Differential effects of inhibiting chitin and 1,3-{beta}-D-glucan synthesis in ras and calcineurin mutants of Aspergillus fumigatus

Abstract

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-beta-d-glucan content. Echinocandin treatment further decreased the levels of 1,3-beta-d-glucan for all strains tested yet also partially corrected the hyphal growth defect of the DeltarasA strain. The inhibition of glucan synthesis caused an increase in chitin content for wild-type, dominant-active rasA, and DeltarasA strains. However, this important compensatory response was diminished in the calcineurin pathway mutants (DeltacnaA and DeltacrzA). 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.

FIG. 1.

Cell wall inhibition, coupled with the loss of Ras or calcineurin signaling, causes morphological aberrancies. Shown is the no treatment (NT) and the nikkomycin Z (NZ), caspofungin (CA), FK506, and nikkomycin Z plus caspofungin (NZ + CA) treatment of the wild-type (Af293 and H237), ΔcnaA, and ΔrasA strains in RPMI 1640 medium at 37°C. Conidia from each strain were incubated in the presence or absence of drug for 48 h, followed by microscopic examination. Arrowheads denote hyphal tips that have swollen or lysed due to caspofungin treatment. Representative fields are demonstrated. Scale bar, 40 μm.

FIG. 2.

Loss of RasA, CnaA, or CrzA signaling causes decreased baseline levels of 1,3-β-d-glucan. Analysis of 1,3-β-d-glucan levels of the ΔcnaA and ΔcrzA (A) and DArasA and ΔrasA (B) strains and those of their respective parental wild-type strains. All analyses were performed in biological triplicate, and results are normalized to values for the control strain (untreated wild type). Results are presented as the means ± standard deviations. NT, no treatment.

FIG. 3.

CnaA and CrzA, but not RasA, contribute to the compensatory response of chitin to 1,3-β-d-glucan inhibition. Analysis of chitin content of the ΔcnaA and ΔcrzA (A) and DArasA and ΔrasA (B) strains and that of their respective parental wild-type strains. A total of 10⁶ conidia/ml were inoculated into RPMI 1640 medium and grown in the presence or absence of 1 μg/ml of nikkomycin Z (NZ), caspofungin (CA), or a combination of both drugs (NZ + CA) for 48 h at 37°C and 250 rpm. (C) Analysis of the chitin content of the wild-type and ΔcnaA strains in the presence of anidulafungin (1 μg/ml) or micafungin (1 μg/ml). All analyses were performed in biological triplicate. Results and error bars indicate the means ± standard deviations. NT, no treatment.

FIG. 4.

Caspofungin treatment increases calcofluor white staining of wild-type A. fumigatus. A total of 10³ conidia/ml of the Af293, ΔcnaA, or ΔcrzA strains were inoculated onto coverslips immersed in RPMI 1640 medium and grown in the presence or absence of caspofungin (1 μg/ml) or nikkomycin Z (1 μg/ml) for 16 h at 37°C. Coverslips were removed and stained with calcofluor white. BF, brightfield; UV, ultraviolet; NT, no treatment. Scale bar, 20 μm.

FIG. 5.

Response of chitin biosynthesis to high-level nikkomycin Z treatment in A. fumigatus. Analysis of the chitin content of the ΔcnaA and ΔcrzA (A) and DArasA and ΔrasA (B) strains and that of their respective parental wild-type strains when treated with ascending doses of nikkomycin Z. A total of 10⁶ conidia/ml were inoculated into RPMI 1640 medium and grown in the presence or absence of the indicated amount of nikkomycin Z for 48 h at 37°C and 250 rpm. All analyses were performed in biological triplicate. Results and error bars indicate the means ± standard deviations.