Aspergillus fumigatus is the most important airborne fungal pathogen causing life-threatening infections in immunocompromised patients. Macrophages and neutrophils are known to kill conidia, whereas hyphae are killed mainly by neutrophils. Since hyphae are too large to be engulfed, neutrophils possess an array of extracellular killing mechanisms including the formation of neutrophil extracellular traps (NETs) consisting of nuclear DNA decorated with fungicidal proteins. However, until now NET formation in response to A. fumigatus has only been demonstrated in vitro, the importance of neutrophils for their production in vivo is unclear and the molecular mechanisms of the fungus to defend against NET formation are unknown. Here, we show that human neutrophils produce NETs in vitro when encountering A. fumigatus. In time-lapse movies NET production was a highly dynamic process which, however, was only exhibited by a sub-population of cells. NETosis was maximal against hyphae, but reduced against resting and swollen conidia. In a newly developed mouse model we could then demonstrate the existence and measure the kinetics of NET formation in vivo by 2-photon microscopy of Aspergillus-infected lungs. We also observed the enormous dynamics of neutrophils within the lung and their ability to interact with and phagocytose fungal elements in situ. Furthermore, systemic neutrophil depletion in mice almost completely inhibited NET formation in lungs, thus directly linking the immigration of neutrophils with NET formation in vivo. By using fungal mutants and purified proteins we demonstrate that hydrophobin RodA, a surface protein making conidia immunologically inert, led to reduced NET formation of neutrophils encountering Aspergillus fungal elements. NET-dependent killing of Aspergillus-hyphae could be demonstrated at later time-points, but was only moderate. Thus, these data establish that NET formation occurs in vivo during host defence against A. fumigatus, but suggest that it does not play a major role in killing this fungus. Instead, NETs may have a fungistatic effect and may prevent further spreading. The fungus Aspergillus fumigatus grows on decaying organic matter and produces large numbers of spores, called conidia, which are constantly inhaled by humans. This is harmless, because we have a functioning defence system of immune cells called neutrophil granulocytes, but people with too few or non-functioning neutrophils can die of Aspergillus infections. Neutrophils invade the lung, engulf/phagocytose and thereby kill conidia. Dying neutrophils can also throw their nuclear DNA on hyphal elements as NETs (Neutrophil Extracellular Traps) that are decorated with antimicrobial proteins. Thus, larger fungal amounts, including tissue-invading hyphae, can still be controlled. However, until today the formation of NETs has not been demonstrated in Aspergillus-infected lungs, the role of neutrophils for this process was unknown and whether the fungus has anti-NET defence strategies on its own was not clear. We demonstrate here the existence of NETs in Aspergillus-infected lungs, show that neutrophils produce these structures and that they phagocytose fungal elements within the lung tissue. Furthermore, we show that Aspergillus camouflages its spores by means of the surface protein hydrophobin RodA, which is able to strongly prevent NET formation by neutrophils. These studies shed new light on the dynamics and molecular mechanisms of this key process of host-pathogen interaction. Although these data establish that NET formation occurs in vivo during host defence against A. fumigatus, we suggest that NET formation does not play a major role in killing this fungus.

Resistance to infection is critically dependent on the ability of pattern recognition receptors to recognize microbial invasion and induce protective immune responses. One such family of receptors are the C-type lectins, which are central to antifungal immunity¹. These receptors activate key effector mechanisms upon recognition of conserved fungal cell-wall carbohydrates. However, several other immunologically active fungal ligands have been described; these include melanin^2,3, for which the mechanism of recognition is hitherto undefined. Here we identify a C-type lectin receptor, melanin-sensing C-type lectin receptor (MelLec), that has an essential role in antifungal immunity through recognition of the naphthalene-diol unit of 1,8-dihydroxynaphthalene (DHN)-melanin. MelLec recognizes melanin in conidial spores of Aspergillus fumigatus as well as in other DHN-melanized fungi. MelLec is ubiquitously expressed by CD31⁺ endothelial cells in mice, and is also expressed by a sub-population of these cells that co-express epithelial cell adhesion molecule and are detected only in the lung and the liver. In mouse models, MelLec was required for protection against disseminated infection with A. fumigatus. In humans, MelLec is also expressed by myeloid cells, and we identified a single nucleotide polymorphism of this receptor that negatively affected myeloid inflammatory responses and significantly increased the susceptibility of stem-cell transplant recipients to disseminated Aspergillus infections. MelLec therefore recognizes an immunologically active component commonly found on fungi and has an essential role in protective antifungal immunity in both mice and humans.

Aspergillus and Fusarium species are important causes of fungal infections worldwide. Airborne spores (conidia) of these filamentous fungi express a surface protein that confers hydrophobicity (hydrophobin) and covers cell wall components that would otherwise induce a host immune cell response. Using a mutant Aspergillus fumigatus strain (ΔrodA) that does not express the RodA hydrophobin, and Aspergillus and Fusarium conidia from clinical isolates that were treated with hydrofluoric acid (which removes the A. fumigatus RodA protein), we observed increased surface exposure of β1,3-glucan and α-mannose on Aspergillus and Fusarium conidia. We also found that ΔrodA and hydrofluoric acid–treated conidia stimulate significantly higher NF-κB p65 nuclear translocation and cytokine production by macrophages from C57BL/6, but not from Dectin-1^−/− or Dectin-2^−/− mice. Using a murine model of A. fumigatus corneal infection, we showed that ΔrodA conidia induced significantly higher cytokine production, neutrophil infiltration, and more rapid fungal clearance from C57BL/6 corneas compared with the parent G10 strain, which was dependent on Dectin-1 and Dectin-2. Together, these findings identify the hydrophobin RodA as a virulence factor that masks Dectin-1 and Dectin-2 recognition of conidia, resulting in impaired neutrophil recruitment to the cornea and increased fungal survival and clinical disease.

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ΔhacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ΔhacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ΔhacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy. The pathogenic mold Aspergillus fumigatus is the leading cause of airborne fungal infections in immunocompromised patients. The fungus normally resides in compost, an environment that challenges the organism to obtain nutrients by degrading complex organic polymers. This is accomplished by secreted enzymes, some of which may also contribute to nutrient acquisition during infection. Extracellular enzymes are folded in the endoplasmic reticulum (ER) prior to secretion. If the folding capacity of the ER is overwhelmed by increased secretory demand, the resulting ER stress triggers an adaptive response termed the unfolded protein response (UPR). In this study, we uncover a previously unknown function for the master transcriptional regulator of the UPR, HacA, in fungal virulence. In the absence of HacA, A. fumigatus was unable to secrete high levels of proteins and had reduced virulence in mice. In addition, loss of HacA caused a cell wall defect and increased susceptibility to two major classes of antifungal drugs used for the treatment of aspergillosis. These findings demonstrate that A. fumigatus relies on HacA for growth in the host environment and suggest that therapeutic targeting of the UPR could have merit against A. fumigatus, as well as other eukaryotic pathogens with highly developed secretory systems.