Bernhard Jahn

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease and resulting in high mortality. In order to determine the importance of the cAMP signaling pathway for virulence, three genes encoding putative elements of the pathway have been cloned and characterized: the adenylate cyclase gene acyA, and gpaA and gpaB, both of which encode α subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the ΔacyA mutant producing very few conidia. The growth rate of the ΔacyA mutant was also reduced, in contrast to that of the ΔgpaB mutant. Addition of 10 mM dibutyryl-cAMP to the culture medium completely restored the wild-type phenotype in both mutant strains. To study the influence of GPAB on the expression of the gene pksP, which encodes a virulence factor that is involved in pathogenicity, a pksPp- lacZ gene fusion was generated and integrated as a single copy at the pyrG gene locus of both the parental strain and the ΔgpaB mutant strain. The ΔgpaB mutant showed reduced expression of the pksPp-lacZ reporter gene relative to that in the parental strain. In mycelia of both the parental strain and the ΔgpaB mutant pksPp-lacZ expression was increased when isobutyl-methyl-xanthine, an inhibitor of intracellular phosphodiesterases, was added to the medium. The survival rate of conidia after ingestion by human monocyte-derived macrophages was also determined. The killing rate for conidia from ΔacyA and ΔgpaB strains was significantly higher than that for wild-type conidia. Taken together, these findings suggest that cAMP triggers a system that protects A. fumigatus from the effects of immune effector cells of the host.

Previously, we described the isolation of an Aspergillus fumigatus mutant producing non-pigmented conidia, as a result of a defective polyketide synthase gene, pksP (polyketide synthase involved in p...

An immunocompromised child developed necrotizing pneumonia with BAL cultures growing Legionella pneumophila resistant to treatment, including erythromycin and rifampicin. Ciprofloxacin and clarithromycin reversed the clinical course; their use as first-line drugs is justifiable and a high index of suspicion for the occurrence of legionellosis is warranted.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. To be able to analyze the expression of putative virulence-associated genes of A. fumigatus, the use of the enhanced green fluorescent protein (EGFP) as a reporter was established. Two 5′ sequences, containing the putative promoters of thepyrG gene, encoding orotidine-5′-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence ofA. fumigatus, were fused with the egfpgene. The PpksP-egfp construct was integrated via homologous recombination into the genomicpksP locus. EGFP production was analyzed by fluorescence spectrometry, Western blot analysis, and fluorescence microscopy. Differential gene expression in A. fumigatus was observed. Fluorescence derived from the PYRG-EGFP fusion protein was detected during all developmental stages of the fungus, i.e., during germination, during vegetative growth, in conidiophores, and weakly in conidia. In addition, it was also detected in germinating conidia when isolated from the lungs of immunocompromised mice. By contrast, PKSP-EGFP-derived fluorescence was not found in hyphae or stalks of conidiophores but was found in phialides and conidia in vitro when the fungus was grown under standard conditions, indicating a developmentally controlled expression of the gene. Interestingly,pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice. This finding indicates that thepksP gene can also be expressed in hyphae under certain conditions and, furthermore, that the pksP gene might also contribute to invasive growth of the fungus.

A defect in the pksP gene of Aspergillus fumigatus is associated with the loss of conidial pigmentation, a profound change of the conidial surface structure, and reduced virulence. The structural change of the conidial surface structure was not observed in similar A. nidulans wA mutants. Our data indicate that the pigment of both species is important for scavenging reactive oxygen species and for protection of conidia against oxidative damage.

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here, we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47–73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model.

A cytofluorometric assay that allowed assessment of damage to phagocytosed Aspergillus fumigatus conidia at the single-cell level was developed. After ingestion by monocyte-derived macrophages (MDMs), conidia were reisolated by treatment of the cells with streptolysin O, a pore-forming toxin with lytic properties on mammalian cells but not on fungi. The counts obtained by staining of damaged conidia with propidium iodide and quantification by cytofluorometry correlated with colony counts. By the use of this method, we demonstrate that MDMs differentiated in vitro by low-dose granulocyte-macrophage colony-stimulating factor and gamma interferon have only a limited capacity to damageAspergillus conidia in vitro. The killing rate 12 h after phagocytosis was found to be only 10 to 15%. However, intracellular loading of the phagocytes with amphotericin B (AmB) dose dependently enhanced the anticonidial activity. Preincubation of macrophages with only 1 μg of AmB per ml resulted in an uptake of 18 fg of AmB/cell, leading to killing rates of 50 to 60%. The experimental protocol provides a new tool for the rapid quantification of anticonidial activity against A. fumigatus in vitro. Intracellular accumulation of AmB may represent an important factor underlying the efficacy of this antifungal drug in the prophylaxis and treatment ofAspergillus infections.