Calcineurin target CrzA regulates conidial germination, hyphal growth, and pathogenesis of Aspergillus fumigatus

Abstract

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 DeltacnaA (calcineurin A) strain. Like the DeltacnaA strain, the DeltacrzA 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.

FIG. 1.

Construction of A. fumigatus ΔcrzA and ΔcrzA + crzA complemented strains. (A) Schematic of crzA gene replacement in A. fumigatus strain 293.1. The wild-type crzA locus (2.3 kb) was replaced with the 3.1-kb A. parasiticus pyrG gene from plasmid pJW24. (B) Southern blot analysis of SmaI-digested genomic DNA with a 1.2-kb digoxigenin-labeled probe revealed successful gene replacement of crzA with a single copy of pyrG in a transformant (KO) and successful ectopic reintroduction of the wild-type (WT) crzA gene (2.7-kb band) in the crzA mutant background (Comp). Lane L, ladder.

FIG. 2.

Multiple alignment of Saccharomyces cerevisiae Crz1p (YNL027W) and putative Aspergillus fumigatus CrzA (Afu1g06900), created with CLUSTALW using the BLOSUM62 scoring matrix and default parameters. Conserved residues are in black. *, serine-rich region; ^, NLS site in Crz1p; $, calcineurin interaction site present in Crz1p but absent in CrzA; #, C2H2 zinc finger domain region.

FIG. 3.

The calcineurin pathway is required for in vitro conidium germination. Conidium germination of the wild-type (AF293), ΔcnaA, ΔcrzA, and ΔcrzA + crzA strains was examined at 37°C in RPMI 1640 liquid medium over a 48-h time course. Significant differences in both the rate and quantity of conidia germination are seen among the wild-type, the crzA complemented strain, and the cnaA and crzA mutant strains. Percent germination is based on analysis of 100 conidia under light microscopy (magnification, ×400), repeated in triplicate.

FIG. 4.

(A and B) CrzA is necessary for in vitro hyphal growth. (A) In vitro culture morphology of the wild-type (AF293), ΔcrzA, and ΔcrzA + crzA strains at 37°C on glucose minimal medium after 4 days of growth. (B) The ΔcrzA strain displayed a 68% decrease in radial growth over a 5-day time course compared with the wild-type and complemented strains (P < 0.0001). (C) CrzA is required for the production of asexual conidia in A. fumigatus. Conidia were harvested from glucose minimal medium plates grown at 37°C as described in Materials and Methods. Conidia were quantified with a hemacytometer. The ΔcrzA strain had a 73% decrease in conidia produced per mm² of colony hyphal growth compared to the wild-type strain (P < 0.0001). Error bars indicate standard errors.

FIG. 5.

The ΔcrzA strain has malformed hyphae. Scanning electron microscopy of conidia from the three A. fumigatus strains grown at 37°C in glucose minimal medium broth for 48 h is shown. The crzA mutant strain shows abnormal, malformed hyphal tips that form abnormal mycelia. Levels of magnification are as shown. Bars, 100 μm (top row) and 50 μm (bottom row).

FIG. 6.

Conidia of the ΔcrzA strain show abnormal conidial surface morphology. Scanning electron microscopy of sputter-coated A. fumigatus conidia from glucose minimal medium plates is shown. Levels of magnification are as shown. Bars, 10 μm (top row) and 2 μM (middle and bottom rows). ΔcrzA strain conidia lack the bumpy and echinulate surface observed on wild-type and reconstituted strains.

FIG. 7.

Cell wall β-1,3-glucan is decreased in calcineurin mutants. Aniline blue fluorescence, measuring β-1,3-glucan in the cell wall, shows statistically significant decreases in β-1,3-glucan content in the ΔcnaA strain compared with the wild-type strain AF293 (P = 0.03). In addition, treatment with the β-1,3-glucan synthase inhibitor caspofungin results in a further decrease of β-1,3-glucan content in A. fumigatus strains in the wild-type strain (P = 0.03), in the absence of calcineurin A (P < 0.0001), and in the absence of CrzA (P = 0.011). Decreases in β-1,3-glucan content in the ΔcrzA strain were not statistically significant compared to the wild-type strain (P = 0.09). Treatment of the strains with the chitin inhibitor nikkomycin Z resulted in a compensatory increase in the ΔcnaA strain (P < 0.0001), indicating that calcineurin-independent mechanisms of β-1,3-glucan biosynthesis exist in A. fumigatus. Asterisks indicate statistical significance (P < 0.05). Error bars indicate standard errors.

FIG. 8.

Enhanced effects of β-1,3-glucan and chitin synthesis inhibition on the wild-type, ΔcnaA, and ΔcrzA strains. Strains were grown on glucose minimal medium for 96 h with the indicated treatments. The effects of the antifungal agents are enhanced with genetic replacement of calcineurin or crzA and are most evident with combination treatment with β-1,3-glucan (caspofungin) and chitin synthesis (nikkomycin Z) inhibitors. Importantly, treatment of the ΔcrzA strain with the calcineurin inhibitor FK506 resulted in a phenotype virtually identical to that of the ΔcnaA strain, indicating that CrzA has little or no function in a cell lacking calcineurin activity.

FIG. 9.

CrzA is required for A. fumigatus pathogenesis in a persistently neutropenic inhalational murine model of IPA. (A) Kaplan-Meier survival curve. Mice inoculated with the wild-type (AF293) and crzA complemented strains displayed significant mortality by 14 days following infection, while the mice infected with the ΔcrzA strain all survived (P = 0.0011). Each experimental arm consisted of 20 mice. (B) Lung histopathology. Top row, Gomori's methenamine silver staining demonstrated extensive hyphal proliferation in the lung tissue of mice infected with the wild-type or crzA complemented strain, while mice infected with the ΔcrzA strain displayed a limited amount of hyphal proliferation. Magnification, ×400. Bottom row, hematoxylin and eosin staining shows significant necrosis and inflammation in mice infected with the wild-type and crzA complemented strains compared with mice infected with the ΔcrzA strain. Magnification, ×400.