The activity of the plant defensin NaD1 against human fungal pathogens

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Biochemistry, School of Molecular Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The plant defensin NaD1 is a small cysteine-rich peptide that has potent antifungal activity against pathogens of plants. Its mechanism of action has been partially defined against the plant pathogen Fusarium oxysporum f. sp. vasinfectum (Fov). It has a three step mechanism which involves binding to the hyphal cell wall, disruption of the plasma membrane and entry into the cytoplasm, where reactive oxygen species are produced. The primary aim of this thesis was to evaluate whether NaD1 has potential for the treatment of human fungal diseases and to shed more light on the mechanism of action. This study revealed that NaD1 inhibits the growth of a number of fungal pathogens which affect human health, including Candida albicans, which can cause invasive infections of mucosal tissues, and species of Cryptococcus and Aspergillus, which can cause meningitis and infect the lungs. NaD1's activity against yeast allowed the use of several experimental techniques to which yeast cells are more amenable than filamentous fungi, including time-lapse microscopy and flow cytometry. Furthermore, libraries of mutants and advanced genomic data are available. This led to the discovery that NaD1's three step mechanism of action initially observed in Fov is conserved in C. albicans, so findings in yeast are also likely to apply to filamentous fungal species. Using confocal microscopy, cellular inhibitors and deletion mutants it was revealed that NaD1 enters the cytoplasm of C. albicans, and most likely does so by endocytosis. After entering the cytoplasm, retrograde transport is not required for NaD1's antifungal activity. Screening of a partial C. albicans deletion mutant library revealed that the high-osmolarity glycerol stress response pathway protects the yeast from the deleterious effects of NaD1. The role of various stress pathways in enhancing tolerance to antifungal peptides and fungicides was reviewed and led to the hypothesis that manipulation of these stress response pathways could be used to enhance the activity of antifungal molecules.