Abstract
The secondary metabolome provides pathogenic fungi with a plethoric and versatile panel of molecules that can be deployed during host ingress. While powerful genetic and analytical chemistry methods have been developed to identify fungal secondary metabolites (SMs), discovering the biological activity of SMs remains an elusive yet critical task. Here, we describe a process for identifying the immunosuppressive properties of Aspergillus SMs developed by coupling a cost-effective microfluidic neutrophil chemotaxis assay with an in vivo zebrafish assay. The microfluidic platform allows the identification of metabolites inhibiting neutrophil recruitment with as little as several nano-grams of compound in microliters of fluid. The zebrafish assay demonstrates a simple and accessible approach for performing in vivo studies without requiring any manipulation of the fish. Using this methodology we identify the immunosuppressive properties of a fungal SM, endocrocin. We find that endocrocin is localized in Aspergillus fumigatus spores and its biosynthesis is temperature-dependent. Finally, using the Drosophila toll deficient model, we find that deletion of encA, encoding the polyketide synthase required for endocrocin production, yields a less pathogenic strain of A. fumigatus when spores are harvested from endocrocin permissive but not when harvested from endocrocin restrictive conditions. The tools developed here will open new “function-omic” avenues downstream of the metabolomics, identification, and purification phases.
Highlights
The secondary metabolome provides filamentous fungi with a biologically active panel of molecules, deployed in the presence of competing/host organisms or specific microenvironmental factors, and increasingly found to afford both physical and competitive fitness to the producing fungus [1]
secondary metabolites (SMs) are of particular interest in medical research as the genus is genetically accessible, produces a plethora of bioactive compounds [5], and contains several opportunistic pathogenic species including A. fumigatus and A. nidulans whose SMs are assessed in this study [6,7]
Genome assessment of human pathogenic Aspergillus species indicates these fungi have the capabilities of producing hundreds of SMs, most of which are currently not characterized for their effect on human health and the immune system
Summary
The secondary metabolome provides filamentous fungi with a biologically active panel of molecules, deployed in the presence of competing/host organisms or specific microenvironmental factors, and increasingly found to afford both physical and competitive fitness to the producing fungus [1]. As the methods for identifying SM gene clusters and the compounds they produce are becoming well established [4], there is an increasing need for improved assays, compatible with the fungal metabolomics process, that can reveal the biological activity of metabolites produced and break a bottleneck in scientific advancement. As the innate immune response is the primary line of defense against fungal spores in the lung, inhibition of essential functions of these cells may confer to the fungi an ability to evade immune clearance, and increase its pathogenicity. These findings highlight the necessity of mapping the interactome between fungi and host organisms to establish the pathomechanism of fungal diseases as well as to bioprospect
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