Disruption of the Aspergillus fumigatus gene encoding nucleolar protein CgrA impairs thermotolerant growth and reduces virulence

Abstract

Aspergillus fumigatus CgrA is the ortholog of a yeast nucleolar protein that functions in ribosome synthesis. To determine how CgrA contributes to the virulence of A. fumigatus, a Delta cgrA mutant was constructed by targeted gene disruption, and the mutant was reconstituted to wild type by homologous introduction of a functional cgrA gene. The Delta cgrA mutant had the same growth rate as the wild type at room temperature. However, when the cultures were incubated at 37 degrees C, a condition that increased the growth rate of the wild-type and reconstituted strains approximately threefold, the Delta cgrA mutant was unable to increase its growth rate. The absence of cgrA function caused a delay in both the onset and rate of germination at 37 degrees C but had little effect on germination at room temperature. The Delta cgrA mutant was significantly less virulent than the wild-type or reconstituted strain in immunosuppressed mice and was associated with smaller fungal colonies in lung tissue. However, this difference was less pronounced in a Drosophila infection model at 25 degrees C, which correlated with the comparable growth rates of the two strains at this temperature. To determine the intracellular localization of CgrA, the protein was tagged at the C terminus with green fluorescent protein, and costaining with propidium iodide revealed a predominantly nucleolar localization of the fusion protein in living hyphae. Together, these findings establish the intracellular localization of CgrA in A. fumigatus and demonstrate that cgrA is required for thermotolerant growth and wild-type virulence of the organism.

FIG. 1.

Disruption and reconstitution of the cgrA gene in A. fumigatus. (A) Schematic representation of the predicted ΔcgrA mutation (downward arrow) resulting from homologous recombination (×) between the plasmid containing a disrupted cgrA allele (p413) and the wt cgrA locus. The solid boxes represent exons of the cgrA gene, and the horizontal arrows indicate the transcriptional orientations of phleo and cgrA. The restriction sites are KpnI (K), SstI (S), Bsu36I (B), and HincII (H). (B) Schematic representation of the predicted reconstitution of cgrA (r-wt) in the ΔcgrA mutant, resulting from a single crossover between plasmid p393 (linearized at the HincII site) and the ΔcgrA mutant allele. (C) Southern blot analysis of SstI-digested genomic DNA from the wt, ΔcgrA, and r-wt strains using the 2.3-kb SstI fragment containing the cgrA gene as a probe.

FIG. 2.

Impaired radial-growth rate of the ΔcgrA mutant. (A) Colony morphologies of the wt, ΔcgrA, and r-wt strains after 7 days of growth at room temperature (22°C) or 3 days of growth at 37°C. (B and C) Conidia from the wt, ΔcgrA, and r-wt strains were spotted into the center of a plate of Aspergillus minimal medium, and the change in the diameter of the colony with time was measured at 22 (B) and 37°C (C). The first measurement was taken on day 1, 24 h after inoculation. The experiment was performed in triplicate, and the values shown are averages ± standard errors of the mean.

FIG. 3.

(A) Comparison of radial growth rates at 22 to 48°C. The radial growth rates of the wt, r-wt, and ΔcgrA strains on minimal medium are shown for the indicated temperatures. The experiment was performed in triplicate, and the values shown are averages ± standard errors of the mean. (B) Hygromycin sensitivity of the ΔcgrA mutant. Conidia from the wt, r-wt, and ΔcgrA mutant were spotted onto a yeast-peptone-dextrose plate in the absence (−H) or presence (+H) of 62.5 μg of hygromycin/ml and incubated at 37°C for 8 days.

FIG. 4.

Impaired germination of the ΔcgrA mutant. The percentages of swollen conidia that had initiated a germ tube after the indicated periods of growth in liquid cultures in YG medium are shown for the wt, r-wt, and ΔcgrA strains at 22 and 37°C. The experiment was performed in triplicate, and the values shown are averages ± standard errors of the mean.

FIG. 5.

Subcellular localization of CgrA. (A and B) wt A. fumigatus expressing GFP alone is shown in a differential interference contrast image (A) and a corresponding fluorescent image (B). (C to F) Hypha from a wt strain expressing the CgrA-GFP fusion protein and counterstained with the DNA-specific dye PI. (C) Differential interference contrast image. (D) GFP fluorescence. (E) PI fluorescence. (F) Overlay of the PI and GFP fluorescence showing GFP localization to the PI-excluded nucleolar region.

FIG. 6.

Complementation of the thermosensitive ΔcgrA phenotype by a cgrA-gfp fusion gene. The radial growth of the wt, ΔcgrA and the ΔcgrA strain expressing cgrA-gfp from an ectopically integrated transgene are compared at 22 (A) and 37°C (B). The growth rate of the wt strain expressing the cgrA-gfp fusion gene was indistinguishable from that of the wt (data not shown).

FIG. 7.

Virulence of wt and ΔcgrA conidia in two infection models. (A) Groups of 20 to 22 immunosuppressed mice were infected with 10⁵ conidia and monitored for 2 weeks. (B) Groups of 40 to 70 adult spz flies were inoculated with 1,500 conidia and monitored for 120 h at 25°C. P is <0.05 for both experiments.

FIG. 8.

Histologic analysis of lung tissue from infected mice. At 2 and 4 days following inoculation, lung sections from mice infected with wt A. fumigatus or the ΔcgrA mutant were stained with methenamine silver nitrate to reveal the morphologies of fungal colonies. Only sporadic germlings could be identified in lungs from mice infected with the ΔcgrA mutant on day 2 (arrows).