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.

Fungal pathogens can be recognized by the immune system via their β-glucan, a potent proinflammatory molecule that is present at high levels but is predominantly buried beneath a mannoprotein coat and invisible to the host. To investigate the nature and significance of “masking” this molecule, we characterized the mechanism of masking and consequences of unmasking for immune recognition. We found that the underlying β-glucan in the cell wall of Candida albicans is unmasked by subinhibitory doses of the antifungal drug caspofungin, causing the exposed fungi to elicit a stronger immune response. Using a library of bakers' yeast (Saccharomyces cerevisiae) mutants, we uncovered a conserved genetic network that is required for concealing β-glucan from the immune system and limiting the host response. Perturbation of parts of this network in the pathogen C. albicans caused unmasking of its β-glucan, leading to increased β-glucan receptor-dependent elicitation of key proinflammatory cytokines from primary mouse macrophages. By creating an anti-inflammatory barrier to mask β-glucan, opportunistic fungi may promote commensal colonization and have an increased propensity for causing disease. Targeting the widely conserved gene network required for creating and maintaining this barrier may lead to novel broad-spectrum antimycotics. Opportunistic fungal pathogens such as Candida albicans often cause fatal infections in patients with a compromised immune system. Unfortunately, current drugs often fail to halt fungal disease, are ineffective against drug-resistant strains, and have severe side effects. Despite the clear clinical significance of fungal infections, it is still not understood how fungi are recognized by the immune system. Candida has high levels of the structural molecule β-glucan in its cell wall, but the majority of its β-glucan is masked by a mannoprotein coat and is therefore invisible to the immune system. Masking of β-glucan may be a fungal virulence factor, because exposed β-glucan provokes a proinflammatory response that is important for mounting an effective immune response against the fungus and clearing the infection. By surveying the genome of the model fungus Saccharomyces cerevisiae (bakers' yeast), the authors discovered a genetic network required for masking β-glucan from the immune system. Mutation of genes in this network in C. albicans caused unmasking of β-glucan and an increased immune response to the fungus. The authors also found that sublethal doses of the antifungal drug caspofungin cause unmasking and lead to a greater immune response. Drugs targeting this fungally conserved masking network may provide new tools to fight fungal infections.

Candida albicans, a clinically important dimorphic fungal pathogen that can evade immune attack by masking its cell wall β-glucan from immune recognition, mutes protective host responses mediated by the Dectin-1 β-glucan receptor on innate immune cells. Although the ability of C. albicans to switch between a yeast- or hyphal-form is a key virulence determinant, the role of each morphotype in β-glucan masking during infection and treatment has not been addressed. Here, we show that during infection of mice, the C. albicans β-glucan is masked initially but becomes exposed later in several organs. At all measured stages of infection, there is no difference in β-glucan exposure between yeast-form and hyphal cells. We have previously shown that sub-inhibitory doses of the anti-fungal drug caspofungin can expose β-glucan in vitro, suggesting that the drug may enhance immune activity during therapy. This report shows that caspofungin also mediates β-glucan unmasking in vivo. Surprisingly, caspofungin preferentially unmasks filamentous cells, as opposed to yeast form cells, both in vivo and in vitro. The fungicidal activity of caspofungin in vitro is also filament-biased, as corroborated using yeast-locked and hyphal-locked mutants. The uncloaking of filaments is not a general effect of anti-fungal drugs, as another anti-fungal agent does not have this effect. These results highlight the advantage of studying host–pathogen interaction in vivo and suggest new avenues for drug development. Candida is a common human commensal but disseminated candidiasis is a serious clinical problem, especially among immunocompromised patients. The innate immune system controls Candida infection, in part through the germline-encoded β-glucan receptor Dectin-1. However, during in vitro growth, Candida albicans mutes Dectin-1 recognition by cloaking its β-glucan underneath a layer of mannan. Bridging these two seemingly contradictory observations, we demonstrate that C. albicans masks β-glucan early during infection, but it becomes exposed later, allowing Dectin-1 to recognize the fungi and mediate immunity. Remarkably, treatment of mice with sub-therapeutic doses of the antifungal drug caspofungin causes exposure of β-glucan on C. albicans even when it would not be exposed naturally. We introduce a new technique for monitoring of epitope exposure during infection, which can be used to monitor the availability of any epitope for immune recognition. This technique allowed us to show that natural unmasking of β-glucan is not morphotype-specific, but drug-mediated unmasking is biased towards the invasive filamentous form of C. albicans. These results highlight the unexplored area of dynamic epitope exposure during infection and therapy, which might be targetable to enhance immune recognition and fungal clearance.

Systemic Candida albicans infection causes high morbidity and mortality and is associated with neutropenia; however, the roles of other innate immune cells in pathogenesis are poorly defined. Here, using a mouse model of systemic candidiasis, we found that resident macrophages accumulated in the kidney, the main target organ of infection, and formed direct contacts with the fungus in vivo mainly within the first few hours after infection. Macrophage accumulation and contact with Candida were both markedly reduced in mice lacking chemokine receptor CX₃CR1, which was found almost exclusively on resident macrophages in uninfected kidneys. Infected Cx3cr1^–/– mice uniformly succumbed to Candida-induced renal failure, but exhibited clearance of the fungus in all other organs tested. Renal macrophage deficiency in infected Cx3cr1^–/– mice was due to reduced macrophage survival, not impaired proliferation, trafficking, or differentiation. In humans, the dysfunctional CX₃CR1 allele CX₃CR1-M280 was associated with increased risk of systemic candidiasis. Together, these data indicate that CX₃CR1-mediated renal resident macrophage survival is a critical innate mechanism of early fungal control that influences host survival in systemic candidiasis.