Antifungal Protein PAF Severely Affects the Integrity of the Plasma Membrane ofAspergillus nidulansand Induces an Apoptosis-Like Phenotype

Abstract

The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the ␤-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.A large number of small, basic, cysteine-rich antimicrobial proteins are produced by organisms throughout all kingdoms. They display a great variety in their primary structure, in species specificity, and in the mechanism of action.Few ascomycetes secrete strongly related antifungal proteins, which do not show any sequence homology with other antimicrobial proteins, but most of these proteins exhibit structural similarities (46): a net positive charge and numerous cysteine residues that are involved in disulfide bond formation. These properties contribute to a compact tertiary structure and a high stability against environmental impact and finally support the model of a membrane-disturbing nature. The antifungal protein PAF from P. chrysogenum and AFP from A. giganteus are the two most intensively studied peptides in the group of antifungals from ascomycetes, but the information available on their exact mechanism of action is still rather limited (32,55,68,69). PAF inhibits the growth of various important plant pathogenic and zoopathogenic filamentous fungi, e.g., Aspergillus fumigatus, A. niger, A. nidulans, and Botrytis cinerea. Previous studies revealed the induction of multifactorial detrimental effects on target organisms that include growth inhibition, reduction of cellular metabolism, severe changes in hyphal morphology, increased K ϩ efflux, and the generation of intracellular reactive oxygen species (ROS) (32, 48). PAF was found to...

Transcriptional profiling and functional analysis of heterokaryon incompatibility in Neurospora crassa reveals that reactive oxygen species, but not metacaspases, are associated with programmed cell death

“…Hyphal death during HI shows some features that are similar to apoptosis in mammalian cells, including shrinkage of plasma membrane, membrane-bound vesicle formation, DNA condensation and TUNEL-positive nuclei (Biella et al, 2002;Jacobson et al, 1998;Leslie & Zeller, 1996;Marek et al, 2003). Markers associated with apoptosis have also been reported upon exposure of various filamentous fungi to treatment with H 2 O 2 , acetic acid, farnesol, sphingolipids and amphotericin B (Castro et al, 2008;Chen & Dickman, 2005;Cheng et al, 2003;Leiter et al, 2005;Mousavi & Robson, 2004;Phillips et al, 2003;Semighini et al, 2006).…”

In vivo application of a small molecular weight antifungal protein of Penicillium chrysogenum (PAF)

“…In fungal colonies treated by PAF the number of cells showing apoptotic signs including increased phosphatidylserine externalization and DNA fragmentation are increased compared to control colonies while the proportion of the necrotic cells remains the same (10).…”

“…It causes an increased production of reactive oxygen radicals, reduces the metabolism of the fungal cells, and initiates an increased potassium efflux (8). PAF enters the cells via an active transport mechanism (16) and acts intracellularly, probably through heterotrimeric G-protein signaling (10,14). It causes the hyperpolarisation of the membranes of the fungal cells, which leads to the disintegration of the membranes and, consequently, an apoptosis-like phenotype appears.…”

Production of the biotechnologically relevant AFP from Aspergillus giganteus in the yeast Pichia pastoris

“…Concerning AFP, it has been reported that this protein enters the fungal cells by its ability to interact with phospholipid membranes [11] thus causing membrane permeabilization in the target fungal cell [15,23]. In other studies, it was reported that AFP binds fungal cell wall chitin [24].…”

“…Apparently, the AFP protein enters into the fungal cell and targets the nucleus, as revealed by co-localization experiments of Alexa-labeled AFP with the SYTOX Green dye [15]. AFP would bind fungal nucleic acids promoting charge neutralization and condensation of DNA [15,20], maybe triggering apoptosis [23]. The available data suggest that it is this combination of fungal cell permeabilization, cell-penetrating ability and nucleic acid-binding activity of AFP that determines its potent antifungal activity.…”