The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus

Abstract

To increase the frequency of homologous recombination, we inactivated the KU80 homologue in Aspergillus fumigatus (named akuB(KU80)). Homologous integration reached about 80% for both calcineurin A (calA) and polyketide synthase pksP (alb1) genes in the akuB(KU80) mutant to 3 and 5%, respectively, when using a wild-type A. fumigatus strain. Deletion of akuB(KU80) had no influence on pathogenicity in a low-dose murine infection model.

FIG. 1.

ΔakuB^KU80 strain displays increased sensitivity to MMS. (A) Growth phenotype of the A. fumigatus wild-type and KU80ΔpyrG strains grown for 48 h at 37°C in YUU medium in the presence or absence of different concentrations of MMS. (B) Southern blot analysis of the KU80ΔpyrG strain. At the left panel, genomic DNA from the wild-type and KU80ΔpyrG strains was isolated and digested with EcoRI and the entire akuB^KU80 open reading frame was used as a hybridization probe. The akuB^KU80 gene probe recognized a single band of approximately 14.0 kb only in the wild-type strain, indicating the akuB^KU80 gene was deleted in the KU80ΔpyrG strain. In the right panel, genomic DNA from the wild-type and KU80ΔpyrG strains was isolated and cleaved with the enzyme KpnI and the pyrG gene was used as a hybridization probe. The pyrG gene recognized one band in the wild-type strain (approximately 4.0 kb) and two bands in the akuB^KU80 deletion strain (around 4.0 and 9.0 kb). This additional band in the KU80ΔpyrG strain indicates a single event of integration of the transforming deletion cassette.

FIG. 2.

Calcineurin A deletion mutant (ΔcalA) showed increased branching and a phenotype of reduced growth. (A) Southern blot analysis of the ΔcalA strain. Genomic DNA from wild-type and four ΔcalA strains (T3, T9, T10, and T11) was isolated and digested with SfiI and the entire calA open reading frame was used as a hybridization probe. The calA gene probe recognized a single band of approximately 15.0 kb only in the wild-type strain, indicating the calA gene was deleted in all four transformants. (B) Growth phenotype of the A. fumigatus wild-type (left panel) and ΔcalA (right panel) strains grown for 48 h at 30, 37, and 44°C in YUU medium. (C) Conidia of the wild-type and ΔcalA strains were inoculated onto coverslips in YG+UU medium. After 10 h incubation at 37°C, coverslips with adherent germlings were transferred to fixative solution (3.7% formaldehyde, 50 mM sodium phosphate buffer, pH 7.0, 0.2% Triton X-100) for 30 min at room temperature. Then, they were briefly rinsed with PBS buffer (140 mM NaCl, 2 mM KCl, 10 mM NaHPO₄, 1.8 mM KH₂PO₄, pH 7.4) and incubated for 5 min in a solution with 100 ng/ml of DAPI (4′,6′-diamino-2-phenylindole, Sigma Chemical Co.). After incubation with the dyes, they were washed with phosphate-buffered saline for 5 min at room temperature and then rinsed in distilled water and mounted on the slides. The material was photographed using a Zeiss epifluorescence microscope. The number of nuclei was assessed by DAPI staining. Graph bars indicate 10 μm.

FIG. 3.

Virulence of A. fumigatus KU80Δ mutant strain. Survival of mice infected intranasally with conidia of A. fumigatus CEA17pyrG+ and KU80Δ was determined. As shown by d'Enfert et al. (3) uracil auxotrophic A. fumigatus strains show attenuated virulence. Therefore, the uracil prototroph strain CEA17pyrG+ was isolated from strain CEA17 by spontaneous reversion of the point mutation in the pyrG locus. CEA17pyrG+ shows the same virulence in comparison to the A. fumigatus wild-type strain ATCC 46645 (data not shown). For the same reason the uracil prototrophic strain KU80Δ was used instead of KU80ΔpyrG. In brief, female BALB/c mice (Harlan Winkelmann, Borchen, Germany) of 18 to 20 g body weight were immunosuppressed by intraperitoneal injection of cyclophosphamide (100 mg/ml; Sigma, Taufkirchen, Germany) on days −4, −1, 2, 5, 8, 11, and 14 prior to and after infection on day 0. A single dose of cortisone acetate (200 mg/kg of body weight; Sigma) was injected subcutaneously on day −1. Suspensions of A. fumigatus conidia were harvested with phosphate-buffered saline containing 0.1% (vol/vol) Tween 80 (Merck) and filtered through a 40-μm nylon cell strainer (BD Biosciences Europe, Belgium). Mice were anesthetized by intraperitoneal injection of 200 μl of 1% (vol/vol) ketamine (Velonarcon, Berlin Chemie, Germany) and 0.02% (vol/vol) xylazine (Rompun, Bayer Leverkusen, Germany) and intranasally infected with a 25-μl drop of a fresh suspension containing 2 × 10⁴ conidia. Survival was monitored daily, and moribund animals were sacrificed by intraperitoneal injection of 200 μl of 3.2% (vol/vol) narcoren (Rhone Merieux, Laupheim, Germany). The drinking water was supplemented with 0.5 mg of tetracycline (Sigma) per ml to prevent opportunistic bacterial infections. Mice were tested in cohorts of 10 animals. A control group (inhalation of phosphate-buffered saline [PBS]) remained uninfected to monitor the influence of the immunosuppression procedure on vitality. Survival was monitored for 16 days. Data are representative of several independent experiments.