Cytolytic proteins and peptide toxins are classical virulence factors of several bacterial pathogens which disrupt epithelial barrier function, damage cells and activate or modulate host immune responses. Such toxins have not been identified previously in human pathogenic fungi. Here we identify the first, to our knowledge, fungal cytolytic peptide toxin in the opportunistic pathogen Candida albicans. This secreted toxin directly damages epithelial membranes, triggers a danger response signalling pathway and activates epithelial immunity. Membrane permeabilization is enhanced by a positive charge at the carboxy terminus of the peptide, which triggers an inward current concomitant with calcium influx. C. albicans strains lacking this toxin do not activate or damage epithelial cells and are avirulent in animal models of mucosal infection. We propose the name ‘Candidalysin’ for this cytolytic peptide toxin; a newly identified, critical molecular determinant of epithelial damage and host recognition of the clinically important fungus, C. albicans.

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.

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans , iron represses iron acquisition via the GATA factor SreA, and induces iron‐dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron‐dependent pathways (e.g., heme biosynthesis) are repressed during iron‐depleted conditions by physical interaction of HapX with the CCAAT‐binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron‐depleted and iron‐replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron‐depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron‐depleted conditions. Remarkably, CBC‐mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans , compared with Saccharomyces cerevisae .