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Gliotoxin is an immunosuppressive mycotoxin long suspected to be a potential virulence factor of Aspergillus fumigatus. Recent studies using mutants lacking gliotoxin production, however, suggested that the mycotoxin is not important for pathogenesis of A. fumigatus in neutropenic mice resulting from treatment with cyclophosphomide and hydrocortisone. In this study, we report on the pathobiological role of gliotoxin in two different mouse strains, 129/Sv and BALB/c, that were immunosuppressed by hydrocortisone alone to avoid neutropenia. These strains of mice were infected using the isogenic set of a wild type strain (B-5233) and its mutant strain (gliP⌬) and the the glip reconstituted strain (gliP R ). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliP⌬ strain was significantly less virulent than strain B-5233 or gliP R in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliP⌬, and gliP R strains showed the following: (i) deletion of gliP abrogated gliotoxin production, as determined by high-performance liquid chromatography analysis; (ii) unlike the CFs from strains B-5233 and gliP R , gliP⌬ CFs failed to induce proapoptotic processes in EL4 thymoma cells, as tested by Bak conformational change, mitochondrial-membrane potential disruption, superoxide production, caspase 3 activation, and phosphatidylserine translocation. Furthermore, superoxide production in human neutrophils was strongly inhibited by CFs from strain B-5233 and the gliP R strain, but not the gliP⌬ strain. Our study confirms that gliotoxin is an important virulence determinant of A. fumigatus and that the type of immunosuppression regimen used is important to reveal the pathogenic potential of gliotoxin.

The genus Aspergillus contains etiologic agents of aspergillosis. The clinical manifestations of the disease range from allergic reaction to invasive pulmonary infection. Among the pathogenic aspergilli, Aspergillus fumigatus is most ubiquitous in the environment and is the major cause of the disease, followed by Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus nidulans, and several species in the section Fumigati that morphologically resemble A. fumigatus. Patients that are at risk for acquiring aspergillosis are those with an altered immune system. Early diagnosis, species identification, and adequate antifungal therapy are key elements for treatment of the disease, especially in cases of pulmonary invasive aspergillosis that often advance very rapidly. Incorporating knowledge of the basic biology of Aspergillus species to that of the diseases that they cause is fundamental for further progress in the field.

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.

Chitin synthesis contributes to cell wall biogenesis and is essential for invasion of solid substrata and pathogenicity of filamentous fungi. In contrast to yeasts, filamentous fungi contain up to 10 chitin synthases (CHS), which might reflect overlapping functions and indicate their complex lifestyle. Previous studies have shown that a class VI CHS of the maize anthracnose fungus Colletotrichum graminicola is essential for cell wall synthesis of conidia and vegetative hyphae. Here, we report on cloning and characterization of three additional CHS genes, CgChsI, CgChsIII, and CgChsV, encoding class I, III, and V CHS, respectively. All CHS genes are expressed during vegetative and pathogenic development. DeltaCgChsI and DeltaCgChsIII mutants did not differ significantly from the wild-type isolate with respect to hyphal growth and pathogenicity. In contrast, null mutants in the CgChsV gene, which encodes a CHS with an N-terminal myosin-like motor domain, are strongly impaired in vegetative growth and pathogenicity. Even in osmotically stabilized media, vegetative hyphae of DeltaCgChsV mutants exhibited large balloon-like swellings, appressorial walls appeared to disintegrate during maturation, and infection cells were nonfunctional. Surprisingly, DeltaCgChsV mutants were able to form dome-shaped hyphopodia that exerted force and showed host cell wall penetration rates comparable with the wild type. However, infection hyphae that formed within the plant cells exhibited severe swellings and were not able to proceed with plant colonization efficiently. Consequently, DeltaCgChsV mutants did not develop macroscopically visible anthracnose disease symptoms and, thus, were nonpathogenic.

Background Aspergillus fumigatus is the most common etiologic agent of invasive aspergillosis in immunocompromised patients. Several studies have addressed the mechanism involved in host defense but only few have investigated the pathogen's response to attack by the host cells. To our knowledge, this is the first study that investigates the genes differentially expressed in conidia vs hyphae of A. fumigatus in response to neutrophils from healthy donors as well as from those with chronic granulomatous disease (CGD) which are defective in the production of reactive oxygen species.Methodology/Principal FindingsTranscriptional profiles of conidia and hyphae exposed to neutrophils, either from normal donors or from CGD patients, were obtained by using the genome-wide microarray. Upon exposure to either normal or CGD neutrophils, 244 genes were up-regulated in conidia but not in hyphae. Several of these genes are involved in the degradation of fatty acids, peroxisome function and the glyoxylate cycle which suggests that conidia exposed to neutrophils reprogram their metabolism to adjust to the host environment. In addition, the mRNA levels of four genes encoding proteins putatively involved in iron/copper assimilation were found to be higher in conidia and hyphae exposed to normal neutrophils compared to those exposed to CGD neutrophils. Deletants in several of the differentially expressed genes showed phenotypes related to the proposed functions, i.e. deletants of genes involved in fatty acid catabolism showed defective growth on fatty acids and the deletants of iron/copper assimilation showed higher sensitivity to the oxidative agent menadione. None of these deletants, however, showed reduced resistance to neutrophil attack.ConclusionThis work reveals the complex response of the fungus to leukocytes, one of the major host factors involved in antifungal defense, and identifies fungal genes that may be involved in establishing or prolonging infections in humans.

Gliotoxin is a member of the epipolythiodioxopiperazine class of toxins and is both the major and the most potent toxin produced by Aspergillus fumigatus. Since the discovery of the putative gliotoxin biosynthetic 12-gene cluster in the genome of A. fumigatus, five different laboratories have attempted to determine the role of this toxin in the virulence of A. fumigatus. The genes in the cluster that have been disrupted to study the pathobiological importance of gliotoxin include gliZ that encodes a transcription factor and gliP that encodes a nonribosomal peptide synthase. Two of the five laboratories have reported gliotoxin to be an important virulence determinant of A. fumigatus, while the other three laboratories have shown it to be unimportant. Comparisons of the data generated among the five laboratories revealed that the immunosuppressive regimen used for mice was the key factor that contributed to the observed disparity. Regardless of either the mouse strains used or the route of infection, immunosuppression with a combination of cyclophosphamide and corticosteroids (neutropenic mice) showed gliotoxin to be unimportant. The mice immunosuppressed with corticosteroids alone, however, revealed that gliotoxin is an important virulence determinant of A. fumigatus. These studies indicate that the neutropenic mice model is inadequate to reveal the pathobiological importance of fungal secondary metabolites in invasive pulmonary aspergillosis.

Agrobacterium tumefaciens was used to transform Aspergillus fumigatus by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via Agrobacterium tumefaciens-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of A. fumigatus conidia and Agrobacterium (1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10 7 conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose alb1, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of ⌬alb1 mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing alb1 disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in A. fumigatus.Aspergillus fumigatus, an airborne fungal pathogen, is the major cause of allergic bronchopulmonary aspergillosis, aspergilloma, and invasive aspergillosis. Of particular importance is the invasive aspergillosis that starts with inhalation of conidia and progresses to life-threatening infection in immunocompromised patients (11). Due to the rising prevalence of cancer, organ transplantation, and other causes of immunosuppression, the number of patients at risk of invasive aspergillosis is on the rise. Despite aggressive antifungal therapy, the overall mortality of invasive aspergillosis remains high, and new strategies to prevent and treat this disease are urgently needed (7). Several factors such as polyketide synthase, which is involved in melanin synthesis (12,17), and a Ras-related protein, RhbA, presumably involved in nutrient sensing (15), have been reported to be associated with A. fumigatus virulence. Further identification of the genes necessary for virulence will enable us to address the key pathobiological questions for this fungus and provide a foundation for better strategies to manage aspergillosis.As genomic sequencing of A. fumigatus is in its final stages, identification of potential virulence genes can be more rapidly assessed. Investigation of the functions of such genes can be accomplished via mutational analysis either by reverse or forward genetic approaches. Transformation of fungi with DNA that does not possess homology with the fungal genome results in random integration into the fungal genome and can cause gene disruption as an insertional mutagen. A decisive advantage of insertional mutagenesis over chemical or radiation mutagenesis is that the mutated genes are tagged by the transforming DNA (T-DNA), which can be used to identify the disrupted genes. Although recombinational analysis by classical genetics is not possible in A. fum...