Structure and function of the antifungal proteins PAFB and 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 that lead to severe medical problems and enormous economic losses strongly demand for the development of new antifungal strategies. The novel antifungal proteins PAFB of Penicillium chrysogenum and NFAP of Neosartorya fischeri NRRL 181 have great potential for application in these fields. However, the solution structure, the structure-function relation and the antifungal mechanisms of action of PAFB and NFAP have not been investigated in detail so far. The proposed project aims at (1) investigating the solution structure, dynamics, folding/unfolding of PAFB and NFAP, (2) identifying core elements that are responsible for the correct folding and the interaction of the proteins with their fungal targets and (3) making first steps towards the characterization of potential fungal targets. We will address these aims by applying sophisticated and state-of-the-art NMR-based technology and by performing functional tests. To this end recombinant PAFB and NFAP will be generated in well-established fungal expression systems. Our approach will enable us to compare in detail the specific features of PAFB and NFAP with those of already characterized antifungal proteins and define similarities and differences in the mode of antifungal action. The bilateral collaboration between the Hungarian and Austrian expert team promise the successful execution of the proposed objectives by applying a truly interdisciplinary approach. The generated data of this study will significantly contribute to understand the diverse mechanisms of action and the structure-based biological function of this novel group of antifungal proteins. The development of new antifungal strategies, the design and patent registration of new, synthetic, effective, target specific antifungal compounds with low or negligible side-effects can be expected.

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 fungi are eukaryotes and their cellular structure and physiology resembles that of the host. Or they become ineffective due to a fast resistance development of the pathogens. Therefore, new antifungal compounds exhibiting a different mechanism of action to the existing ones and well tolerable by the infected host are urgently needed. Moulds are a rich source for small, cysteine-rich and cationic antimicrobial proteins (AMPs) that represent promising candidates for new antifungal strategies. It is of crucial importance, however, to dissect in detail the structural and functional nature of AMPs to take advantage of their antifungal potential and improve their efficacy by rational design. The aim of this project was to characterize new AMPs from moulds, investigate in more detail the relation between the structure and function of already identified AMPs and provide a proof-of-principle of their applicability. This necessitates the generation of high AMP yields of high quality. Therefore, a protein expression system was developed based on the AMP producer Penicillium chrysogenum. This system allows easy purification of the recombinant expressed AMPs from the culture supernatant and enables the generation of AMP variants for the investigation of the impact of distinct amino acids on the structure-function. We produced and studied the AMPs PAFB from P. chrysogenum, NFAP and NFAP2 from Neosartorya fischeri, AfpB from Penicillium digitatum and variants of the P. chrysogenum PAF. The analysis of the solution structure of these AMPs by nuclear magnetic resonance revealed a common characteristic tertiary structure resembling the known 3D structure of PAF. For full antifungal activity, the charge distribution at the protein surface is of crucial importance and guarantees the interaction of the positively charged AMP with the negatively charged membrane of the fungal target cell. We further characterized the structure-function of AfpB, defined structural motifs important for antifungal activity and determined the species specificity. Finally, we successfully proved the applicability of NAFP2 in a vulvovaginitis mouse model to reduce the load of the human-pathogen Candida albicans. In search for AMP interaction molecules which may be considered as new antifungal targets for drug development, we analysed the composition of the membrane lipids of the AMP-sensitive model fungus Neurospora crassa and documented the membrane lipid role in fungal fitness and susceptibility to PAF and PAFB, both showing a different mode of action. Our study proved to have major socio-economic impact on human life as it significantly contributed to understand the structure-function relation of AMPs and paved the way to the development of new antifungal strategies.