Aspergillus fumigatus AcuM regulates both iron acquisition and gluconeogenesis.

Relatively few transcription factors that govern the virulence of Aspergillus fumigatus are known. We constructed 11 A. fumigatus transcription factor mutants and screened them for altered virulence in Galleria mellonella larvae. We discovered that the zinc cluster transcription factor, AcuM, is essential for maximal virulence in this model, as well as in murine models of haematogenously disseminated and invasive pulmonary aspergillosis. Transcriptional profiling experiments suggested that AcuM suppresses sreA and induces hapX to stimulate expression of genes involved in both reductive iron assimilation and siderophore-mediated iron uptake. Consistent with these results, a ΔacuM mutant had reduced iron incorporation, decreased extracellular siderophore production and impaired capacity to grow under iron-limited conditions. Interestingly, an Aspergillus nidulansΔacuM mutant had normal extracellular siderophore production and growth under iron-limited conditions, indicating that AcuM does not govern iron acquisition in this organism. A. fumigatus AcuM also regulated genes involved in gluconeogenesis, and the ΔacuM mutant had impaired growth on gluconeogenic carbon sources. Deletion of sreA in the ΔacuM mutant restored iron uptake, extracellular siderophore production and virulence, but not the defect in gluconeogenesis. Thus, AcuM represses SreA and thereby induces iron acquisition, a process that is essential for the maximal virulence of A. fumigatus.

Aspergillus fumigatus MedA governs adherence, host cell interactions and virulence.

In medically important fungi, regulatory elements that control development and asexual reproduction often govern the expression of virulence traits. We therefore cloned the Aspergillus fumigatus developmental modifier MedA and characterized its role in conidiation, host cell interactions and virulence. As in the model organism Aspergillus nidulans, disruption of medA in A. fumigatus dramatically reduced conidiation. However, the conidiophore morphology was markedly different between the two species. Further, gene expression analysis suggested that MedA governs conidiation through different pathways in A. fumigatus compared with A. nidulans. The A. fumigatusDeltamedA strain was impaired in biofilm production and adherence to plastic, as well as adherence to pulmonary epithelial cells, endothelial cells and fibronectin in vitro. The DeltamedA strain also had reduced capacity to damage pulmonary epithelial cells, and stimulate pro-inflammatory cytokine mRNA and protein expression. Consistent with these results, the A. fumigatusDeltamedA strain also exhibited reduced virulence in both an invertebrate and a mammalian model of invasive aspergillosis. Collectively, these results suggest that the downstream targets of A. fumigatus MedA mediate virulence, and may provide novel therapeutic targets for invasive aspergillosis.

Aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease.

Invasive aspergillosis is characterized by hyphal invasion of the blood vessels, which contributes to the pathogenesis of this disease. During this angioinvasion, Aspergillus fumigatus interacts with the endothelial cell lining of the blood vessels. We investigated the response of vascular endothelial cells to A. fumigatus infection in vitro and in mouse models of invasive pulmonary aspergillosis. Infection with hyphae, but not with conidia, stimulated endothelial cells to synthesize E-selectin, vascular cell adhesion molecule 1 (VCAM-1), interleukin 8, and tumor necrosis factor alpha (TNF-alpha) in vitro. Killed hyphae induced approximately 40% less stimulation than did live hyphae. Endothelial cell stimulation required contact between the hyphae and endothelial cells but not endocytosis of the organisms. Studies with DeltagliP and DeltastuA null mutants of A. fumigatus indicated that the extent of endothelial cell stimulation was not influenced by gliotoxin or other StuA-dependent factors synthesized by A. fumigatus. In neutropenic mice infected with wild-type A. fumigatus, increased pulmonary expression of E-selectin, cytokine-induced neutrophil chemoattractant (KC), and TNF-alpha occurred only when neutropenia had resolved. In nonneutropenic mice immunosuppressed with corticosteroids, A. fumigatus stimulated earlier pulmonary expression of E-selectin, VCAM-1, and KC, while expression of intercellular adhesion molecule 1 and TNF-alpha was suppressed. In both mouse models, expression of E-selectin and KC was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells. Therefore, the expression of leukocyte adhesion molecules and secretion of proinflammatory cytokines by endothelial cells in response to A. fumigatus could enhance the host defense against this organism by contributing to the recruitment of activated leukocytes to sites of angioinvasion.

In vivo analysis of Aspergillus fumigatus developmental gene expression determined by real-time reverse transcription-PCR.

Very little is known about the developmental stages of Aspergillus fumigatus during invasive aspergillosis. We performed real-time reverse transcription-PCR analysis on lung samples from mice with invasive pulmonary aspergillosis to determine the expression of A. fumigatus genes that are expressed at specific stages of development. In established infection, A. fumigatus exhibited mRNA expression of genes specific to developmentally competent hyphae, such as stuA. In contrast, mRNA of genes expressed by conidia and precompetent hyphae was not detected. Many genes required for mycotoxin synthesis, including aspHS, gliP, mitF, and metAP, are known to be expressed by developmentally competent hyphae in vitro. Interestingly, each of these genes was expressed at significantly higher levels during invasive infection than in vitro. The expression of gliP mRNA in vitro was found to be highly dependent on culture conditions. Furthermore, gliP expression was found to be dependent on the transcription factor StuA both in vitro and in vivo. Therefore, developmentally competent hyphae predominate during established invasive infection, and many mycotoxin genes are expressed at high levels in vivo. These results highlight the importance of the evaluation of putative virulence factors expressed by competent hyphae and analysis of gene expression levels during invasive infection rather than in vitro alone.

Gliotoxin production in Aspergillus fumigatus contributes to host-specific differences in virulence.

BACKGROUND: Gliotoxin is a epipolythiodioxopiperazine toxin that is made by the filamentous fungus Aspergillus fumigatus. Gliotoxin has a wide range of effects on metazoan cells in culture, including induction of apoptosis through inhibition of Nf-kappaB, and inhibition of superoxide production by phagocytes. These activities have led to the proposal that gliotoxin contributes to pathogenesis during invasive aspergillosis. We tested this hypothesis by creating isogenic strains of gliotoxin-producing and nonproducing strains. METHODS: We deleted gliP, the gene that encodes the nonribosomal peptide synthetase GliP. GliP catalyzes the first biosynthetic step in the synthesis of gliotoxin. We then tested for gliotoxin production and virulence in different animal models. RESULTS: Deletion of gliP resulted in strains that were wild type for growth, but they did not synthesize gliotoxin. Transformation of gliP deletion mutants with a full copy of gliP restored gliotoxin production. The gliP deletion strain had attenuated virulence in nonneutropenic mice immunosuppressed with corticosteroids, but had normal virulence in neutropenic mice. It also had reduced virulence in a Drosophila melanogaster model. CONCLUSIONS: Gliotoxin only contributes to the virulence of A. fumigatus in nonneutropenic mice and in fruit flies with functional phagocytes. These results suggest that the principal targets of gliotoxin are neutrophils or other phagocytes.

Candida albicans protein kinase CK2 governs virulence during oropharyngeal candidiasis.

To identify Candida albicans genes whose proteins are necessary for host cell interactions and virulence, a collection of C. albicans insertion mutants was screened for strains with reduced capacity to damage endothelial cells in vitro. This screen identified CKA2. CKA2 and its homologue CKA1 encode the catalytic subunits of the protein kinase CK2. cka2delta/cka2delta strains of C. albicans were constructed and found to have significantly reduced capacity to damage both endothelial cells and an oral epithelial cell line in vitro. Although these strains invaded endothelial cells similarly to the wild-type strain, they were defective in oral epithelial cell invasion. They were also hypersusceptible to hydrogen peroxide, but not to high salt or to cell wall damaging agents. A cka1delta/cka1delta mutant caused normal damage to both endothelial cells and oral epithelial cells, and it was not hypersusceptible to hydrogen peroxide. However, overexpression of CKA1 in a cka2delta/cka2delta strain restored wild-type phenotype. Although the cka2delta/cka2delta mutant had normal virulence in the mouse model of haematogenously disseminated candidiasis, it had significantly attenuated virulence in the mouse model of oropharyngeal candidiasis. Therefore, Cka2p governs the interactions of C. albicans with endothelial and oral epithelial cells in vitro and virulence during oropharyngeal candidiasis.

Efficacy of ambruticin analogs in a murine model of invasive pulmonary aspergillosis.

Ambruticins are a family of polyketides. The antifungal activity of an ambruticin, KOSN-2079, was tested in the mouse model of invasive aspergillosis. KOSN-2079 significantly reduced pulmonary fungal burdens and improved survival over that with the vehicle control. These results support the continued development of ambruticins as antifungal agents.

Standardization of an experimental murine model of invasive pulmonary aspergillosis.

Evaluating new therapeutic agents for invasive aspergillosis requires animal models that are reproducible among different laboratories. We therefore evaluated a murine model of aerosol infection in two independent laboratories and found a high level of both intra- and interlaboratory reproducibility of survival, fungal burden over time, and the efficacy of liposomal amphotericin B.

The Aspergillus fumigatus StuA protein governs the up-regulation of a discrete transcriptional program during the acquisition of developmental competence.

Members of the Asm1p, Phd1p, Sok2p, Efg1p, and StuAp (APSES) family of fungal proteins regulate morphogenesis and virulence in ascomycetes. We cloned the Aspergillus fumigatus APSES gene encoding StuAp and demonstrated that stuA transcription is markedly up-regulated after the acquisition of developmental competence. A. fumigatus DeltastuA mutants were impaired in their ability to undergo asexual reproduction. Conidiophore morphology was markedly abnormal, and only small numbers of dysmorphic conidia were produced, which exhibited precocious germination. Whole genome transcriptional analysis during the onset of developmental competence was performed and identified a subset of developmentally regulated genes that were stuA dependent, including a cluster of putative secondary metabolite biosynthesis genes, genes encoding proteins implicated in the regulation of morphogenesis, and genes encoding allergens and other antigenic proteins. Additionally, hyphae of the DeltastuA mutant displayed reduced expression of the catalase gene CAT1 and were hypersusceptible to hydrogen peroxide.

Novel inhalational murine model of invasive pulmonary aspergillosis.

We developed a novel model of invasive aspergillosis (IA) that recapitulates human disease. Mice were immunosuppressed with cyclophosphamide and cortisone acetate and then infected in an aerosol chamber. This procedure reproducibly delivered 1 x 10(3) to 3 x 10(3) conidia to the lungs. Lethal pulmonary IA developed over 2 weeks and was prevented by amphotericin B.