The gamma-core motif of antifungal proteins of Ascomycetes

Fungal infections pose a severe threat to our health and food supply. Small, cysteine-rich, cationic antifungal proteins (AFPs) from molds (belonging to the Ascomycetes) are suitable to combat fungal infections in plants and humans/animals. A detailed structural and functional analysis is essential for their potential application as novel antifungal agents and in new therapies. AFPs contain a structurally conserved motif that resembles the Greek letter : the -core. The consensus sequence is evolutionary highly conserved. This points towards a central structural and functional role of this motif, which we want to investigate in this study. We hypothesise that (i) the -core motif determines the mode of action of AFPs; (ii) the function of AFPs can be improved by the modification of the amino acid sequence of the -core motif; (iii) short synthetic peptides spanning only the -core motif have antifungal activity per se; (iv) the activity of synthetic -core peptides can be improved by rational design; (v) improved AFPs and synthetic -core peptides are suitable to combat fungal infections in plants and humans/animals. We will address our hypotheses on the basis of two intensively studied AFPs, the PAF from Penicillium chrysogenum and the NFAP from Neosartorya fischeri. We plan to change the amino acid sequence of the -core motif of these two proteins to investigate the impact of the mutations on the structure and mode of action of PAF and NFAP and to improve their antifungal efficiency. We will further design and synthesise -core peptides with enhanced antifungal activity and characterize their function. Finally we will provide a proof-of-principle for the applicability of the best candidates of engineered AFPs and rationally designed -core peptides to inhibit plant- pathogen infection and fungal toxin (mycotoxin) production in crops and fungal infection of the human skin (dermatophytosis). To this end the application of plant infection models and human skin models is planned. We will achieve our goals with a multidisciplinary approach that integrates state-of-the-art technology including bio-informatics, peptide design and synthesis, genetic engineering, biophysics, biochemistry, cell biology, antifungal and cell toxicity validation. The accomplishment of our objectives will allow important new insights into the functional and structural role of the highly conserved -core motif and provide the first steps towards a biotechnological application by developing bioactive molecules with worldwide economic and societal impact.

The incidence of fungal infections in humans, animals and plants has been rising dramatically for the last decades. Only a limited number of licensed drugs are available to combat fungal infections. Some of them exhibit severe side effects, because the cellular structure and physiology of fungi - being eukaryotes - resemble that of the host. Furthermore, fungal pathogens often develop resistance mechanisms against these drugs, which then become ineffective in the treatment of fungal infection. Therefore, new antifungal compounds with new modes of action that are well tolerated by the infected host are urgently needed. Moulds are a rich source for small, cysteine-rich and cationic antimicrobial proteins (APs) that represent promising candidates for new antifungal strategies. It is of crucial importance, however, to dissect in detail the structural and functional nature of APs to be able take advantage of their antifungal potential and improve their efficacy by rational design. The aim of this project was to study the role of an evolutionary conserved structural motif (-core) in the structure-function relation of APs of fungal origin. The exchange of specific amino acids in the -core of the P. chrysogenum antifungal proteins PAF, PAFB, and PAFC, and the Neosartorya fischeri antifungal proteins NFAP and NFAP2, revealed that this motif is essential for the correct folding of the APs which is a prerequisite for full protein function. For full antifungal activity, the charge distribution at the protein surface is of crucial importance and guarantees the interaction of the positively charged APs with the negatively charged membrane of the fungal target cell. The analysis of the physicochemical properties and the antifungal activity of chemically synthesized short peptides (Ps) spanning the -core-motif and other parts of the APs proved that - depending on the APs' classification - this motif specifically determines the APs' antifungal activity. APs and Ps were successfully applied in in vitro and in vivo test models to prevent or treat fungal infections in plants, fruits, and animals. A breakthrough in the screening of APs and Ps for applicability was the use of 3D reconstructed human skin models that help to reduce breeding costs and the number of animals sacrificed, an approach which overcomes ethical, regulatory, practical or economic limitations that are linked with animal experiments. Our study might have major socio-economic impact on human life as it significantly contributed to a comprehensive unterstanding of the structure, function and applicability of APs from moulds and newly designed Ps thereof which may pave the way to the development of new antifungal strategies.