Tackling the multifactorial nature of virulence and antifungal drug resistance in A. fumigatus requires the mechanistic interrogation of a multitude of genes, sometimes across multiple genetic backgrounds. Classical fungal gene replacement systems can be laborious and time-consuming and, in wild-type isolates, are impeded by low rates of homologous recombination. Our simple and universal CRISPR-Cas9 system for gene manipulation generates efficient gene targeting across different genetic backgrounds of A. fumigatus. We anticipate that our system will simplify genome editing in A. fumigatus, allowing for the generation of single- and multigene knockout libraries. In addition, our system will facilitate the delineation of virulence factors and antifungal drug resistance genes in different genetic backgrounds of A. fumigatus.

Attenuated activity of echinocandin antifungals at high concentrations, known as the "paradoxical effect," is a well-established phenomenon in Candida albicans and Aspergillus fumigatus. In the yeast C. albicans, upregulation of chitin biosynthesis via the protein kinase C (PKC), high-osmolarity glycerol response (HOG), and Ca 2؉ /calcineurin signaling pathways is an important cell wall stress response that permits growth in the presence of high concentrations of echinocandins. However, nothing is known of the molecular mechanisms regulating the mold A. fumigatus and its paradoxical response to echinocandins. Here, we show that the laboratory strain of A. fumigatus and five of seven clinical A. fumigatus isolates tested display various magnitudes of paradoxical growth in response to caspofungin. Interestingly, none of the eight strains showed paradoxical growth in the presence of micafungin or anidulafungin. Treatment of the ⌬cnaA and ⌬crzA strains, harboring gene deletions of the calcineurin A subunit and the calcineurin-dependent transcription factor, respectively, with high concentrations of caspofungin revealed that the A. fumigatus paradoxical effect is calcineurin pathway dependent. Exploring a molecular role for CnaA in the compensatory chitin biosynthetic response, we found that caspofungin treatment resulted in increased chitin synthase gene expression, leading to a calcineurin-dependent increase in chitin synthase activity. Taken together, our data suggest a mechanistic role for A. fumigatus calcineurin signaling in the chitin biosynthetic response observed during paradoxical growth in the presence of high-dose caspofungin treatment.The echinocandin antifungals inhibit 1,3-␤-D-glucan synthesis, and all three currently available echinocandins (caspofungin, anidulafungin, and micafungin) have activity against both yeasts and molds. However, in vitro and in vivo studies with this antifungal class have revealed an interesting "paradoxical effect" on growth, evidenced by a recurrent increase in the growth of the fungal organism at drug concentrations above a certain threshold (36).The paradoxical effect was first described for caspofungin treatment of Candida albicans and was not found to be due to mutation or increased expression of glucan synthase activity (30,32). Further studies showed that this paradoxical effect on growth occurred in many Candida species, appearing in both planktonic and biofilm cells (22). Early efforts to elucidate a mechanism revealed increased chitin content in the cell wall upon caspofungin treatment (31). Initial studies with the Saccharomyces cerevisiae FKS1 deletion mutant, a strain defective in the 1,3-␤-D-glucan synthase, showed that chitin biosynthesis is upregulated in response to glucan depletion (14). Furthermore, at the molecular level, several signal transduction pathways have been implicated in the regulation of the C. albicans paradoxical effect, including protein kinase C (PKC), highosmolarity glycerol response (HOG), and calcineurin signaling events (25,34,35). Alt...

Aspergillus fumigatus must be able to properly form hyphae and maintain cell wall integrity in order to establish invasive disease. Ras proteins and calcineurin each have been implicated as having roles in these processes. Here, we further delineate the roles of calcineurin and Ras activity in cell wall biosynthesis and hyphal morphology using genetic and pharmacologic tools. Strains deleted for three genes encoding proteins of these pathways, rasA (the Ras protein), cnaA (calcineurin), or crzA (the zinc finger transcription factor downstream of calcineurin), all displayed decreased cell wall 1,3-␤-D-glucan content. Echinocandin treatment further decreased the levels of 1,3-␤-D-glucan for all strains tested yet also partially corrected the hyphal growth defect of the ⌬rasA strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active rasA, and ⌬rasA strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (⌬cnaA and ⌬crzA). Taken together, our data suggest that the Ras and calcineurin pathways act in parallel to regulate cell wall formation and hyphal growth. Additionally, the calcineurin pathway elements cnaA and crzA play a major role in proper chitin and glucan incorporation into the A. fumigatus cell wall.

ABSTRACTHeat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance ofCandida albicansto azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogenAspergillus fumigatus. We investigated the role of Hsp90 inA. fumigatusby genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain ofhsp90suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (pniiA-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factorsbrlA,wetA, andabaA. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.

Summary Calcineurin, a heterodimer composed of the catalytic (CnaA) and regulatory (CnaB) subunits, plays key roles in growth, virulence, and stress responses of fungi. To investigate the contribution of CnaA and CnaB to hyphal growth and septation, ΔcnaB and ΔcnaA ΔcnaB strains of A. fumigatus were constructed. CnaA co-localizes to the contractile actin ring early during septation and remains at the center of the mature septum. While CnaB's septal localization is CnaA-dependent, CnaA's septal localization is CnaB-independent but CnaB is required for CnaA's function at the septum. Catalytic null mutations in CnaA caused stunted growth despite septal localization of the calcineurin complex, indicating the requirement of calcineurin activity at the septum. Compared to the ΔcnaA and ΔcnaB strains, the ΔcnaA ΔcnaB strain displayed more defective growth and aberrant septation. While three Ca2+-binding motifs in CnaB were sufficient for its association with CnaA at the septum, the amino-terminal arginine-rich domains (16-RRRR-19 and 44-RLRKR-48) are dispensable for septal localization, yet required for complete functionality. Mutation of the 51-KLDK-54 motif in CnaB causes its mislocalization from the septum to the nucleus, suggesting it is a nuclear export signal sequence. These findings confirm a cooperative role for calcineurin complex in regulating hyphal growth and septation.

Autophagy is the major cellular pathway for bulk degradation of cytosolic material and is required to maintain viability under starvation conditions. To determine the contribution of autophagy to starvation stress responses in the filamentous fungus Aspergillus fumigatus, we disrupted the A. fumigatus atg1 gene, encoding a serine/threonine kinase required for autophagy. The ⌬Afatg1 mutant showed abnormal conidiophore development and reduced conidiation, but the defect could be bypassed by increasing the nitrogen content of the medium. When transferred to starvation medium, wild-type hyphae were able to undergo a limited amount of growth, resulting in radial expansion of the colony. In contrast, the ⌬Afatg1 mutant was unable to grow under these conditions. However, supplementation of the medium with metal ions rescued the ability of the ⌬Afatg1 mutant to grow in the absence of a carbon or nitrogen source. Depleting the medium of cations by using EDTA was sufficient to induce autophagy in wild-type A. fumigatus, even in the presence of abundant carbon and nitrogen, and the ⌬Afatg1 mutant was severely growth impaired under these conditions. These findings establish a role for autophagy in the recycling of internal nitrogen sources to support conidiophore development and suggest that autophagy also contributes to the recycling of essential metal ions to sustain hyphal growth when exogenous nutrients are scarce.Nutrient limitation is one of the most significant stresses encountered by microorganisms in nature. Autophagy is a catabolic membrane trafficking system that counters such nutrient stress by initiating a process of limited intracellular digestion to support the organism during periods of reduced nutrient availability (26,32,37,60). The process begins with the formation of isolation membranes within the cytoplasm, whose origin is not entirely clear. These membranes progressively expand, nonselectively encapsulating cytosolic material into a doublemembrane vesicle called the autophagosome. The autophagosome fuses its outer membrane with a vacuole, releasing the autophagic body and its contents into the vacuolar lumen for degradation by resident hydrolases. Autophagy is upregulated in response to starvation stress, resulting in the generation of a pool of recycled molecules that provide the building blocks for continued synthesis of essential components until nutrient conditions improve (26,37,39,60). However, some autophagy remains constitutively active at low levels, even under nutrientreplete conditions, where it serves as a mechanism to remove old or damaged proteins and organelles (17,25,28,46,54). This provides an important form of quality control that combats the toxic accumulation of abnormal cytoplasmic components.The degradative functions of autophagy contribute to several important aspects of cell physiology, including autophagydependent programmed cell death (2, 14), cellular remodeling during development and differentiation (21,36,40,49,57), maintenance of endoplasmic reticulum homeostasis (4, 30, 6...

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The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The ⌬rasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The ⌬rasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The ⌬rasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.Aspergillus fumigatus is an opportunistic fungal pathogen that causes a variety of diseases, ranging from allergic reactions to invasive pulmonary infection. The pathogenicity of this organism has been attributed to many factors, including thermotolerance, rapid growth of hyphae, and small conidial size (16). The conidia of A. fumigatus, considered the infective propagule, are ubiquitous in nature and are inhaled and cleared by immunocompetent hosts. However, in immunosuppressed patients, inhaled conidia survive and initiate growth. After germination and apical extension, the organism penetrates the pulmonary epithelium and vascular endothelium. In the vessel lumen, shear forces sever portions of the hyphae and deposit fungus-laden emboli in areas rich in microvasculature, leading to dissemination of the infection. Once dissemination has occurred, the mortality rate ranges from 30 to 85%, depending on the patient population (20).