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Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-β1-transporting vesicles in response to the pathogenic fungus Candida albicans . Soluble β-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-β1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-β1 to the TGF-β receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-β1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-β1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway. Extracellular vesicles can carry immunoregulatory cytokines such as TGF-β. Here the authors use CD11b-deficient mice and macrophages to show that such vesicles carrying TGF-β are produced in response to Candida albicans infections and can limit the proinflammatory response partly via a positive feedback on TGF-β production by endothelial cells.

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 . This study identifies a cytolytic peptide toxin in the opportunistic human fungal pathogen Candida albicans —the peptide is both a crucial virulence factor that permeabilizes the host cell plasma membrane and a key signal that triggers a host danger response pathway. A toxin identified in a human fungal pathogen This study identifies and characterizes a cytolytic peptide toxin in the opportunistic human fungal pathogen Candida albicans . The peptide, termed Candidalysin, acts both as a crucial virulence factor that permeabilizes the host cell plasma membrane and as a key signal that triggers a host danger-response pathway.

Protein kinases are key components of many signaling pathways that regulate cellular activities, and some of them are indispensable for the viability of cells. We used inducible gene deletion to assess the importance of a set of putative essential protein kinases for growth and viability of the pathogenic yeast Candida albicans and to get clues about the functions of uncharacterized essential kinases. We found that bud32 Δ, ctk1 Δ, rio1 Δ, and rio2 Δ mutants were viable but grew very slowly, explaining previous failures to generate homozygous deletion mutants. PTK2 was essential, but under certain conditions ptk2 Δ mutants remained viable and over time could acquire suppressor mutations in the Ptk2-dependent plasma membrane ATPase Pma1 that restored growth. Deletion of the uncharacterized orf19.5376 was lethal and the null mutants formed pseudohyphae that lacked normal septa and eventually lysed, a phenotype that was phenocopied by auxin-induced protein depletion. The mutants were defective in septin organization, indicating that the orf19.5376 -encoded kinase is functionally similar to the nonessential kinase Elm1 of Saccharomyces cerevisiae , but is indispensable for viability in C. albicans . Mutants lacking orf19.3456 , which does not have a homolog in S. cerevisiae , were also nonviable and grew as aseptate, sometimes multinucleate hyphae before cell death. Co-immunoprecipitation followed by liquid chromatography-mass spectrometry identified a protein, encoded by the uncharacterized orf19.193 , as a candidate regulatory subunit of the orf19.3456 -encoded kinase, as mutants lacking this protein exhibited the same terminal phenotype as orf19.3456 mutants. These results provide strong evidence that instead of using a mitotic exit network (MEN) with only two kinases (Cdc15 and Dbf2), as was previously thought, C. albicans regulates septum formation and cytokinesis via a septation initiation network (SIN), known from fission yeast and filamentous fungi, which contains a protein kinase cascade consisting of the upstream kinase Cdc15, the orf19.3456 -encoded kinase, and the downstream kinase Dbf2.

Another important protein on the surface of A. fumigatus conidia is the Mep1p metalloprotease, which is released from spores in the mammalian lung to cleave host complement proteins and enhance infection, similar to the Alp1p serine protease released from mycelia for the same purpose [44]. In particular, detection of fungal conidia from environmental samples might provide an early warning to those suffering from lung conditions like asthma or chronic obstructive pulmonary disease, in which patients show a heightened susceptibility to allergic exacerbations due to fungal sensitization [46]. [...]conidial surface proteins play an integral role at the interface between normal fungal function and pathogenesis while offering a wealth of potential biomarkers and novel therapeutic targets. Levdansky E, Kashi O, Sharon H, Shadkchan Y, Osherov N. The Aspergillus fumigatus cspA gene encoding a repeat-rich cell wall protein is important for normal conidial cell wall architecture and interaction with host cells.

Breathe easy: why inhaled fungal spores don't provoke an immune reaction Every day we inhale thousands of tiny fungal spores (conidia), originating from many different fungal species. Yet although these spores are packed with antigens and allergens, their inhalation does not continuously activate our innate immune cells or provoke inflammatory responses. A series of immunological, biochemical and genetic experiments shows why: immune recognition of these spores is prevented by a hydrophobic layer of rodlet proteins covering the conidial surface. If this layer is removed, spores activate the immune system. A pathogenic spore equipped with this defensive layer might lie dormant beyond host defences until conditions are suitable for germination. Therapeutically the robust nature of the rodlet proteins might be exploited to generate nanoparticles containing embedded molecules targeted to a specific location in the body, or optimized for sustained delivery. Fungal spores are ubiquitous in the air we breathe and contain many antigens and allergens, and yet they neither continuously activate the host innate immune cells nor induce detrimental inflammatory responses after their inhalation. Here, the surface layer on dormant spores is shown to mask their recognition by the immune system and hence prevent an immune response. The air we breathe is filled with thousands of fungal spores (conidia) per cubic metre, which in certain composting environments can easily exceed 10 9 per cubic metre. They originate from more than a hundred fungal species belonging mainly to the genera Cladosporium , Penicillium , Alternaria and Aspergillus 1 , 2 , 3 , 4 . Although these conidia contain many antigens and allergens 5 , 6 , 7 , it is not known why airborne fungal microflora do not activate the host innate immune cells continuously and do not induce detrimental inflammatory responses following their inhalation. Here we show that the surface layer on the dormant conidia masks their recognition by the immune system and hence prevents immune response. To explore this, we used several fungal members of the airborne microflora, including the human opportunistic fungal pathogen Aspergillus fumigatus , in in vitro assays with dendritic cells and alveolar macrophages and in in vivo murine experiments. In A. fumigatus , this surface ‘rodlet layer’ is composed of hydrophobic RodA protein covalently bound to the conidial cell wall through glycosylphosphatidylinositol-remnants. RodA extracted from conidia of A. fumigatus was immunologically inert and did not induce dendritic cell or alveolar macrophage maturation and activation, and failed to activate helper T-cell immune responses in vivo . The removal of this surface ‘rodlet/hydrophobin layer’ either chemically (using hydrofluoric acid), genetically (Δ rodA mutant) or biologically (germination) resulted in conidial morphotypes inducing immune activation. All these observations show that the hydrophobic rodlet layer on the conidial cell surface immunologically silences airborne moulds.

Candida albicans is an opportunistic fungal pathogen that colonizes mucosal surfaces of most humans. Only in rare cases, C. albicans isolates are found in the environment. This study investigated whether environmental isolates differ in their virulence potential from clinical strains and how adaptation to a human diet influences key virulence attributes. We examined three C. albicans isolates from oak trees in the United Kingdom, and observed that one exhibited high host cell damage, increased hypha formation, invasion capacity, and candidalysin production, along with an intrinsic resistance to amphotericin B. The other two showed lower virulence which was still similar to most tested clinical isolates. All oak tree isolates showed an increased resistance to fluconazole. To mimic the more recent evolution of C. albicans to a sugar-rich diet, we evolved a low-damaging isolate in sugar-rich medium, which unexpectedly enhanced its metabolic flexibility, epithelial damage potential, and antifungal resistances, including a new resistance to amphotericin B. These findings suggest that C. albicans isolates can develop high virulence potential and antifungal resistance in the environment, and that adaptation of C. albicans to sugar-rich diets, as in westernized countries, can affect fungal pathogenicity and drug resistance.

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.

Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD + pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa . The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca 2+ -binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms. Some bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD + pool, thereby causing rapid cell death. Here, Strømland et al. identify NADases on the surface of fungal spores, and show that the enzymes display unique biochemical and structural properties.

In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Filamentous fungi of the genus Aspergillus are of particular interest for biotechnological applications due to their natural capacity to secrete carbohydrate-active enzymes (CAZy) that target plant biomass. The presence of easily metabolizable sugars such as glucose, whose concentrations increase during plant biomass hydrolysis, results in the repression of CAZy-encoding genes in a process known as carbon catabolite repression (CCR), which is undesired for the purpose of large-scale enzyme production. To date, the C 2 H 2 transcription factor CreA has been described as the major CC repressor in Aspergillus spp., although little is known about the role of posttranslational modifications in this process. In this work, phosphorylation sites were identified by mass spectrometry on Aspergillus nidulans CreA, and subsequently, the previously identified but uncharacterized site S262, the characterized site S319, and the newly identified sites S268 and T308 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was investigated. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 was not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. All sites were shown to be important for glycogen and trehalose metabolism. This study highlights the importance of CreA phosphorylation sites for the regulation of CCR. These sites are interesting targets for biotechnological strain engineering without the need to delete essential genes, which could result in undesired side effects. IMPORTANCE In filamentous fungi, the transcription factor CreA controls carbohydrate metabolism through the regulation of genes encoding enzymes required for the use of alternative carbon sources. In this work, phosphorylation sites were identified on Aspergillus nidulans CreA, and subsequently, the two newly identified sites S268 and T308, the previously identified but uncharacterized site S262, and the previously characterized site S319 were chosen to be mutated to nonphosphorylatable residues before their effect on CCR was characterized. Sites S262, S268, and T308 are important for CreA protein accumulation and cellular localization, DNA binding, and repression of enzyme activities. In agreement with a previous study, site S319 is not important for several here-tested phenotypes but is key for CreA degradation and induction of enzyme activities. This work characterized novel CreA phosphorylation sites under carbon catabolite-repressing conditions and showed that they are crucial for CreA protein turnover, control of carbohydrate utilization, and biotechnologically relevant enzyme production.

Efficient adaptation to iron starvation is an essential virulence determinant of the most common human mold pathogen, Aspergillus fumigatus. Here, we demonstrate that the cytosolic monothiol glutaredoxin GrxD plays an essential role in iron sensing in this fungus. Our studies revealed that (i) GrxD is essential for growth; (ii) expression of the encoding gene, grxD, is repressed by the transcription factor SreA in iron replete conditions and upregulated during iron starvation; (iii) during iron starvation but not iron sufficiency, GrxD displays predominant nuclear localization; (iv) downregulation of grxD expression results in de-repression of genes involved in iron-dependent pathways and repression of genes involved in iron acquisition during iron starvation, but did not significantly affect these genes during iron sufficiency; (v) GrxD displays protein-protein interaction with components of the cytosolic iron-sulfur cluster biosynthetic machinery, indicating a role in this process, and with the transcription factors SreA and HapX, which mediate iron regulation of iron acquisition and iron-dependent pathways; (vi) UV-Vis spectra of recombinant HapX or the complex of HapX and GrxD indicate coordination of iron-sulfur clusters; (vii) the cysteine required for iron-sulfur cluster coordination in GrxD is in vitro dispensable for interaction with HapX; and (viii) there is a GrxD-independent mechanism for sensing iron sufficiency by HapX; (ix) inactivation of SreA suppresses the lethal effect caused by GrxD inactivation. Taken together, this study demonstrates that GrxD is crucial for iron homeostasis in A. fumigatus.