Variations in yeast plasma‐membrane lipid composition affect killing activity of three families of insect antifungal peptides

Abstract

Naturally occurring antimicrobial peptides and their synthetic analogues are promising candidates for new antifungal drugs. We focused on three groups of peptides isolated from the venom of bees and their synthetic analogues (lasioglossins, halictines and hylanines), which all rapidly permeabilised the plasma membrane. We compared peptides' potency against six pathogenic Candida species (C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, C. krusei and C. dubliniensis) and the non‐pathogenic model yeast Saccharomyces cerevisiae. Their activity was independent of the presence of the multidrug‐resistant pumps of C. glabrata but was influenced by the lipid composition of cell plasma membranes. Although the direct interaction of the peptides with ergosterol was negligible in comparison with amphotericin B, the diminished ergosterol content after terbinafine pretreatment resulted in an increased resistance of C. glabrata to the peptides. The tested peptides strongly interacted with phosphatidylglycerol, phosphatidic acid and cardiolipin and partly with phosphatidylinositol and phosphatidylethanolamine. The interactions between predominantly anionic phospholipids and cationic peptides indicated a mainly electrostatic binding of peptides to the membranes. The results obtained also pointed to a considerable role of the components of lipid rafts (composed from sphingolipids and ergosterol) in the interaction of yeast cells with the peptides.

Squalene lipotoxicity in a lipid droplet‐less yeast mutant is linked to plasma membrane dysfunction

“…The probe can thus distinguish between complete permeabilization of the plasma membrane and subtle r changes in the membrane structure. Differences in diS‐C 3 (3) accumulation indicated by fluorescence spectral shifts have been utili z ed for the assessment of the activity of membrane‐active drugs (Kodedová, Sigler, Lemire, & Gášková, ), effect of sterol composition on the properties of yeast plasma membrane (Kodedová & Sychrová, ) , or for studies of the activity of antimicrobial peptides (Kodedová, Valachovič, Csáky, & Sychrová, ). We applied this method to analyse the effect of squalene accumulation on the generation and maintenance of membrane potential in the WT and QM cells.…”

Styrylpyridinium Derivatives as New Potent Antifungal Drugs and Fluorescence Probes

“…The diS-C 3 (3) assay monitors the rate of influx of this cationic probe into cells, which is driven by the internal negative membrane potential in intact cells. A very rapid increase in staining suggests that the integrity of plasma membrane is disrupted and the probe enters cells independently of the membrane potential (Kodedová et al, 2011(Kodedová et al, , 2019. Moreover, comparing the changes in staining curves of wild-type and cdr1 cells upon the addition of the tested compounds should confirm the results of the growth test, i.e., the ability of Cdr1 to eliminate the tested styrylpyridinium compounds from cells.…”

“…The relative membrane potential and integrity of the yeast cell plasma membrane was estimated by a fluorescence assay based on the potential-dependent redistribution of the fluorescence probe diS-C 3 (3) (3,3 -dipropylthiacarbocyanine iodide) (Denksteinová et al, 1997;Gášková et al, 1998), as described in Kodedová et al (2019). The probe was added to a final concentration of 0.02 µM to yeast suspensions in CP buffer, and samples were occasionally gently stirred.…”

“…Washed C. glabrata DSY565 cells were resuspended in deionized water, stained with 2 µM styrylpyridinium derivatives and after cca 10 min observed with a Leica TCS SP8 WLL SMD confocal fluorescence microscope (objective HC PL APO CS2 63×/NA 1.20 with water immersion, λ ex = 405 nm, green fluorescence λ em = 480-650 nm, red fluorescence λ em = 650-800 nm). For the demonstration of the different accumulation of styrylpyridinium derivatives in yeast cells with damaged and intact plasma membranes, C. glabrata cells were resuspended in deionized water to OD 600 = 0.2 and treated with 3 µM octenidine dihydrochloride (ODDC), causing full permeabilization of yeast cells within 15 min (Kodedová et al, 2019). Then the cells were washed with deionized water and used as a model of killed cells for styrylpyridinium staining.…”

“…We observed larger growth inhibition zones formed in the presence of terbinafine combined with styrylpyridinium derivatives possessing longer aliphatic chains (-C 12 H 25 of compounds 2 and 3, -C 10 H 21 of drug 6) than only an octyl substituent (5). Styrylpyridinium derivatives with longer alkyl chains are probably more tightly anchored between the surrounding lipids in C. glabrata membranes, because this yeast incorporates almost identical proportions of lipids with C16 and C18 fatty acids, in contrast to other Candida species that predominantly contain C18 fatty acids in membrane lipids (Kodedová et al, 2019). Terbinafine treatment initiates a drop in C18 fatty acids content in favor of C16 and an increase in the saturation of fatty acids in C. glabrata (Kodedová et al, 2019), which could even strengthen the interaction between similarly long chains of styrylpyridinium compounds and membrane lipids.…”