No abstract

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.

Aspergillus fumigatus is an important pathogen of immunocompromised hosts, causing pneumonia and invasive disseminated disease with high mortality. The factors contributing to the predominance of A. fumigatus as an opportunistic pathogen are largely unknown. Since the survival of conidia in the host is a prerequisite for establishing disease, we have been attempting to identify factors which are associated with conidia and, simultaneously, important for infection. Therefore, an A. fumigatus mutant strain (white [W]) lacking conidial pigmentation was isolated. Scanning electron microscopy revealed that conidia of the W mutant also differed in their surface morphology from those of the wild type (WT). Mutant (W) and WT conidia were compared with respect to their capacities to stimulate an oxidative response in human phagocytes, their intracellular survival in human monocytes, and virulence in a murine animal model. Luminol-dependent chemiluminescence was 10-fold higher when human neutrophils or monocytes were challenged with W conidia compared with WT conidia. Furthermore, mutant conidia were more susceptible to killing by oxidants in vitro and were more efficiently damaged by human monocytes in vitro than WT conidia. In a murine animal model, the W mutant strain showed reduced virulence compared with the WT. A reversion analysis of the W mutant demonstrated that all phenotypes associated with the W mutant, i.e., altered conidial surface, amount of reactive oxygen species release, susceptibility to hydrogen peroxide, and reduced virulence in an murine animal model, coreverted in revertants which had regained the ability to produce green spores. This finding strongly suggests that the A. fumigatus mutant described here carries a single mutation which caused all of the observed phenotypes. Our results suggest that the conidium pigment or a structural feature related to it contributes to fungal resistance against host defense mechanisms in A. fumigatus infections.

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 alpha subunits of heterotrimeric G proteins. The acyA and gpaB genes were each deleted in A. fumigatus. Both mutants showed reduced conidiation, with the deltaacyA mutant producing very few conidia. The growth rate of the deltaacyA mutant was also reduced, in contrast to that of the deltagpaB 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 deltagpaB mutant strain. The deltagpaB 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 deltagpaB 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 deltaacyA and deltagpaB 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.

SummaryPreviously, 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 pigment biosynthesis). The virulence of the pksP mutant was attenuated in a murine animal infection model and its conidia showed enhanced susceptibility towards damage by monocytes in vitro. Because macrophagemediated killing is critical for host resistance to aspergillosis, the interaction of both grey-green wildtype conidia and white pksP mutant conidia with human monocyte-derived macrophages (MDM) was studied with respect to intracellular processing of ingested conidia. After phagocytosis, the percentage of wild-type conidia residing in an acidic environment was approximately fivefold lower than that observed for non-pigmented pksP mutant conidia. The phagolysosome formation, as assessed by co-localization of LAMP-1 and cathepsin D with ingested conidia, was significantly lower for wild-type conidia compared with pksP mutant conidia. Furthermore, the intracellular kill of pksP mutant conidia was significantly higher than of wild-type conidia, which was markedly increased by chloroquine, a known enhancer of phagolysosome fusion. Taken together, these findings suggest that the presence of a functional pksP gene in A. fumigatus conidia is associated with an inhibition of phagolysosome fusion in human MDM. These data show for the first time that a fungus has the capability to inhibit the fusion of the phagosome with the lysosome. This finding might help explain the attenuated virulence of pksP mutant strains in a murine animal model and provides a conceptual frame to understand the virulence of A. fumigatus .

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 spp. are the predominant causative agents of invasive pulmonary aspergillosis (IPA), an often lethal infection of the immunocompromised host (4,10,13,16). Since conidia are the infectious agent in IPA, recent studies focused on the elucidation of conidial factors contributing to pathogenicity (8,17). Previously, we and others have shown that conidia lacking pigmentation due to the defective polyketide synthase gene pksP were less resistant to the attack by monocytes in vitro and showed reduced virulence in a murine animal model (8,9,17,18). During these studies, it became apparent that coincubation of human phagocytes with pksP mutant conidia resulted in a marked increase in the release of reactive oxygen species (ROS) compared with wild-type (wt) conidia (8). Since a defective pksP gene not only impaired conidial pigmentation but concomitantly resulted in profound alterations of the conidial surface (8, 9), the question arose as to whether the large amounts of ROS detected after incubation of phagocytes with pksP mutant conidia were due to a change in the activation pattern of the cells or, alternatively, reflected the lack of ROS quenching capacity caused by the loss of conidial pigment. To address this question, conidia of wt strains of both Aspergillus fumigatus and the nonpathogenic fungus Aspergillus nidulans were compared with their respective pigmentless mutant strains.The WA mutant of A. nidulans (strain WG370; wA3 bgaO biA1) lacking the conidial pigment due to a defective polyketide synthase gene (wA) was constructed by a sexual cross of appropriate parental strains (12) using standard genetic techniques (14). The wA gene product might have a function similar to that of the pksP gene product of A. fumigatus, although major differences between the pigment biosyntheses of the two Aspergillus species exist (2, 18; this study).As previously reported, conidia of the A. fumigatus wt strain showed a rough surface; i.e., they had an ornamentation which was lacking in the pksP mutant strain (8, 9) ( Fig. 1A and B). The A. nidulans wt conidia showed a similar ornamentation (Fig. 1C). In contrast to the pigmentless pksP mutant strain of A. fumigatus, however, similar pigmentless conidia of A. nidulans (wA) still exhibited the ornamentation characteristic of wild-type conidia (Fig. 1D). Taken together, the difference in surface structure between the A. fumigatus pksP mutant and the A. nidulans wA mutant further supports the assumption that different pathways exist for either conidial pigment biosynthesis or pigment deposition in the two species (1). As was noted p...

We describe a simple microtiter method for determining the susceptibility of Candida albicans and hyphal forms of Aspergillus fumigatus against antifungal agents. The assay measures mitochondrial respiration by determining reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) to formazan, a process that is enhanced in the presence of menadione. C. albicans or conidial suspensions of A. fumigatus are seeded into microtiter plates. Hyphal outgrowth of Aspergillus spp. was achieved by a 12 to 14-h culture at 30؇C. Antifungal agents (amphotericin B, fluconazole, itraconazole) were added to the cultures for 24 h. Thereafter, incubations were continued for 3 h in the presence of MTT plus 0.1 mM menadione. Formazan formation was quantified photometrically after extraction of the formazan with acid isopropanol. Well-defined dose-response curves reflecting impairment of mitochondrial function by the antifungal agents were obtained. With C. albicans, the results correlated excellently with the MIC determinations performed according to the standard macrodilution procedure. In confirmation of a recent report, it was found that fluconazole was unable to exert its fungistatic action on a sensitive C. albicans strain in the presence of serum. The presented method can easily be integrated in the standard repertoire of a diagnostic microbiology laboratory and should prove useful as a means to assess the antifungal action of various agents on yeasts and filamentous fungi in the presence and absence of serum proteins or body fluids.

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 virulenceassociated 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 the pyrG gene, encoding orotidine-5-phosphate decarboxylase, and the pksP gene, encoding a polyketide synthase involved in both pigment biosynthesis and virulence of A. fumigatus, were fused with the egfp gene. The PpksP-egfp construct was integrated via homologous recombination into the genomic pksP 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 the pksP 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.