Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The ⌬cnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the ⌬cnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the ⌬cnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the ⌬cnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

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...

The calcineurin pathway is a critical signal transduction pathway in fungi that mediates growth, morphology, stress responses, and pathogenicity. The importance of the calcineurin pathway in fungal physiology creates an opportunity for the development of new antifungal therapies that target this critical signaling pathway. In this study, we examined the role of the zinc finger transcription factor Crz1 homolog (CrzA) in the physiology and pathogenicity of the opportunistic human fungal pathogen Aspergillus fumigatus. Genetic replacement of the crzA locus in A. fumigatus resulted in a strain with significant defects in conidial germination, polarized hyphal growth, cell wall structure, and asexual development that are similar to but with differences from defects seen in the A. fumigatus ⌬cnaA (calcineurin A) strain. Like the ⌬cnaA strain, the ⌬crzA strain was incapable of causing disease in an experimental persistently neutropenic inhalational murine model of invasive pulmonary aspergillosis. Our results suggest that CrzA is an important downstream effector of calcineurin that controls morphology in A. fumigatus, but additional downstream effectors that mediate calcineurin signal transduction are likely present in this opportunistic fungal pathogen. In addition, the importance of CrzA to the production of disease is critical, and thus CrzA is an attractive fungus-specific antifungal target for the treatment of invasive aspergillosis.

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.

Calcineurin mutation or inhibition enhanced the antifungal morphological effect of cell wall inhibitors caspofungin or nikkomycin Z against Aspergillus fumigatus. Quantification of 1,3-␤-D-glucan revealed decreased amounts in the calcineurin A (⌬cnaA) mutant. Calcineurin can be an excellent adjunct therapeutic target in combination with other cell wall inhibitors against A. fumigatus.Invasive aspergillosis (IA) is a leading cause of infectious mortality in the growing number of immunocompromised patients (7). Our previous in vitro work demonstrated anti-Aspergillus activity of the calcineurin inhibitors FK506 and cyclosporine (11,12). We subsequently showed with a calcineurin A (cnaA) mutant that calcineurin is critical for A. fumigatus hyphal growth, tissue invasion, and pathogenicity (10). Here we further examine potential enhanced antifungal involvement of calcineurin and cell wall synthesis inhibitors against A. fumigatus. Our results lend support to our hypothesis that the calcineurin pathway is an innovative target for more efficacious strategies against IA.Patients generally possess blood levels of FK506 from 5 to 25 ng/ml for the prevention of organ rejection. We utilized radial growth assays (8) and found patient achievable levels of FK506 (20 ng/ml) led to significantly stunted A. fumigatus growth similar to the untreated ⌬cnaA mutant and more effective than therapeutically relevant and greater (300 ng to 1 g/ml) concentrations of the echinocandin antifungal caspofungin. The higher dose of caspofungin did lead to a phenotypically identical growth arrest as the ⌬cnaA mutant, suggesting a possible similar or linked mechanism of action on Aspergillus growth. These findings were confirmed with scanning electron microscopy, which demonstrated absent hyphal growth in the ⌬cnaA mutant or after FK506 (20 ng/ml) treatment and superior antifungal potency compared to caspofungin (1 g/ml) treatment.Impact on early fungal growth was also examined. Wild-type A. fumigatus conidia (10 6 conidia/ml) were incubated for 14 h in RPMI 1640, and subsequent addition of caspofungin (1 g/ml) resulted in well-described dysmorphic hyphae (6).FK506 acted in a similar fashion to caspofungin, supporting a potentially similar mechanism or pathway (Fig. 1A). Ungerminated wild-type conidia treated with FK506 showed significant delay in germ-tube extension, including growth retardation at only 20 ng/ml. Germination into hyphae was complete by 12 h in the untreated controls, but germination had only begun in the calcineurin inhibitor-treated strains after 60 h (Fig. 1B). Thus, inhibiting the calcineurin pathway caused a profound initial delay in conidial germination.Given the link between cell wall morphogenesis and hyphal growth in filamentous fungi, we next used the aniline blue fluorescence assay (8) to quantify 1,3-␤-D-glucan after calcineurin inhibition. Measurement was obtained as relative fluorescence units (RFU) normalized to mycelial mass and analyzed using an unpaired t test with significance reported as a two-tailed P value o...

Calcineurin is a conserved protein phosphatase that plays a critical role in Ca2؉ signaling and stress responses. Previously, a new class of conserved calcineurin-binding proteins, the calcipressins, was identified. However, the role of these proteins remains controversial, and both inhibitory and stimulatory effects on calcineurin were observed. In this study, we investigate the role of CbpA, the Aspergillus fumigatus member of the calcipressin family, and report that deletion of the cbpA gene resulted in reduced hyphal growth and limited attenuated virulence. Interestingly, under high-calcium-level conditions, the ⌬cbpA strain displayed improved Ca 2؉ tolerance compared to the wild-type strain and revealed increased expression of vcxA, chsA, and cnaA, which encode the vacuolar Ca 2؉ /H ؉ exchanger VcxA, chitin synthase A, and the calcineurin catalytic subunit CnaA, respectively. The increased transcript levels of these three genes were reversed in the presence of the calcineurin inhibitor FK506, indicating a calcineurin-dependent mechanism. Overexpression of cbpA resulted in decreased transcription of vcxA, chsA, and cnaA, associated with wild-type sensitivity to Ca 2؉ . Taken together, our study highlights the importance of CbpA in the regulation of hyphal growth and calcium adaptation of A. fumigatus and provides evidence that CbpA may serve as a feedback inhibitor in some aspects of calcineurin functions.

The Aspergillus fumigatus ⌬pmrA (Golgi apparatus Ca 2؉ /Mn 2؉ P-type ATPase) strain has osmotically suppressible basal growth defects and cationic tolerance associated with increased expression of calcineurin pathway genes. Despite increased ␤-glucan and chitin content, it is hypersensitive to cell wall inhibitors but remains virulent, suggesting a role for PmrA in cation homeostasis and cell wall integrity.

A functional calcineurin A fusion to enhanced green fluorescent protein (EGFP), CnaA-EGFP, was expressed in the Aspergillus fumigatus ⌬cnaA mutant. CnaA-EGFP localized in actively growing hyphal tips, at the septa, and at junctions between the vesicle and phialides in an actin-dependent manner. This is the first study to implicate calcineurin in septum formation and conidiophore development of a filamentous fungus.Calcineurin, a Ca 2ϩ -calmodulin-dependent protein phosphatase (PP2B), regulates diverse processes, including morphogenesis, ion homeostasis, virulence, and stress responses in fungi (1, 3-5, 9, 12, 13, 16-18, 28). In Aspergillus fumigatus, calcineurin is required for hyphal growth, cell wall integrity, conidial morphology, PO 4 Ϫ transport, and pathogenicity via its downstream target, CrzA (2,6,(20)(21)(22)(23)(24). However, the specific molecular mechanisms by which the calcineurin signal transduction pathway regulates hyphal extension or cell wall biosynthesis remain largely unknown.To elucidate calcineurin roles in hyphal growth, its localization pattern was analyzed during growth by fusion to enhanced green fluorescent protein (EGFP). The A. fumigatus cnaA cDNA, encoding the calcineurin A catalytic subunit, was inserted into the pUCGH plasmid (a gift from Axel Brakhage) (14). The cnaA cDNA was cloned at the N terminus of egfp to obtain the cnaA-egfp fusion allele (pUCGH-cnaA) under the control of the synthetic otef promoter. The A. fumigatus ⌬cnaA mutant was transformed with pUCGH-cnaA, as previously described (21), and transformants were selected by resistance to hygromycin B. After single spore isolation, Southern analysis indicated that one of the six transformants contained a singlecopy cnaA-egfp cassette integrated ectopically, and we have designated that as the cnaA-egfp expression strain (Fig. 1A, lane 4).Because the ⌬cnaA mutant exhibits stunted hyphal growth (6, 21), we next examined whether the cnaA-egfp expression strain returned to wild-type growth. As shown in Fig. 1B, radial growth, quantified as previously described (21), indicated that cnaA-egfp expression complemented the cnaA deletion and restored the wild-type phenotype. The cnaAegfp expression strain was also grown in the presence of FK506, a specific inhibitor of calcineurin, to reconfirm that hyphal growth was stunted after FK506 treatment in a similar manner to that of the wild-type strain (Fig. 1C, plate assay). Microscopic examination of the wild-type and cnaAegfp expression strains grown in the presence of FK506 also revealed a highly branched phenotype similar to the ⌬cnaA mutant (Fig. 1D, lower panel). Taken together, these results indicate that the cnaA-egfp construct is functional after placement in the ⌬cnaA mutant.Next, in order to observe the localization patterns of CnaA-EGFP, the cnaA-egfp expression strain was grown on slide cultures for 24 h. While fluorescence microscopy revealed a general distribution of the CnaA-EGFP fusion protein in the cytoplasm, dot-like structures were observed closer to the cell wall...