PAF, an antifugal protein, secreted by the filamentous fungus Penicillium chrysogenum

Molds are found ubiquitously in nature and contribute to the decay of organic material. They play an important role in the spoilage of nutrients as well as in the infestation of plants. At the same time, these fungi, mainly Aspergilli, represent dangerous pathogenic agents, which may damage inner organs, e.g. the lungs, in people with an impaired immune system. During the last years the incidence of life-threatening opportunistic fungal infections increased dramatically. The rise in cases has been particularly apparent in immunocompromised patients, e.g. people with HIV infection or with immunosuppressive medication, e.g. for organ transplantations or in cancer therapies. These systemic fungal infections are difficult to treat and may be fatal, as only a restricted number of antimycotic drugs are available so far. Our scientific group works on the identification of new biotechnologically useful proteins and agents, secreted by filamtentous fungi. One of the most important organism in this respect is Penicillium chrysogenum, which is used in the production of antibiotics, i.e. Penicillin. We have been able to isolate a protein from the culture supernatant of this organism, which shows strong growth inhibitory properties towards molds, such as Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger. So far, no information on the exact mode of action of this protein is available. However, due to its activity it might be used as a new antimycotic drug as well as for food preservation or for the generation of plants, resistant to phytopathogenic fungi. Therefore, we will investigate the structure and function of the protein by genetical, biochemical and cell biological methods. The knowledge of the exact way of action is an important prerequisite to consider this protein as a promising candidate for medical applications or for the development of new strategies against fungal infection and other harmful fungal effects.

The Penicillin-producing filamentous fungus Penicillium chryosgenum abundantly secretes the small, compact and highly stable protein PAF into the supernatant. PAF inhibits the germination of conidia as well as the growth of hyphae of numerous plant- and zoopathogenic molds, i.e. Botrytis cinerea, Aspergillus (A.) fumigatus, A. niger, A. nidulans. Growth inhibition at sublethal concentrations is associated with a severe change in morphology which becomes apparent as crippled hyphae with atypical branching. An A. nidulans strain carrying a mutated protein (heterotrimeric G-protein) that plays a central role in the signal transduction cascade (G-protein mediated signalling) shows reduced sensitivity towards PAF. This strengthens our hypothesis that specific receptors exist which are resident in the outer cellular layer (plasma membrane) and mediate specificity and toxicity of PAF. Further investigations revealed that growth inhibition in sensitive organisms is associated with a strongly regulated mechansim which leads to a programmed cell death (apoptosis) similar to that observed in mammalian cells. We proved the intracellular accumulation of oxidative radicals after the application of PAF. This could be one major reason for the induction of apoptosis apart from the involvement of the signal transduction cascade mentioned above. Oxidative radicals damage biomolecules, i.e. proteins, lipids, nucleotides, which in consequence results in the malfunction and damage of cellular structures, such as the nucleus, mitochondria, plasma membrane. Furthermore, we detected a change in distict properties of the plasma membrane after incubation with PAF. We measured an increased leakage for potassion ions which could explain a significant change of the plasma membrane potential (hyperpolarization). Finally, we showed that the antifungal protein PAF was actively internalized by sensitive fungi. This uptake- mechanism was enery (ATP) dependent and resembled the receptor-mediated uptake mechansims of various substances and molecules in mammalian cells (receptor-mediated endocytosis). Due to the fact that PAF is highly stable and that we did not detect any detrimental effects in cultured mammalian cells, renders PAF a promising candidate for the development of a novel antifungal therapy applicable in medicine as well as in agriculture and in the food industry to prevent and treat fungal infections. Importantly, new antifungal drugs are urgently needed since the incidence for life-threatening fungal infections has been rising dramatically during the last decades. On the one hand this is due to improvements in life-saving intensive care at the cost of the immune system which in consequence elevates the predisposition for microbial infections. On the other hand, pathogenic fungi develop resistance towards conventionally applied antifungal drugs. Therefore, the detailed characterization of the mode of action of PAF is of crucial importance and a prerequisite for its successful future application or for the development of new therapeutic approaches.