Ulrike Binder

The Penicillium chrysogenum antifungal protein PAF, a promising tool for the development of new antifungal therapies and fungal cell biology studies

Abstract.In recent years the interest in antimicrobial proteins and peptides and their mode of action has been rapidly increasing due to their potential to prevent and combat microbial infections in all areas of life. A detailed knowledge about the function of such proteins is the most important requirement to consider them for future application. Our research in recent years has been focused on the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum, which inhibits the growth of opportunistic zoo-pathogens including Aspergillus fumigatus, numerous plant-pathogenic fungi and the model organism Aspergillus nidulans. So far, the experimental results indicate that PAF elicits hyperpolarization of the plasma membrane and the activation of ion channels, followed by an increase in reactive oxygen species in the cell and the induction of an apoptosis-like phenotype. Detailed knowledge about the molecular mechanism of action of antifungal proteins such as PAF contributes to the development of new antimicrobial strategies that are urgently needed.

Functional aspects of the solution structure and dynamics of PAF – a highly‐stable antifungal protein from Penicillium chrysogenum

Penicillium antifungal protein (PAF) is a promising antimycotic without toxic effects on mammalian cells and therefore may represent a drug candidate against the often lethal Aspergillus infections that occur in humans. The pathogenesis of PAF on sensitive fungi involves G‐protein coupled signalling followed by apoptosis. In the present study, the solution structure of this small, cationic, antifungal protein from Penicillium chrysogenum is determined by NMR. We demonstrate that PAF belongs to the structural classification of proteins fold class of its closest homologue antifungal protein from Aspergillus giganteus. PAF comprises five β‐strands forming two orthogonally packed β‐sheets that share a common interface. The ambiguity in the assignment of two disulfide bonds out of three was investigated by NMR dynamics, together with restrained molecular dynamics calculations. The clue could not be resolved: the two ensembles with different disulfide patterns and the one with no S–S bond exhibit essentially the same fold. 15N relaxation dispersion and interference experiments did not reveal disulfide bond rearrangements via slow exchange. The measured order parameters and the 3.0 ns correlation time are appropriate for a compact monomeric protein of this size. Using site‐directed mutagenesis, we demonstrate that the highly‐conserved and positively‐charged lysine‐rich surface region enhances the toxicity of PAF. However, the binding capability of the oligosaccharide/oligonucleotide binding fold is reduced in PAF compared to antifungal protein as a result of less solvent‐exposed aromatic regions, thus explaining the absence of chitobiose binding. The present study lends further support to the understanding of the documented substantial differences between the mode of action of two highly homologous antifungal proteins.

The Antifungal Activity of the Penicillium chrysogenum Protein PAF Disrupts Calcium Homeostasis in Neurospora crassa

ABSTRACT The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca2+ signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca2+ concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca2+ ([Ca2+]c). The Ca2+ signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid], a Ca2+ selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca2+ homeostasis. These results indicate that extracellular Ca2+ was the major source of these PAF-induced effects. The L-type Ca2+ channel blocker diltiazem disrupted Ca2+ homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca2+ signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca2+]c resting level. However, experiments with an aequorin-expressing Δcch-1 deletion strain of N. crassa indicated that the L-type Ca2+ channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca2+]c resting level. This study is the first demonstration of the perturbation of fungal Ca2+ homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.

The antifungal protein PAF interferes with PKC/MPK and cAMP/PKA signalling of Aspergillus nidulans.

The Penicillium chrysogenum antifungal protein PAF inhibits polar growth and induces apoptosis in Aspergillus nidulans. We report here that two signalling cascades are implicated in its antifungal activity. PAF activates the cAMP/protein kinase A (Pka) signalling cascade. A pkaA deletion mutant exhibited reduced sensitivity towards PAF. This was substantiated by the use of pharmacological modulators: PAF aggravated the effect of the activator 8‐Br‐cAMP and partially relieved the repressive activity of caffeine. Furthermore, the Pkc/mitogen‐activated protein kinase (Mpk) signalling cascade mediated basal resistance to PAF, which was independent of the small GTPase RhoA. Non‐functional mutations of both genes resulted in hypersensitivity towards PAF. PAF did not increase MpkA phosphorylation or induce enzymes involved in the remodelling of the cell wall, which normally occurs in response to activators of the cell wall integrity pathway. Notably, PAF exposure resulted in actin gene repression and a deregulation of the chitin deposition at hyphal tips of A. nidulans, which offers an explanation for the morphological effects evoked by PAF and which could be attributed to the interconnection of the two signalling pathways. Thus, PAF represents an excellent tool to study signalling pathways in this model organism and to define potential fungal targets to develop new antifungals.

The Aspergillus giganteus antifungal protein AFPNN5353 activates the cell wall integrity pathway and perturbs calcium homeostasis

BackgroundThe antifungal protein AFPNN5353 is a defensin-like protein of Aspergillus giganteus. It belongs to a group of secretory proteins with low molecular mass, cationic character and a high content of cysteine residues. The protein inhibits the germination and growth of filamentous ascomycetes, including important human and plant pathogens and the model organsims Aspergillus nidulans and Aspergillus niger.ResultsWe determined an AFPNN5353 hypersensitive phenotype of non-functional A. nidulans mutants in the protein kinase C (Pkc)/mitogen-activated protein kinase (Mpk) signalling pathway and the induction of the α-glucan synthase A (agsA) promoter in a transgenic A. niger strain which point at the activation of the cell wall integrity pathway (CWIP) and the remodelling of the cell wall in response to AFPNN5353. The activation of the CWIP by AFPNN5353, however, operates independently from RhoA which is the central regulator of CWIP signal transduction in fungi.Furthermore, we provide evidence that calcium (Ca2+) signalling plays an important role in the mechanistic function of this antifungal protein. AFPNN5353 increased about 2-fold the cytosolic free Ca2+ ([Ca2+]c) of a transgenic A. niger strain expressing codon optimized aequorin. Supplementation of the growth medium with CaCl2 counteracted AFPNN5353 toxicity, ameliorated the perturbation of the [Ca2+]c resting level and prevented protein uptake into Aspergillus sp. cells.ConclusionsThe present study contributes new insights into the molecular mechanisms of action of the A. giganteus antifungal protein AFPNN5353. We identified its antifungal activity, initiated the investigation of pathways that determine protein toxicity, namely the CWIP and the Ca2+ signalling cascade, and studied in detail the cellular uptake mechanism in sensitive target fungi. This knowledge contributes to define new potential targets for the development of novel antifungal strategies to prevent and combat infections of filamentous fungi which have severe negative impact in medicine and agriculture.

The metalloreductase FreB is involved in adaptation of Aspergillus fumigatus to iron starvation.

Highlights ► Characterization of a major ferrireductase encoding gene, FreB. ► Crucial role of FreB in adaptation to iron starvation. ► Link between iron and copper metabolism. ► Siderophore system increases resistance against copper starvation.

New insights into invasive aspergillosis--from the pathogen to the disease.

Disease manifestations with Aspergillus spp. are very diverse and dependent on interaction between the fungus and the host. Invasive aspergillosis (IA) is the most severe form of Aspergillus - associated disease found in immunocompromised hosts. Infections are mainly due to Aspergillus (A.) fumigatus, an air-borne opportunistic pathogen that causes 90% of IA. Mortality rate of this disease is still very high (50-95%), partly because of diagnostic difficulties, limited antifungal treatment options, weak conditions of patients at risk; but also in part because understanding of virulence factors involved in A. fumigatus pathogenicity and interactions of the pathogen with the host immune system is still poor. This review focuses on properties of A. fumigatus in terms of putative virulence factors and interactions of the pathogen with a main focus on the innate immune system.

Histidine biosynthesis plays a crucial role in metal homeostasis and virulence of Aspergillus fumigatus

Abstract Aspergillus fumigatus is the most prevalent airborne fungal pathogen causing invasive fungal infections in immunosuppressed individuals. The histidine biosynthetic pathway is found in bacteria, archaebacteria, lower eukaryotes, and plants, but is absent in mammals. Here we demonstrate that deletion of the gene encoding imidazoleglycerol-phosphate dehydratase (HisB) in A. fumigatus causes (i) histidine auxotrophy, (ii) decreased resistance to both starvation and excess of various heavy metals, including iron, copper and zinc, which play a pivotal role in antimicrobial host defense, (iii) attenuation of pathogenicity in 4 virulence models: murine pulmonary infection, murine systemic infection, murine corneal infection, and wax moth larvae. In agreement with the in vivo importance of histidine biosynthesis, the HisB inhibitor 3-amino-1,2,4-triazole reduced the virulence of the A. fumigatus wild type and histidine supplementation partially rescued virulence of the histidine-auxotrophic mutant in the wax moth model. Taken together, this study reveals limited histidine availability in diverse A. fumigatus host niches, a crucial role for histidine in metal homeostasis, and the histidine biosynthetic pathway as being an attractive target for development of novel antifungal therapy approaches.

Galleria mellonella as a host model to study Aspergillus terreus virulence and amphotericin B resistance

The aim of this study was to investigate if the alternative in vivo model Galleria mellonella can be used (i) to determine differences in pathogenicity of amphotericin B (AMB) resistant and susceptible A. terreus isolates, (ii) to evaluate AMB efficacy in vivo (iii) and to correlate outcome to in vitro susceptibility data. Larvae were infected with 2 A. terreus AMB resistant (ATR) and 3 AMB susceptible (ATS) isolates and survival rates were correlated to physiological attributes and killing ability of larval haemocytes. Additionally, infected larvae were treated with different concentrations of L-AMB. Haemocyte density were ascertained to evaluate the influence of L-AMB on the larval immune cells. Larvae were sensitive to A. terreus infection in an inoculum-size and temperature dependent manner. In vitro susceptibility to L-AMB correlated with in vivo outcome of antifungal treatment, defining an AMB susceptible strain cluster of A. terreus. Susceptibility to L-AMB increased virulence potential in the larval model, but this increase was also in accordance with faster growth and less damage caused by larval haemocytes. L-AMB treatment primed the larval immune response by increasing haemocyte density. G. mellonella provides a convenient model for the in vivo screening of A. terreus virulence and treatment options, contributing to the generation of a hypothesis that can be further tested in refined experiments in mammalian models.

Blocking Hsp70 Enhances the Efficiency of Amphotericin B Treatment against Resistant Aspergillus terreus Strains

ABSTRACT The polyene antifungal amphotericin B (AmB) is widely used to treat life-threatening fungal infections. Even though AmB resistance is exceptionally rare in fungi, most Aspergillus terreus isolates exhibit an intrinsic resistance against the drug in vivo and in vitro. Heat shock proteins perform a fundamental protective role against a multitude of stress responses, thereby maintaining protein homeostasis in the organism. In this study, we elucidated the role of heat shock protein 70 (Hsp70) family members and compared resistant and susceptible A. terreus clinical isolates. The upregulation of cytoplasmic Hsp70 members at the transcriptional as well as translational levels was significantly higher with AmB treatment than without AmB treatment, particularly in resistant A. terreus isolates, thereby indicating a role of Hsp70 proteins in the AmB response. We found that Hsp70 inhibitors considerably increased the susceptibility of resistant A. terreus isolates to AmB but exerted little impact on susceptible isolates. Also, in in vivo experiments, using the Galleria mellonella infection model, cotreatment of resistant A. terreus strains with AmB and the Hsp70 inhibitor pifithrin-μ resulted in significantly improved survival compared with that achieved with AmB alone. Our results point to an important mechanism of regulation of AmB resistance by Hsp70 family members in A. terreus and suggest novel drug targets for the treatment of infections caused by resistant fungal isolates.