Characterization of the toxicity of PAF

Our research in recent years has been focused on the investigation of the molecular mechanism of action of the low molecular weight, cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum which belongs with other peptides from P. nalgiovense (NAF), Aspergillus niger (ANAFP) and A. giganteus (AFP) to a new group of antimicrobial proteins. The model organism A. nidulans, the opportunistic zoo-pathogen A. fumigatus and numerous plant-pathogenic fungi belong to the most PAF-sensitive molds. Thus PAF bears great potential for the development of novel antifungal therapies applicable in medicine as well as in agriculture and in the food industry to prevent and treat fungal infections. Apart from its biotechnological potential, PAF turned out to be a valuable tool for studies in fungal cell biology. We could show that growth inhibition in sensitive organisms is associated with a strongly regulated mechanism which leads to a programmed cell death (apoptosis) that is preceeded by the hyperpolarization of the membrane at the hyphal tips, the activation of ion channels and the generation of elevated levels of reactive oxygen species. The interaction of PAF with the target organism A. nidulans involves heterotrimeric G-protein signaling and is accompanied by the active internalization of the protein. Furthermore, PAF leads to a severe change of the hyphal morphology at sublethal concentrations and evokes a hyperbranched phenotype. In the course of further investigations we could detect a PAF-specific elevation of the intracellular calcium concentration in the target organism A. nidulans. This could elicit the perturbation of the intracellular calcium gradient which may account for the grave consequences for the morphology and the survival of the target organism. In addition, we proved a calcium-dependent neutralization of the toxicity of the antifungal protein which underlines a central role of calcium in PAF-activity. So far, calcium signaling has been studied only in few fungal species and detailed knowledge is lacking. A detailed and complete characterization of the interaction of PAF with target organisms is an important prerequisite to understand its mechanism of action and to develop new strategies for an efficient antifungal therapy. Based on our preliminary results, we propose in the present project to further characterize the PAF-elicited changes in the intracellular calcium concentration by using A. nidulans expressing the calcium sensitive photoprotein aequorin. Furthermore, we will perform a random mutagenic screen for PAF-resistant A. nidulans mutants to identify and characterize further molecular targets of PAF. The PAF-induced differential gene expression will be studied in detail by a genome-wide gene expression analysis (microarray analysis). Finally, we plan to analyze the tertiary structure of PAF and of mutated protein forms of PAF to identify those structural motifs that determine its antifungal toxicity.

The antibiotic producing filamentous fungus Penicillium chrysogenum abundantly secretes the small, cationic and cysteine-rich protein PAF into the supernatant. PAF has antifungal activity and inhibits the conidial germination and hyphal growth of numerous plant-, animal- and human-pathogenic molds. The goal of the completed project was to study the solution structure of PAF and to identify protein motifs that regulate the interaction of PAF with target fungi.Furthermore, we investigated signalling pathways involved in the transduction of a PAF-specific response with special emphasis on the characterization of the PAF effect on the fungal calcium (Ca2+) homeostasis. Finally we started to identify gene markers that render yeasts PAF resistant by screening a Saccharomyces cerevisiae mutant library and we analysed the regulation of gene transcription in response to PAF by microarray analysis to determine and further characterize putative fungal targets in the PAF-sensitive human-pathogen Aspergillus fumigatus. Both experiments are still in progress.We successfully analysed the solution structure of PAF by Nuclear Magnetic Resonance (NMR) spectroscopy. PAF shows a compact structure that is stabilized by three disulfide bonds formed between six cysteine residues. The compact structure contributes to high protein stability under extreme environmental conditions (high temperature, wide pH range and protease digestion). A positively charged lysine-rich motif on the PAF surface is essential for full biological activity.By the analysis of fungal cell signalling mutants and by a pharmacological approach we demonstrated that PAF activates the protein kinase A mediated signalling cascade which is directly involved in Ca2+ signalling and programmed cell death and thus contributes to the antifungal activity of PAF. We intensified our studies by analysing the detrimental effect of PAF on the intracellular Ca2+ homeostasis in sensitive fungi and proved that PAF triggers a sustained elevation of the intracellular Ca2+ concentration that is directly linked with fungal growth inhibition. We could show that PAF related antifungal proteins similarly affect cell signalling and Ca2+ homeostasis, but we also reported differences in their antifungal mechanism of action compared to PAF. The antifungal protein AFPNN5353 from Aspergillus giganteus, for example, activates the cell wall integrity pathway (CWIP) in sensitive fungi that leads to the fortification of the cell wall.This effect could not be verified for PAF. PAF does not trigger the CWIP, but its elementary activity mediates basic resistance towards low PAF concentrations.Bioinformatical analysis of the genome wide gene expression analysis of PAF-exposed A. fumigatus revealed the deregulation of genes involved in metabolism, genetic information processing, environmental information processing, mitochondrial function, fatty acid and lipid metabolism and cellular processes (cell cycle, growth and cell death). The effect of PAF on the mitochondrial function was further investigated and indicated that PAF negatively affects fungal respiration and ATP production.Finally, we started to characterize a new, so far un-described function of small, secreted and cysteine-rich proteins. PAF and the Aspergillus nidulans Anisin1 are involved in the regulation of asexual development and stress response in the producing fungi themselves. Thus such