The leucine biosynthetic pathway is crucial for adaptation to iron starvation and virulence in Aspergillus fumigatus.

In contrast to mammalia, fungi are able to synthesize the branched-chain amino acid leucine de novo. Recently, the transcription factor LeuB has been shown to cross-regulate leucine biosynthesis, nitrogen metabolism and iron homeostasis in Aspergillus fumigatus, the most common human mold pathogen. Moreover, the leucine biosynthetic pathway intermediate α-isopropylmalate (α-IPM) has previously been shown to posttranslationally activate LeuB homologs in S. cerevisiae and A. nidulans. Here, we demonstrate that in A. fumigatus inactivation of both leucine biosynthetic enzymes α-IPM synthase (LeuC), which disrupts α-IPM synthesis, and α-IPM isomerase (LeuA), which causes cellular α-IPM accumulation, results in leucine auxotrophy. However, compared to lack of LeuA, lack of LeuC resulted in increased leucine dependence, a growth defect during iron starvation and decreased expression of LeuB-regulated genes including genes involved in iron acquisition. Lack of either LeuA or LeuC decreased virulence in an insect infection model, and inactivation of LeuC rendered A. fumigatus avirulent in a pulmonary aspergillosis mouse model. Taken together, we demonstrate that the lack of two leucine biosynthetic enzymes, LeuA and LeuC, results in significant phenotypic consequences indicating that the regulator LeuB is activated by α-IPM in A. fumigatus and that the leucine biosynthetic pathway is an attractive target for the development of antifungal drugs.

Contribution of ATPase copper transporters in animal but not plant virulence of the crossover pathogen Aspergillus flavus.

The ubiquitous fungus Aspergillus flavus is notorious for contaminating many important crops and food-stuffs with the carcinogenic mycotoxin, aflatoxin. This fungus is also the second most frequent Aspergillus pathogen after A. fumigatus infecting immunosuppressed patients. In many human fungal pathogens including A. fumigatus, the ability to defend from toxic levels of copper (Cu) is essential in pathogenesis. In A. fumigatus, the Cu-fist DNA binding protein, AceA, and the Cu ATPase transporter, CrpA, play critical roles in Cu defense. Here, we show that A. flavus tolerates higher concentrations of Cu than A. fumigatus and other Aspergillus spp. associated with the presence of two homologs of A. fumigatus CrpA termed CrpA and CrpB. Both crpA and crpB are transcriptionally induced by increasing Cu concentrations via AceA activity. Deletion of crpA or crpB alone did not alter high Cu tolerance, suggesting they are redundant. Deletion of both genes resulted in extreme Cu sensitivity that was greater than that following deletion of the regulatory transcription factor aceA. The ΔcrpAΔcrpB and ΔaceA strains were also sensitive to ROI stress. Compared to wild type, these mutants were impaired in the ability to colonize maize seed treated with Cu fungicide but showed no difference in virulence on non-treated seed. A mouse model of invasive aspergillosis showed ΔcrpAΔcrpB and to a lesser degree ΔaceA to be significantly reduced in virulence, following the greater sensitivity of ΔcrpAΔcrpB to Cu than ΔaceA.

Aspergillus fumigatus Copper Export Machinery and Reactive Oxygen Intermediate Defense Counter Host Copper-Mediated Oxidative Antimicrobial Offense.

The Fenton-chemistry-generating properties of copper ions are considered a potent phagolysosome defense against pathogenic microbes, yet our understanding of underlying host/microbe dynamics remains unclear. We address this issue in invasive aspergillosis and demonstrate that host and fungal responses inextricably connect copper and reactive oxygen intermediate (ROI) mechanisms. Loss of the copper-binding transcription factor AceA yields an Aspergillus fumigatus strain displaying increased sensitivity to copper and ROI in vitro, increased intracellular copper concentrations, decreased survival in challenge with murine alveolar macrophages (AMΦs), and reduced virulence in a non-neutropenic murine model. ΔaceA survival is remediated by dampening of host ROI (chemically or genetically) or enhancement of copper-exporting activity (CrpA) in A. fumigatus. Our study exposes a complex host/microbe multifactorial interplay that highlights the importance of host immune status and reveals key targetable A. fumigatus counter-defenses.

Ultrashort peptide bioconjugates are exclusively antifungal agents and synergize with cyclodextrin and amphotericin B.

Many natural broad-spectrum cationic antimicrobial peptides (AMPs) possess a general mode of action that is dependent on lipophilicity and charge. Modulating the lipophilicity of AMPs by the addition of a fatty acid has been an effective strategy to increase the lytic activity and can further broaden the spectrum of AMPs. However, lipophilic modifications that narrow the spectrum of activity and exclusively direct peptides to fungi are less common. Here, we show that short peptide sequences can be targeted to fungi with structured lipophilic biomolecules, such as vitamin E and cholesterol. The conjugates were active against Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans but not against bacteria and were observed to cause membrane perturbation by transmission electron microscopy and in membrane permeability studies. However, for C. albicans, selected compounds were effective without the perturbation of the cell membrane, and synergism was seen with a vitamin E conjugate and amphotericin B. Moreover, in combination with ß-cyclodextrin, antibacterial activity emerged in selected compounds. Biocompatibility for selected active compounds was tested in vitro and in vivo using toxicity assays on erythrocytes, macrophages, and mice. In vitro cytotoxicity experiments led to selective toxicity ratios (50% lethal concentration/MIC) of up to 64 for highly active antifungal compounds, and no in vivo murine toxicity was seen. Taken together, these results highlight the importance of the conjugated lipophilic structure and suggest that the modulation of other biologically relevant peptides with hydrophobic moieties, such as cholesterol and vitamin E, generate compounds with unique bioactivity.

PrtT-regulated proteins secreted by Aspergillus fumigatus activate MAPK signaling in exposed A549 lung cells leading to necrotic cell death.

Aspergillus fumigatus is the most commonly encountered mold pathogen of humans, predominantly infecting the respiratory system. Colonization and penetration of the lung alveolar epithelium is a key but poorly understood step in the infection process. This study focused on identifying the transcriptional and cell-signaling responses activated in A549 alveolar carcinoma cells incubated in the presence of A. fumigatus wild-type and ΔPrtT protease-deficient germinating conidia and culture filtrates (CF). Microarray analysis of exposed A549 cells identified distinct classes of genes whose expression is altered in the presence of germinating conidia and CF and suggested the involvement of both NFkB and MAPK signaling pathways in mediating the cellular response. Phosphoprotein analysis of A549 cells confirmed that JNK and ERK1/2 are phosphorylated in response to CF from wild-type A. fumigatus and not phosphorylated in response to CF from the ΔPrtT protease-deficient strain. Inhibition of JNK or ERK1/2 kinase activity substantially decreased CF-induced cell damage, including cell peeling, actin-cytoskeleton damage, and reduction in metabolic activity and necrotic death. These results suggest that inhibition of MAPK-mediated host responses to treatment with A. fumigatus CF decreases cellular damage, a finding with possible clinical implications.