Psd1 is a basic, cysteine-rich plant defensin isolated from Pisum sativumseeds which inhibits the growth of a broad range fungi species. Defensins are also non-toxic to mammalian cells, highlighting their potential as antifungal drugs. We have shown that FITC-labelled Psd1 was internalized in F. solani hyphae, interacting with cyclin F and leading to fungal cell cycle arrest. This internalization seemed to be dependent of glucosylceramide (CMH, of cerebroside monohexoside), once C. albicans cells lacking the ceramide synthase are 25 % less susceptible to Psd1 than the parental strain. Fungal and mammalian CMH are structurally divergent, as the former presents a C8-unsaturation and C9-methylation on the sphingoid base, which could possibly drive Psd1 selectivity. In this work, we investigated the cell death mechanisms triggered by Psd1 in Aspergillus nidulans and the contribution of CMH structure to Psd1-induced fungal death. We characterized, through fluorescence microscopy, several apoptotic events, such as intense formation of reactive oxygen species (ROS), metacaspase activation and DNA strand breaks. Although A. nidulans hyphae treated with 20 mM Psd1 for 24 hours exhibited severe cell injury, no apoptosis-phenotype was observed. We also investigated whether Psd1 incubation would lead to membrane permeabilization typical of a necrotic death. To this, A. nidulans cells were maintained in the presence or absence of the peptide and the membrane damage was evaluated through Propidium Iodide (PI) staining. We observed 15 % PI positive cells in the suspension treated with Psd1, in contrast to 2 % in control culture. To investigate the role of fungal CMH and its structural modifications to Psd1-induced cell death, we constructed strains lacking the glucosylceramide synthase (ANID_08806), sphingolipid DD8-desaturase (ANID_04592) and sphingolipid C9-methylase (ANID_05688 and ANID_07375) genes. Phenotype analysis showed impaired growth of strains deficient in ANID_08806 and ANID_04592 in comparison to the parental strain. Further investigation will be conducted to characterize Psd1 antifungal activity and apoptosis or necrosis induction in the mutant strains. Unraveling the mechanisms of cell death induced by antifungal peptides may lead to the identification of new targets that drive antimycotic selectivity.
Full conference title:
27th Fungal Genetics Conference
- Fungal Genetics Conference 27th (2013)