Psd1 binding affinity toward fungal membrane components as assessed by SPR: The role of glucosylceramide in fungal recognition and entry

Abstract

Psd1 is a plant defensin that has antifungal activity against several pathogenic and nonpathogenic fungi. Previous analysis of Psd1 chemical shift perturbations by nuclear magnetic resonance (NMR) spectroscopy demonstrated that this defensin interacts with phospholipids and the sphingolipid glucosylceramide isolated from Fusarium solani (GlcCer(Fusarium solani)). In this study, these interactions were evaluated by real-time surface plasmon resonance (SPR) analysis. The data obtained demonstrated that Psd1 could bind more strongly to small unilamellar vesicles (SUV)-containing GlcCer(Fusarium solani) than to SUV that was composed of phosphatidylcholine (PC) alone or was enriched with GlcCer that had been isolated from soybeans. An increase in the SPR response after cholesterol or ergosterol incorporation in PC-SUV was detected; however, SUV composed of PC:Erg (7:3; molar:molar) became unstable in the presence of Psd1, suggesting membrane destabilization. We also observed a lack of Psd1 internalization in Candida albicans strains that were deficient in the glucosyl ceramide synthase gene. Together, these data indicate that GlcCer is essential for Psd1 anchoring in the fungal plasma membrane as well as internalization.

Psd1 Effects on Candida albicans Planktonic Cells and Biofilms

“…Experiments were performed in triplicate and values were analyzed with GraphPad Prism 5, using the Gompertz equation for MIC determination (Lambert and Pearson, 2000). Experiments with Ps d1 were performed at 90 μg/ml (20 μM) (de Medeiros et al, 2014). …”

“…crassa , but not against several tested bacteria (Almeida et al, 2000, 2002; Lobo et al, 2007; de Medeiros et al, 2010). Ps d1 at 20 μM has been shown to cause a 100% growth inhibition of C. albicans wild type (WT), while having a 70% inhibition of its corresponding C. albicans gcs -deleted strain (Δ gcs ) (de Medeiros et al, 2014). Recently, we demonstrated through membrane partition studies that Ps d1 has high affinity and specificity for membranes with ergosterol and GlcCer, as in fungal cells (Gonçalves et al, 2012).…”

“…A lack of Ps d1 internalization in C . albicans mutant strain (Δ gcs ) has been shown by confocal microcopy (de Medeiros et al, 2014). Together, these results indicated that GlcCer is important for Ps d1 interaction with the fungal plasma membrane, as well as for its internalization.…”

Antifungal membranolytic activity of the tyrocidines against filamentous plant fungi

“…Binding the GlcCer could have downstream effects on the fungal pathogen, particularly as it has been found that TrcA leads to ROS, as well as membrane permeabilisation in C. albicans [8]. ROS generation has been related to interaction of some plant defensins with sphingolipids, specifically GlcCer in fungal membranes [15,16].…”

“…Several antifungal peptides lead to the generation of ROS and morphological changes in fungi [15e17]. A number of these peptides have been shown to bind to the sphingolipid glucocylceramide (GlcCer), such as the radish defensin RsAFP1 [18] and string bean defensin PvD1 [15]. There is increasing evidence of alternative/additional modes of action and antimicrobial peptides may utilise more than one mode of action [19e23].…”