Janyce A. Sugui

Gliotoxin Is a Virulence Factor of Aspergillus fumigatus: gliP Deletion Attenuates Virulence in Mice Immunosuppressed with Hydrocortisone

ABSTRACT 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 (gliPΔ) and the the glip reconstituted strain (gliPR). The gliP gene encodes a nonribosomal peptide synthase that catalyzes the first step in gliotoxin biosynthesis. The gliPΔ strain was significantly less virulent than strain B-5233 or gliPR in both mouse models. In vitro assays with culture filtrates (CFs) of B-5233, gliPΔ, and gliPR 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 gliPR, gliPΔ 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 gliPR strain, but not the gliPΔ 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.

Human Polymorphonuclear Leukocytes Inhibit Aspergillus fumigatus Conidial Growth by Lactoferrin-Mediated Iron Depletion

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.

Agrobacterium tumefaciens-Mediated Transformation of Aspergillus fumigatus: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption

ABSTRACT 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/107 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 Δalb1 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—What Makes the Species a Ubiquitous Human Fungal Pathogen?

Aspergillus fumigatus, the major cause of life threatening invasive aspergillosis (IA), is a ubiquitous saprophytic fungus to which humans are exposed daily in most parts of the world. The infection is initiated by inhalation of conidia, which are cleared quickly in a normal host but can cause invasive disease in immunocompromised individuals [1], [2]. The following features make A. fumigatus a ubiquitous pathogen: 1) survival and growth in a wide range of environmental conditions, 2) effective dispersal in the air, 3) physical characteristics that allow conidia to reach the distal airways, and 4) swift adaptability to the host environment. The biology, pathogenesis, molecular biology, and virulence factors of A. fumigatus have been exhaustively reviewed [2]–[8]. This brief article focuses on how A. fumigatus is equipped with the features necessary for a ubiquitous pathogen.

Invasive Aspergillosis Due to Neosartorya udagawae

BACKGROUND Invasive aspergillosis (IA) is most commonly caused by the morphospecies Aspergillus fumigatus. However, genetic-based methods indicate that organisms phenotypically identified as A. fumigatus actually constitute a mold complex, designated Aspergillus section fumigati subgenus fumigati. METHODS Multilocus sequencing and analysis was performed on fungi identified as A. fumigatus from the clinical culture collection maintained at the National Institutes of Health from 2000 through 2008, with a focus on the internal transcribed spacer 1 and 2 regions of ribosomal DNA (rDNA), beta-tubulin, and rodlet A genes. We reviewed the medical records, radiology, and histopathology of corresponding patients. To confirm identification of Neosartorya udagawae isolates, mating studies were performed with reference strains. Antifungal susceptibility testing was performed by broth microdilution and read at 48 hours. RESULTS Thirty-six cases of infection attributed to A. fumigatus were identified; 4 were caused by N. udagawae (3 in patients with chronic granulomatous disease and 1 in a patient with myelodysplastic syndrome). Disease due to N. udagawae was chronic, with a median duration of 35 weeks, compared with a median duration of 5.5 weeks for patients with chronic granulomatous disease who had infection due to A. fumigatus sensu stricto (P < .05 , Mann-Whitney U test). Infection spread across anatomical planes in a contiguous manner and was refractory to standard therapy. Two of the 4 patients died. N. udagawae demonstrated relatively higher minimum inhibitory concentrations to various agents, compared with those demonstrated by contemporary A. fumigatus sensu stricto isolates. CONCLUSIONS To our knowledge, this is the first report documenting infection due to N. udagawae. Clinical manifestations were distinct from those of typical IA. Fumigati-mimetics with inherent potential for antifungal resistance are agents of IA. Genetic identification of molds should be considered for unusual or refractory IA.

Aspergillus fumigatus conidial melanin modulates host cytokine response.

Melanin biopigments have been linked to fungal virulence. Aspergillus fumigatus conidia are melanised and are weakly immunogenic. We show that melanin pigments on the surface of resting Aspergillus fumigatus conidia may serve to mask pathogen-associated molecular patterns (PAMPs)-induced cytokine response. The albino conidia induced significantly more proinflammatory cytokines in human peripheral blood mononuclear cells (PBMC), as compared to melanised wild-type conidia. Blocking dectin-1 receptor, Toll-like receptor 4 or mannose receptor decreased cytokine production induced by the albino but not by the wild type conidia. Moreover, albino conidia stimulated less potently, cytokine production in PBMC isolated from an individual with defective dectin-1, compared to the stimulation of cells isolated from healthy donors. These results suggest that β-glucans, but also other stimulatory PAMPs like mannan derivatives, are exposed on conidial surface in the absence of melanin. Melanin may play a modulatory role by impeding the capability of host immune cells to respond to specific ligands on A. fumigatus.

Genes Differentially Expressed in Conidia and Hyphae of Aspergillus fumigatus upon Exposure to Human Neutrophils

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 pathogens 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 Findings Transcriptional 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. Conclusion This 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.

What do we know about the role of gliotoxin in the pathobiology of Aspergillus fumigatus

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.

Role of laeA in the Regulation of alb1, gliP, Conidial Morphology, and Virulence in Aspergillus fumigatus

ABSTRACT The alb1 (pksP) gene has been reported as a virulence factor controlling the pigmentation and morphology of conidia in Aspergillus fumigatus. A recent report suggested that laeA regulates alb1 expression and conidial morphology but not pigmentation in the A. fumigatus strain AF293. laeA has also been reported to regulate the synthesis of secondary metabolites, such as gliotoxin. We compared the role of laeA in the regulation of conidial morphology and the expression of alb1 and gliP in strains B-5233 and AF293, which differ in colony morphology and nutritional requirements. Deletion of laeA did not affect conidial morphology or pigmentation in these strains, suggesting that laeA is not involved in alb1 regulation during conidial morphogenesis. Deletion of laeA, however, caused down-regulation of alb1 during mycelial growth in a liquid medium. Transcription of gliP, involved in the synthesis of gliotoxin, was drastically reduced in B-5233laeAΔ, and the gliotoxin level found in the culture filtrates was 20% of wild-type concentrations. While up-regulation of gliP in AF293 was comparable to that in B-5233, the relative mRNA level in AF293laeAΔ was about fourfold lower than that in B-5233laeAΔ. Strain B-5233laeAΔ caused slower onset of fatal infection in mice relative to that with B-5233. Histopathology of sections from lungs of infected mice corroborated the survival data. Culture filtrates from B-5233laeAΔ caused reduced death in thymoma cells and were less inhibitory to a respiratory burst of neutrophils than culture filtrates from B-5233. Our results suggest that while laeA is not involved in the regulation of alb1 function in conidial morphology, it regulates the synthesis of gliotoxin and the virulence of A. fumigatus.

Neosartorya udagawae (Aspergillus udagawae), an Emerging Agent of Aspergillosis: How Different Is It from Aspergillus fumigatus?

ABSTRACT A recent report on several cases of invasive aspergillosis caused by Neosartorya udagawae suggested distinctive patterns of disease progression between N. udagawae and Aspergillus fumigatus. This prompted us to characterize N. udagawae in comparison to A. fumigatus. Our findings showed that both species exist in two mating types at similar ratios and produce gliotoxin. However, the thermotolerance of the two species differs: while A. fumigatus is able to grow at 55°C but not at 10°C, N. udagawae is able to grow at 10°C but fails to grow at >42°C. Furthermore, compared to A. fumigatus, the conidia of N. udagawae require longer incubation periods to germinate at 37°C and are more susceptible to neutrophil attack as well as hydrogen peroxide; N. udagawae is also less virulent in gp91phox−/− mice. These findings suggest that growth and susceptibility to the host response might account for the reduced virulence of N. udagawae and the subtle distinction in the progression of the disease caused by the two species.