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 ⌬pkaC1, ⌬gpaB, and ⌬acyA mutant strains. gpaB and acyA encode a G protein ␣ subunit involved in cAMP signal transduction and adenylate cyclase, respectively. Addition of cAMP led to PKA activity in crude extracts of both the ⌬gpaB and ⌬acyA strains but not in crude extracts of the ⌬pkaC1 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 ⌬pkaC1 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 ⌬pkaC1 and ⌬gpaB mutant strains were almost avirulent. Taken together, these findings indicate that the cAMP-PKA signal transduction pathway is required for A. fumigatus pathogenicity.Aspergillus fumigatus has become the most important airborne fungal pathogen of humans. Alveolar macrophages form the first line of defense against fungal conidia, the infectious agents, entering the respiratory tract. Neutrophilic granulocytes, the second line of defense, primarily attack hyphae but also attack conidia. An important killing mechanism for both types of immune effector cells consists of the production of reactive oxygen species (ROS) (22,31,42). Improvement in transplant medicine and the therapy of hematological malignancies is often complicated by the threat of invasive aspergillosis. A. fumigatus accounts for approximately 90% of invasive aspergillosis cases. Specific diagnostics are still limited, as are the possibilities of therapeutic intervention, which leads to a high mortality rate (30 to 98%) for invasive aspergillosis (reviewed in references 9 and 31).One of the important questions concerning A. fumigatus is the identification of pathogenicity determinants and their regulation. Recently, the group of J. Kwon-Chung and our group identified a gene that encodes a pathogenicity determinant. This gene was designated pksP (or alternatively alb1) for polyketide synthase involved in pigment biosynthesis (23,24,25,28,54). Conidia of a pksP mutant strain are white. Based on genetic and biochemical data, the conidial pigment consists of dihydroxynaphthalene (DHN)-melanin (7,30,52,53). pksP mutant strains exhibited reduced virulence in a mouse infection model (23,54). pksP mutant conidia were 20-fold more sensitive to ROS than wild-type conidia, suggesting that the pigment is able to scavenge ROS, thereby presumably detoxifying ROS (23,24). Moreover, a key eleme...

Analysis of Promoter Function in Aspergillus fumigatus

“…Development of reporter gene systems was extremely valuable in the progression of the analysis of promoter function in S. cerevisiae (16) as well as Candida albicans (54). Previous studies in A. fumigatus have described the use of lacZ-based reporter systems (4,27,28,50,56) in addition to expression of firefly luciferase as an indicator of the presence of A. fumigatus in infection models (4,5,11,13). Here we detail the generation of both integrating and autonomously replicating luciferase reporter vectors that will allow analysis and dissection of promoter regions from this fungus.…”

The Aspergillus fumigatus Transcriptional Regulator AfYap1 Represents the Major Regulator for Defense against Reactive Oxygen Intermediates but Is Dispensable for Pathogenicity in an Intranasal Mouse Infection Model

“…As the selectable marker, the Hyg r gene was used. A. fumigatus strains were cultivated at 37°C in Aspergillus minimal medium (AMM) as previously described (37). As solid medium, malt extract (2% [wt/vol] malt extract, 0.2% [wt/vol] yeast extract, 1% [wt/vol] glucose, 5 mM ammonium chloride, 1 mM dipotassium hydrogen phosphate) or AMM containing 3% (wt/vol) agar was used.…”

Regulatory Circuitry Governing Fungal Development, Drug Resistance, and Disease

“…The deletion of numerous genes that regulate germination results in morphogenetic defects as well as decreased virulence. For instance, the deletion of key components of the cAMP-PKA pathway results in altered germination as well as decreased virulence in murine models (330,649). Furthermore, in the G. mellonella model of A. fumigatus infection, conidial germination is tightly coupled with virulence (485).…”