Closed projects

The increasing incidence of fungal infections and contaminations due to drug-resistant filamentous fungi in medicine, agriculture and food industry urges the development of new antifungal strategies.

The highly stable, extracellular, cysteine-rich antifungal proteins from filamentous Ascomycetes (AFPs) offer an alternative, safely applicable solution. Our in silico investigations revealed that all isolated AFPs contain an evolutionary conserved [GXC]-[X3-9]-[C] consensus γ-core motif. Our preliminary results demonstrated that the antifungal efficacy of AFPs possibly depends on the physical and chemical properties of the γ-core constituting amino acids.

Based on this we hypothesize that the antifungal efficacy of AFPs is improvable with rational design of the γ-core motif, and the improved AFPs and γ-core peptides are applicable as antifungal drugs, biopesticides and crop preservatives. We address our hypotheses on the basis of the intensively studied AFPs, the PAF from Penicillium chrysogenum and the NFAP from Neosartorya fischeri. We plan to change the amino acid sequence of the γ -core motif of these two proteins to create new AFP variants with improved antifungal efficiency. We further investigate the structure and function of designed synthetic γ -core peptides that show enhanced activity. Finally, we provide a proof-of-principle for the applicability of the best candidates of engineered AFPs and rationally designed γ -core peptides to inhibit plant-pathogen infection and mycotoxin production in crops and dermatophytosis.

The achievements in this project allow further steps towards the biotechnological application of AFPs.


Supported by Hungarian National Research, Development and Innovation Office; and FWF Austrian Science Fund.

Presumably as a consequence of climate changes the occurrence of resistant phyto-, pre- and postharvest pathogenic fungi and the number of mycotoxin contaminated feeds and foods are continuously increasing in Europe in the last years causing loss of billions of Euro and posing severe risks to human and animal health. These facts urge the development of new, and more effective antifungal strategies.

The highly stable, extracellular, cysteine-rich antifungal proteins from filamentous Ascomycetes (crAFPs) could offer an alternative, safely applicable solution. Our in silico investigations revealed that all isolated crAFPs contain an evolutionary conserved [GXC]-[X3-9]-[C] consensus γ-core motif. Our preliminary results demonstrated that the antifungal efficacy of crAFPs depends on the physical and chemical properties of the γ-core constituting amino acids.

Based on this we hypothesize that the antifungal efficacy of crAFPs is improvable with rational design of the γ-core motif, and the improved crAFPs are applicable as biopesticides and crop preservatives in the agriculture and food industry. We address this question by investigating different crAFPs produced by Neosartorya fischeri, their γ-core improved variants, and their γ-core peptides by antifungal susceptibility, haemolytic activity, cytotoxicity tests, plant and crop model experiments.

The achievements in this project allow further steps towards the biotechnological application of crAFPs, and open up a completely new avenue for the development of bioactive proteins and peptides with worldwide economic and societal impact on pest control and crop preservation.


Supported by Hungarian National Research, Development and Innovation.

The increased incidence of severe fungal infections and the fast development of drug resistant filamentous fungi causing mycoses, plant infections or damage of cultural heritages strongly demand for the development of new antifungal strategies.

Small, cysteine-rich, highly stable antifungal proteins secreted by filamentous Ascomycetes have great potential for application in these fields. The antifungal protein NFAP from the Neosartorya fischeri NRRL 181 isolate is a novel representative of this protein group.

In our previous work we demonstrated that NFAP effectively inhibits the growth of numerous filamentous Ascomycetes including potential human and plant pathogens, and its antifungal effect is dose-dependent and strongly influenced by the extracellular mono- and divalent cation concentration. In susceptible fungi, NFAP causes damage to the cell wall by destructing chitin filaments and triggers apoptotic-necrotic pathways by intracellular accumulation of reactive oxygen species. However, we could also show that NFAP differs in its antifungal spectrum, its antifungal mode of action and its tertiary structure from the two most investigated NFAP-related proteins, the Aspergillus giganteus antifungal protein AFP and the Penicillium chrysogenum antifungal protein PAF. Further efforts are needed to characterize in detail the solution structure of NFAP, its structure-function relation, the primary targets and the antifungal mode of action, which have not been investigated in detail so far.

The present project aims to clarify two aspects: (1) the connection between the protein structure and the antifungal properties; and (2) the identification of target molecules of NFAP. We address the first question by investigating the role of structural features of NFAP by nuclear magnetic resonance, thermal unfolding experiments and antimicrobial susceptibility tests. To this end distinct recombinant NFAP protein mutants are generated for structure-function investigations. The second question we address by molecular screening of (i) potential lipid targets of NFAP via in vitro protein-lipid overlay assays and (ii) protein receptors by chemical cross-linking, affinity purification, and differential mass spectrometry.

The results significantly contribute to the understanding of the mode of action and structure-function relation not only of NFAP but also of other cysteine-rich antifungal proteins from Ascomycetes in general. Taking into account the wide distribution of antimicrobial proteins in nature, our project helps to solve problems that are also relevant for related antimicrobial proteins.

A detailed insight into the structure, function, interaction with target molecules and antifungal mechanisms of more members of this new protein group is an essential prerequisite for the identification of protein motifs with specific functions. This ultimately enables the construction of synthetic or chimeric proteins with improved and specific antimicrobial potential for medical therapy, pest control, food preservation and conservation of cultural heritages in the near future and the outcome of our project promises patent registration.


Supported by FWF Austrian Science Fund.

Defensins and similar antimicrobial proteins are widely distributed in the nature. Five similar proteins have been identified among filamentous fungi. These proteins secreted by taxonomical distinct species have different mode of action and species specificity, nevertheless their structure is very similar.

Neosartorya fischeri antimicrobial protein (NFAP) from N. fischeri (anamorph: Aspergillus fischerianus) is a hypothetical antifungal protein derived from genomic database, its presence is confirmed by in silico investigation only. Its cloning, isolating, antifungal spectrum, mode of action and biological role has not been described yet.

The goal of the proposed project is to increase the knowledge of defensin-like antimicrobial proteins derived from filamentous fungi by investigation of NFAP. The main aims of the research project are the followings: (1) determination of the presence of NFAP and its orthologues from Neosartorya fischeri (NRRL 181) and related species; (2) investigation of the antimicrobial properties and mode of the action in case of NFAP; (3) creation a heterologous expression system for production of NFAP; (4) examination of the connection between the structure of NAFP and its efficacy; (5) observation of biological role of NFAP.

The final results provide information about the distribution of defensin-like antimicrobial proteins among filamentous fungi. The results about the structure-efficacy connection will be the bases of further theoretical and practical studies to develop effective antimicrobial drugs. Achievements of this project provide important information in addition to microbiology.


Supported by the Hungarian Scientific Research Fund.