The cyclic AMP-dependent protein kinase a network regulates development and virulence in Aspergillus fumigatus

Abstract

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To determine the importance of the cyclic AMP (cAMP) signaling pathway for virulence, the pkaC1 gene encoding a protein kinase A (PKA) catalytic subunit was cloned and characterized. Deletion of pkaC1 led to reduced conidiation and growth. PKA activity was not detectable in DeltapkaC1, DeltagpaB, and DeltaacyA mutant strains. gpaB and acyA encode a G protein alpha subunit involved in cAMP signal transduction and adenylate cyclase, respectively. Addition of cAMP led to PKA activity in crude extracts of both the DeltagpaB and DeltaacyA strains but not in crude extracts of the DeltapkaC1 strain. These findings provide evidence that PKAC1 represents the predominant form of PKA under the conditions tested, and GPAB and ACYA are members of the cAMP signaling cascade. Analysis of a pksPp-lacZ gene fusion indicated that the expression of the pathogenicity determinant-encoding pksP gene was reduced in DeltapkaC1 mutant strains compared with the expression of the gene fusion in the parental strain. In a low-dose murine inhalation model, conidia of both the DeltapkaC1 and DeltagpaB mutant strains were almost avirulent. Taken together, these findings indicate that the cAMP-PKA signal transduction pathway is required for A. fumigatus pathogenicity.

FIG. 1.

Deletion of A. fumigatus PKA catalytic subunit-encoding gene pkaC1. (A) Schematic map of the pkaC1 knockout plasmid pUCpkaC1hph1. Abbreviations: amp^R, ampicillin resistance gene; hyg^R, hygromycin B phosphotransferase gene hph used as the selection marker gene in A. fumigatus. (B) Schematic map of the pkaC1 knockout plasmid pUCpkaC1Ura1. Abbreviations: amp^R, ampicillin resistance gene; pyrG, orotidine 5′-monophosphate decarboxylase gene of A. niger used as the selection marker gene; neo, neomycin phosphotransferase genes. (C) Southern blot analysis of the pkaC1 deletion strains. Chromosomal DNA of the parental strains ATCC 46645 (lane 1) and CEA17pksP-lacZ (lane 3), as well as mutant strains ΔpkaC1 (lane 2), CEA17ΔpkaC1 (lane 4), and CEA17ΔpkaC1pksP-lacZ1 (lane 5), was cut by BamHI. An 830-bp pkaC1-derived PCR fragment was used as the probe. In the ΔpkaC1, CEA17ΔpkaC1, and CEA17ΔpkaC1pksP-lacZ1 mutant strains, the band characteristic of the wild type (lanes 1 and 3) had disappeared. Instead, the bands characteristic of gene replacement at the pkaC1 locus were detected. The difference in the sizes of the bands in lane 2 (ΔpkaC1) and lane 4 (CEA17ΔpkaC1) is due to the different cassettes, hph and Ura-blaster, used to delete the pkaC1 gene. Strain CEA17ΔpkaC1pksP-lacZ1 (lane 5) resulted from a forced recombination in which the Ura-blaster was lost during selection on medium containing 5-FOA. Consequently, a copy of the neo gene remained at the pkaC1 gene locus, which led to a pkaC1 knockout band that migrated more quickly than the band of strain CEA17ΔpkaC1 (lane 4). (D) Schematic representation of the chromosomal pkaC1 locus of the wild type and the ΔpkaC1 deletion mutants. Restriction endonuclease cleavage sites and the position to which the probe hybridizes are indicated. The pkaC1 genes in mutants ΔpkaC1 and CEA17ΔpkaC1 lack the pkaC1 regions encoding amino acids 252 to 502 and 336 to 502, respectively, as well as 58 bp of the 3′ untranslated region.

FIG. 2.

Phenotypic characterization of mutant strains ΔpkaC1 and CEA17ΔpkaC1 and parental strains ATCC 46645 and CEA17pksP-lacZ. (A) Growth and sporulation on AMM agar plates. Colonies were grown for 72 h at 37°C. (B and C) Growth of A. fumigatus strains on AMM agar plates at 37°C up to 94 h (B) and on malt extract agar plates at 37°C up to 72 h (C). Colony diameters were measured. The data for each strain represent the means for at least 10 independently grown colonies. The standard deviations were in the range from 0.2 to 0.8 mm. (D) Microscopic photographs (magnification, ×200) of A. fumigatus strains grown on AMM agar at 37°C for 24 h. (E) Kinetics of germ tube outgrowth for A. fumigatus conidia incubated in AMM at 37°C. The numbers of conidia showing a germ tube were determined after different times of incubation in at least two microscopic fields. The percentage of germinated conidia (based on the total number of conidia) is shown. The results are representative of the results of two independent experiments.

FIG. 3.

PKA activities of mutant strains ΔpkaC1, CEA17ΔpkaC1, ΔacyA, and CEA17ΔgpaB and parental strains ATCC 46645 and CEA17pksP-lacZ. (A) Enzyme activity as monitored by gel electrophoresis. A phosphorylated substrate migrated toward the anode. (B) Spots shown in panel A quantified by spectrophotometry. Abbreviations: PKI, PKA inhibitor peptide; WT, wild type.

FIG. 4.

Expression of the pksPp-lacZ gene fusion in A. fumigatus ΔpkaC1 and ΔgpaB deletion strains carrying a pksPp-lacZ gene fusion integrated in a single copy at the chromosomal pyrG locus. (A) Southern blot analysis. Chromosomal DNA of the A. fumigatus parental strains ATCC 46645 (lane 1) and CEA17pksP-lacZ (lane 2) and the ΔpkaC1 mutants CEA17ΔpkaC1pksP-lacZ1 (lane 3) and CEA17ΔpkaC1pksP-lacZ2 (lane 4) was digested by BglII. A 450-bp PCR fragment encoding part of the A. fumigatus pyrG gene was used as the probe. Molecular analysis of strain CEA17ΔgpaBpksP-lacZ is shown in Fig. 1. (B) β-Galactosidase specific activities of strains grown in AMM at 37°C for 24 h. The data for each strain and condition are the mean and standard deviation for three independently grown cultures. Abbreviations: WT, parental strain CEA17pksP-lacZ; ΔpkaC1-1, CEA17ΔpkaC1pksP-lacZ1; ΔpkaC1-2, CEA17ΔpkaC1pksP-lacZ2; ΔgpaB, CEA17ΔgpaBpksP-lacZ.

FIG. 5.

Virulence of A. fumigatus wild-type strain ATCC 46645, as well as ΔgpaB (A) and ΔpkaC1 (B) deletion strains, in mice. The members of groups of 10 BALB/c mice were each infected with 5 × 10³ or 10⁴ A. fumigatus conidia, as indicated, by nasal inhalation. Survival was monitored for 15 days.