In immunocompromised individuals, Aspergillus fumigatus causes invasive fungal disease that is often difficult to treat. Exactly how immune mechanisms control A. fumigatus in immunocompetent individuals remains unclear. Here, we use transparent zebrafish larvae to visualize and quantify neutrophil and macrophage behaviors in response to different A. fumigatus strains. We find that macrophages form dense clusters around spores, establishing a protective niche for fungal survival. Macrophages exert these protective effects by inhibiting fungal germination, thereby inhibiting subsequent neutrophil recruitment and neutrophil-mediated killing. Germination directly drives fungal clearance as faster-growing CEA10-derived strains are killed better in vivo than slower-growing Af293-derived strains. Additionally, a CEA10 pyrG-deficient strain with impaired germination is cleared less effectively by neutrophils. Host inflammatory activation through Myd88 is required for killing of a CEA10-derived strain but not sufficient for killing of an Af293-derived strain, further demonstrating the role of fungal-intrinsic differences in the ability of a host to clear an infection. Altogether, we describe a new role for macrophages in the persistence of A. fumigatus and highlight the ability of different A. fumigatus strains to adopt diverse modes of virulence.

No abstract

Microbial secondary metabolites, including isocyanide moieties, have been extensively mined for their repertoire of bioactive properties. Although the first naturally occurring isocyanide (xanthocillin) was isolated from the fungus Penicillium notatum over half a century ago, the biosynthetic origins of fungal isocyanides remain unknown. Here we report the identification of a family of isocyanide synthases (ICSs) from the opportunistic human pathogen Aspergillus fumigatus. Comparative metabolomics of overexpression or knockout mutants of ICS candidate genes led to the discovery of a fungal biosynthetic gene cluster (BGC) that produces xanthocillin (xan). Detailed analysis of xanthocillin biosynthesis in A. fumigatus revealed several previously undescribed compounds produced by the xan BGC, including two novel members of the melanocin family of compounds. We found both the xan BGC and a second ICS-containing cluster, named the copper-responsive metabolite (crm) BGC, to be transcriptionally responsive to external copper levels and further demonstrated that production of metabolites from the xan BGC is increased during copper starvation. The crm BGC includes a novel type of fungus-specific ICS-nonribosomal peptide synthase (NRPS) hybrid enzyme, CrmA. This family of ICS-NRPS hybrid enzymes is highly enriched in fungal pathogens of humans, insects, and plants. Phylogenetic assessment of all ICSs spanning the tree of life shows not only high prevalence throughout the fungal kingdom but also distribution in species not previously known to harbor BGCs, indicating an untapped resource of fungal secondary metabolism.

Filamentous fungi differentiate along complex developmental programs directed by abiotic and biotic signals. Currently, intrinsic signals that govern fungal development remain largely unknown. Here we show that an endogenously produced and secreted fungal oxylipin, 5,8-diHODE, induces fungal cellular differentiation, including lateral branching in pathogenic Aspergillus fumigatus and Aspergillus flavus, and appressorium formation in the rice blast pathogen Magnaporthe grisea. The Aspergillus branching response is specific to a subset of oxylipins and is signaled through G-protein coupled receptors. RNA-Seq profiling shows differential expression of many transcription factors in response to 5,8-diHODE. Screening of null mutants of 33 of those transcription factors identifies three transcriptional regulators that appear to mediate the Aspergillus branching response; one of the mutants is locked in a hypo-branching phenotype, while the other two mutants display a hyper-branching phenotype. Our work reveals an endogenous signal that triggers crucial developmental processes in filamentous fungi, and opens new avenues for research on the morphogenesis of filamentous fungi.

Copper is an essential micronutrient for the opportunistic human pathogen, Aspergillus fumigatus. Maintaining copper homeostasis is critical for survival and pathogenesis. Copper-responsive transcription factors, AceA and MacA, coordinate a complex network responsible for responding to copper in the environment and determining which response is necessary to maintain homeostasis. For example, A. fumigatus uses copper exporters to mitigate the toxic effects of copper while simultaneously encoding copper importers and small molecules to ensure proper supply of the metal for copper-dependent processes such a nitrogen acquisition and respiration. Small molecules called isocyanides recently found to be produced by A. fumigatus may bind copper and partake in copper homeostasis similarly to isocyanide copper chelators in bacteria. Considering that the host uses copper as a microbial toxin and copper availability fluctuates in various environmental niches, understanding how A. fumigatus maintains copper homeostasis will give insights into mechanisms that facilitate the development of invasive aspergillosis and its survival in nature.