Plant defensins are small, cationic peptides with a highly conserved 3D structure. They have been studied extensively in the past decades. Various biological activities have been attributed to plant defensins, such as anti-insect and antimicrobial activities, but they are also known to affect ion channels and display antitumor activity. This review focuses on the structure, biological activity and antifungal mode of action of some well-characterized plant defensins, with particular attention to their fungal membrane target(s), their induced cell death mechanisms as well as their antibiofilm activity. As plant defensins are, in general, not toxic to human cells, show in vivo efficacy and have low frequencies of resistance occurrence, they are of particular interest in the fight against fungal infections.

The incidence of invasive fungal infections is increasing worldwide, resulting in more than 1.6 million deaths every year. Due to growing antifungal drug resistance and the limited number of currently used antimycotics, there is a clear need for novel antifungal strategies. In this context, great potential is attributed to antimicrobial peptides (AMPs) that are part of the innate immune system of organisms. These peptides are known for their broad-spectrum activity that can be directed toward bacteria, fungi, viruses, and/or even cancer cells. Some AMPs act via rapid physical disruption of microbial cell membranes at high concentrations causing cell leakage and cell death. However, more complex mechanisms are also observed, such as interaction with specific lipids, production of reactive oxygen species, programmed cell death, and autophagy. This review summarizes the structure and mode of action of antifungal AMPs, thereby focusing on their interaction with fungal membranes.

HsAFP1, a plant defensin isolated from coral bells (Heuchera sanguinea), is characterized by broad-spectrum antifungal activity. Previous studies indicated that HsAFP1 binds to specific fungal membrane components, which had hitherto not been identified, and induces mitochondrial dysfunction and cell membrane permeabilization. In this study, we show that HsAFP1 reversibly interacts with the membrane phospholipid phosphatidic acid (PA), which is a precursor for the biosynthesis of other phospholipids, and to a lesser extent with various phosphatidyl inositol phosphates (PtdInsP’s). Moreover, via reverse ELISA assays we identified two basic amino acids in HsAFP1, namely histidine at position 32 and arginine at position 52, as well as the phosphate group in PA as important features enabling this interaction. Using a HsAFP1 variant, lacking both amino acids (HsAFP1[H32A][R52A]), we showed that, as compared to the native peptide, the ability of this variant to bind to PA and PtdInsP’s is reduced (≥74%) and the antifungal activity of the variant is reduced (≥2-fold), highlighting the link between PA/PtdInsP binding and antifungal activity. Using fluorescently labelled HsAFP1 in confocal microscopy and flow cytometry assays, we showed that HsAFP1 accumulates at the cell surface of yeast cells with intact membranes, most notably at the buds and septa. The resulting HsAFP1-induced membrane permeabilization is likely to occur after HsAFP1’s internalization. These data provide novel mechanistic insights in the mode of action of the HsAFP1 plant defensin.

Retrieving single cells of interest from an array of microwells for further off-chip analysis is crucial in numerous biological applications. To this end, several single cell manipulation strategies have been developed, including optical tweezers (OT). OT represent a unique approach for contactless cell retrieval, but their performance is often suboptimal due to nonspecific cell adhesion to the microwell surface. In this study, we focused on improving the surface chemistry of microwell arrays to ensure efficient single cell manipulation using OT. For this purpose, the surface of an offstoichiometry thiol-ene-epoxy (OSTE+) microwell array was grafted with polyethylene glycol (PEG) molecules with different molecular weights: PEG 360, PEG 500, PEG 2000, and a PEG Mix (an equimolar ratio of PEG 500 and PEG 2000). Contact angle measurements showed that the PEG grafting process resulted in an increased surface energy, which was stable for at least 16 weeks. Next, cell adhesion of two cell types, baker's yeast (Saccharomyces cerevisiae) and human B cells, to surfaces treated with different PEGs was evaluated by registering the presence of cellular motion inside microwells and the efficiency of optical lifting of cells that display motion. Optimal results were obtained for surfaces grafted with PEG 2000 and PEG Mix, reaching an average fraction of cells with motion of over 93% and an average lifting efficiency of over 96% for both cell types. Upon the integration of this microwell array with a polydimethylsiloxane (PDMS) microfluidic channel, PEG Mix resulted in proper washing of non-seeded cells. We further demonstrated the wide applicability of the platform by manipulating non-responding yeast cells to antifungal treatment and B cells expressing surface IgG antibodies. The combination of the optimized microwell surface with continuous microfluidics results in a powerful and versatile platform, allowing high-throughput single cell studies and retrieval of target cells for off-chip analysis.