Hunting for new antifungal strategies: the antifungal protein PAF

The small, cationic, cysteine-rich and antifungal protein PAF from Penicillium chrysogenum is toxic for plant- and opportunistic zoo- and human-pathogenic filamentous ascomycetes. Nuclear Magnetic Resonance (NMR) spectroscopy data and the use of mutant PAF protein variants indicated that distinct protein motifs on the surface of PAF regulate the interaction with fungi. Our recent data point towards a crucial role of the fungal plasma membrane in binding and internalizing PAF and in triggering signal transduction pathways in response to PAF. Our results further indicate that PAF specifically interacts with lipid components of the plasma membrane and significantly improves the efficacy of licensed membrane perturbing antifungals such as azoles when applied in combination in vitro. The antifungal activity and the fact that no detrimental effects of PAF were observed on primary mammalian cells renders this protein a promising candidate to develop new antifungal strategies applicable in medicine as well as in agriculture and in the food industry to prevent fungal contaminations and medicate fungal infections. Therefore, a detailed knowledge about the mechanism of action of PAF is an essential prerequisite for its future application. Our main objectives in our project are: A. Variation of PAF activity by site-directed mutagenesis - Generation of mutated PAF variants and large-scale expression in a P. chrysogenum paf deletion mutant for protein structure and function studies B. Characterization of the interaction of PAF with sensitive fungi - pharmacological approach to modulate the fungal lipid composition and to investigate the effect on PAF accessibility in test fungi - testing of fungal mutants that have defects in lipid metabolism for changes in PAF sensitivity - protein-lipid overlay assay to investigate the impact of distinct PAF protein motifs for lipid binding capacity in vitro - NMR spectroscopy to analyse the interaction of PAF and mutated PAF variants with fungal membrane factors - Fluorescence studies on the binding and internalization of PAF and mutated PAF variants in fungi C. Testing for increased drug efficiency of licensed antifungals in combination with PAF - in vitro assays to test combinations of PAF and licensed antifungal drugs - testing of PAF/antifungal drug combinations in insect models infected with Aspergillus sp. In addition to a detailed knowledge about the antifungal potential of PAF, our studies will significantly contribute to a better insight into specific aspects of cellular processes that are less well understood in filamentous model ascomycetes compared to other well characterized models like yeasts. Finally, our project will contribute to the improvement and refinement of NMR spectroscopy techniques.

The incidence of fungal infections has been rising dramatically for the last decades. By now a very limited number of licensed drugs are available to prevent and combat fungal infections. Therefore, new cost-effective antifungal strategies that lack severe side effects for the infected host are urgently needed. The Penicillium chrysogenum antifungal protein PAF is secreted by the penicillin producing mold P. chrysogenum and has high potential for the development of new antifungal strategies as it inhibits the growth of human-, zoo- and plant- pathogenic fungi. It is of crucial importance, however, to dissect in detail the structural and functional nature of PAF to take advantage of its antifungal potential or even improve its efficacy by rational design in the future. The aim of this project was to investigate in detail the link between PAF structure and function by analysing PAF variants in which distinct amino acids had been exchanged. To generate high yields of correctly folded PAF variants with high quality necessary for structural and functional analyses, we developed a protein expression system based on the PAF producer P. chrysogenum that allowed easy purification of the recombinant expressed PAF variants from the culture supernatant. The analysis of the protein structure by nuclear magnetic resonance (NMR) spectroscopy-based techniques revealed that the amino acid substitutions did not significantly change the 3D protein structure but had a significant impact on the features of the protein surface (e.g. surface charge) disturbing even protein parts far away from the mutation sites. These alterations of the protein surface affected the binding of PAF variants to the fungal cells, inhibited their uptake and interaction with intracellular signalling cascades and resulted in the loss of antifungal activity. We further observed that the visible structural state of PAF serves as pool for a low level of conformal variants that might be directly involved in protein function. We also investigated in more detail the observation that extracellular calcium ions neutralize PAF activity and found that PAF specifically binds calcium ions present in the test medium, but we could not relate this binding with any modulation of PAF function. The role of this calcium binding ability is still under investigation. In search for PAF target molecules, we analysed in detail the membrane lipid composition of the PAF- sensitive model fungus Neurospora crassa and documented its role in fungal fitness and PAF susceptibility, suggesting new antifungal targets for drug development. Finally, we characterized the structure and function of the antifungal proteins from the citrus-fruit infective molds Penicillium digitatum and Penicillium expansum which allowed to define structural motifs important for antifungal activity and provided a detailed insight into the species specificity of these antifungal proteins which have a high potential for application in food preservation and plant protection. Our results significantly contributed to understand the structure-function relation of PAF and other related antifungal proteins and paved the way to the generation of new antifungal drugs with a mode of action that differs from the existing ones, possibly preventing drug resistance development. Therefore, our research on the potential applicability of antifungal proteins from molds in agriculture and medicine to prevent and combat fungal infections has a major socio- economic impact on human life.