungal pigment xanthocillin binds copper, affects cellular copper content, and possesses significant metal-dependent antimicrobial properties (Raffa et al 2021 ). In summary, microorganisms capable of synthesizing certain pigments deprive competing cells of important resources while antagonizing undesired neighbors.…”