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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., Author summary Immunocompromised patients are susceptible to invasive fungal infections, including aspergillosis. However, healthy humans inhale spores of the fungus Aspergillus fumigatus from the environment every day without becoming sick, and how the immune system clears this infection is still obscure. Additionally, there are many different strains of A. fumigatus, and whether the pathogenesis of these different strains varies is also largely unknown. To investigate these questions, we infected larval zebrafish with A. fumigatus spores derived from two genetically diverse strains. Larval zebrafish allow for visualization of fungal growth and innate immune cell behavior in live, intact animals. We find that differences in the rate of growth between strains directly affect fungal persistence. In both wild-type and macrophage-deficient zebrafish larvae, a fast-germinating strain is actually cleared better than a slow-germinating strain. This fungal killing is driven primarily by neutrophils while macrophages promote fungal persistence by inhibiting spore germination. Our experiments underline different mechanisms of virulence that pathogens can utilize—rapid growth versus dormancy and persistence—and inform future strategies for fighting fungal infections in susceptible immunocompromised patients.

Neutrophils release extracellular traps (NETs) in response to planktonic C. albicans. These complexes composed of DNA, histones, and proteins inhibit Candida growth and dissemination. Considering the resilience of Candida biofilms to host defenses, we examined the neutrophil response to C. albicans during biofilm growth. In contrast to planktonic C. albicans, biofilms triggered negligible release of NETs. Time lapse imaging confirmed the impairment in NET release and revealed neutrophils adhering to hyphae and migrating on the biofilm. NET inhibition depended on an intact extracellular biofilm matrix as physical or genetic disruption of this component resulted in NET release. Biofilm inhibition of NETosis could not be overcome by protein kinase C activation via phorbol myristate acetate (PMA) and was associated with suppression of neutrophil reactive oxygen species (ROS) production. The degree of impaired NET release correlated with resistance to neutrophil attack. The clinical relevance of the role for extracellular matrix in diminishing NET production was corroborated in vivo using a rat catheter model. The C. albicans pmr1Δ/Δ, defective in production of matrix mannan, appeared to elicit a greater abundance of NETs by scanning electron microscopy imaging, which correlated with a decreased fungal burden. Together, these findings show that C. albicans biofilms impair neutrophil response through an inhibitory pathway induced by the extracellular matrix., Author Summary Candida spp. avidly adhere to medical device surfaces, forming resilient, drug-tolerant biofilms which are encased in a protective extracellular matrix. These infections are notoriously difficult to eradicate and little is known about how they evade host defenses. Here we show a mechanism by which C. albicans biofilms inhibit the activity of neutrophils, leukocytes critical for protection from Candida. When exposed to biofilm, neutrophils fail to release neutrophil extracellular traps (NETs), web-like structures of DNA, histones, and proteins capable of fungal killing. The biofilm extracellular matrix appears to have a major role in this inhibition, as disruption of matrix polysaccharides by physical or genetic means reverses the impairment. C. albicans biofilms block neutrophil production of reactive oxygen species (ROS), a signaling pathway involved in NET production. Impaired NET release results in decreased fungal killing in vitro and in an animal model of biofilm infection. This represents a novel mechanism of immune evasion specific to the biofilm mode of growth.

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., Author Summary Several fungal pathogens produce bioactive small molecules, commonly known as secondary metabolites (SMs) that contribute towards disease development in susceptible hosts. 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. This lack of knowledge is directly correlated to the difficulties of obtaining assayable quantities of pure metabolites. To overcome this roadblock in assessing the potential impact of SMs on the immune system, our laboratories have developed a two-tiered cost-effective, high-throughput program utilizing microfluidic platforms and a novel zebrafish model to identify SMs inhibiting neutrophil chemotaxis. Using minimal and physiologically relevant amounts of SMs, this systematic approach has identified the A. fumigatus spore SM, endocrocin, as a potent chemotaxis inhibitor. Interestingly, the production of endocrocin is temperature dependent and virulence studies with the endocrocin null mutant implicates the temperature at which the fungus forms spores as a factor in disease development.