New antifungal strategies: structure and function of NFAP

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 proposed 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 will 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 will be generated for structure-function investigations. The second question we will 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 will 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 will help 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.

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. The NFAP from the mold Neosartorya fischeri (class Ascomycetes) is a member of the small, cysteine-rich, cationic and highly stable antifungal proteins. NFAP shows remarkable antifungal activity against numerous filamentous fungi. Features of NFAP render it exceptionally suitable as potential commercial preservative, bio-pesticide and drug against molds. A detailed study of the structure, structure-function relationship and potential targets of NFAP in sensitive fungi are still missing, although they are essential prerequisites to improve NFAP efficacy by drug design. This project focused on the investigation of these aspects. We developed a novel Penicillium chrysogenum-based expression system, which allows cost-effective bulk production of cysteine-rich antifungal proteins for structural and functional analyses in a microorganism that is generally recognized as safe by the US Food and Drug Administration. From an economic view, the generation of high yields of antifungal proteins by fungal fermentation is more cost-effective than protein synthesis and our system could be easily adapted by the industry. By using this this system, we resolved the compact solution structure of the highly stable and antifungal active NFAP. We further identified the essential structural determinants for the formation of the antifungal active compact structure of the NFAP, which allows to improve the antifungal activity and target-specificity of this bio-molecule by rational protein design or the synthesis of antifungal peptides. Target screening experiments revealed that NFAP might disturb mitochondrial function in sensitive fungi. This finding provides a potential new drug-target candidate for the pharmaceutical industry. A further milestone in the identification of new small, cysteine-rich, antifungal proteins was the isolation and characterization of NFAP2 from Neosartorya fischeri exhibiting high anti-yeast activity against clinically relevant human pathogen Candida species. Thus, NFAP2 represents a highly interesting and promising antifungal compound for the development of new anti-yeast strategies. Our achievements regarding NFAP in this project will be supportive for the detailed analysis of NFAP2 in the near future. Our results significantly contribute to the understanding of the mode of action and structure-function relation not only of NFAP but - considering the structural similarity - also of other cysteine-rich antifungal proteins in general, and pave the way for the development of new antifungal strategies.