The Aspergillus fumigatus P-type Golgi apparatus Ca2+/Mn2+ ATPase PmrA is involved in cation homeostasis and cell wall integrity but is not essential for pathogenesis

Abstract

The Aspergillus fumigatus DeltapmrA (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 beta-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.

Fig. 1.

Construction of the ΔpmrA strain and phenotypic analysis. (A) Schematic representation of the genomic locus of the WT and ΔpmrA strains. The entire coding sequence of the A. fumigatus pmrA gene was replaced with that of the A. parasiticus pyrG gene by homologous recombination. Southern analysis with SacII-digested genomic DNA and the pmrA left flank probe shows the replacement of pmrA with pyrG as a 7.1-kb fragment in the ΔpmrA strain. (B) Culture morphology of the WT and ΔpmrA strains on glucose minimal medium agar (GMM) after 5 days of growth at 37°C. (C) Radial growth of the ΔpmrA strain under different stress conditions, compared to that of the WT strain. A total of 1 × 10⁴ conidia were inoculated onto GMM plates containing different salt concentrations. In addition, growth analysis was also performed on alkaline (pH 9.0) and acidic (pH 4.0) media. Three independent experiments were performed, and the results shown are the means and standard deviations obtained after 4 days of growth at 37°C. (D) RT-PCR expression analysis in response to salt stress. The expression levels of calcineurin catalytic subunit A (cnaA), a calcipressin (cbpA), a Na⁺ ATPase (enaA), and two zinc finger transcription factors (crzA and sltA) were analyzed after 20 min of exposure to 0.8 M NaCl. Three independent experiments were performed, and the results shown are the means and standard deviations.

Fig. 2.

(A) Effect of cell wall inhibitors on the WT and ΔpmrA strains. A total of 10³ conidia/ml of the WT and ΔpmrA strains were inoculated onto coverslips immersed in GMM medium and grown in the presence or absence of the indicated concentrations of nikkomycin Z (NZ) or caspofungin (CA) for 16 to 20 h at 37°C. Coverslips were removed and observed by microscopy. Scale bar, 20 μm. (B) β-1,3-Glucan content was quantified using the aniline blue assay as previously described (8, 25, 26), using curdlan, a β-1,3-glucan analog, as the standard. Values are expressed as relative fluorescence units (RFU) per mg of mycelial tissue. (C) Quantification of chitin was performed based on a modified protocol (8) and reported as glucosamine equivalents (μg/ml).

Fig. 3.

Calcofluor white staining of the WT and ΔpmrA strains. A total of 10³ conidia/ml of the WT and ΔpmrA strains were inoculated onto coverslips immersed in GMM medium and grown for 24 h at 37°C. The coverslips were rinsed in sterile water and inverted onto a 200-μl drop of calcofluor white staining solution for 5 min at room temperature. Coverslips were then rinsed twice for 10 min in sterile water and mounted in Cytoseal 60 mounting medium. To ensure that the results of the calcofluor white staining of different samples were appropriately compared, the ultraviolet light exposure time for each sample was set at 25 ms for the WT untreated control, and the strains were exposed for the same lengths of time. Scale bar, 50 μm.

Fig. 4.

Virulence analysis in a murine inhalational model of invasive aspergillosis. (A) Kaplan-Meier survival curve. At day 14 postinfection, mice infected with the ΔpmrA strain displayed about 70% mortality, which is comparable to that of mice infected with the WT strain (P = 0.1774). (B) Lung histopathology at day 7 postinfection at ×400 magnification. (Top) Gomori's methenamine silver staining demonstrated comparable extensive hyphal proliferation patterns in the lung tissue of mice infected with the WT and ΔpmrA strains. (Bottom) Hematoxylin and eosin staining shows necrosis and inflammation in the tested strains.