The Membrane Activity of the Amphibian Temporin B Peptide Analog TB_KKG6K Sheds Light on the Mechanism That Kills Candida albicans

Abstract

Temporin B (TB) is a 13-amino-acid-long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We recently showed that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low micromolar concentrations and was neither hemolytic nor cytotoxic to mammalian cells in vitro. The present study aimed to shed light into its mechanism of action, with a focus on its fungal cell membrane activity. We utilized different fluorescent dyes to prove that it rapidly induces membrane depolarization and permeabilization. Studies on model membrane systems revealed that the TB analog undergoes hydrophobic and electrostatic membrane interactions, showing a preference for anionic lipids, and identified phosphatidylinositol and cardiolipin as possible peptide targets. Fluorescence microscopy using fluorescein isothiocyanate-labeled TB_KKG6K in the presence of the lipophilic dye FM4-64 indicated that the peptide compromises membrane integrity and rapidly enters C. albicans cells in an energy-independent manner. Peptide-treated cells analyzed by cryo-based electron microscopy exhibited no signs of cell lysis; however, subcellular structures had disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and supports its great potential as a future anti-Candida therapeutic. IMPORTANCE Fungal infections with the opportunistic human pathogen C. albicans are associated with high mortality rates in immunocompromised patients. This is partly due to the yeast's ability to rapidly develop resistance toward currently available antifungals. Small, cationic, membrane-active peptides are promising compounds to fight against resistance development, as many of them effectuate rapid fungal cell death. This fast killing is believed to hamper the development of resistance, as the fungi do not have sufficient time to adapt to the antifungal compound. We previously reported that the synthetic variant of the amphibian TB peptide, TB_KKG6K, rapidly kills C. albicans. In the current study, the mechanism of action of the TB analog was investigated. We show that this TB analog is membrane-active and impairs cell membrane function, highlighting its potential to be developed as an attractive alternative anti-C. albicans therapeutic that may hinder the development of resistance.

Keywords: Candida albicans; TB analog; Temporin B; antifungal peptide; depolarization; leakage; membrane activity; permeabilization; uptake.

FIG 1

Membrane depolarization potential of TB_KKG6K in C. albicans, determined with DiSC₃(5) dye. Filled symbols show results for TB peptide at 0.5 μM (▴), 1 μM (■), and 2 μM (●); open symbols show results with the controls Triton X-100 at 1% (wt/vol) (▽) and PCγ^C-terminal at 32 μM (◇). Compound addition is marked with an arrow, and the depolarization of the cell membrane was monitored over a 60-min time period. The arbitrary fluorescence units (AFU) shown have been normalized by subtracting the background fluorescence values of the C. albicans-DiSC₃(5) combination without compound addition (untreated control). The values represent the means ± standard deviation (SD) of fluorescence values collected from two independent experiments performed in technical duplicates.

FIG 2

Membrane permeabilization activity of TB_KKG6K in C. albicans detected with the SYTOX green uptake assay. Filled symbols show results with TB peptide at 0.5 μM (▴), 1 μM (■), or 2 μM (●); open symbols show results for the controls, octenidine at 2 μM (▽) or PCγ^C-terminal at 32 μM (◇). AFU were normalized by subtracting the background fluorescence of the medium with or without compounds in the absence of cells. AFU values depicted in the graph were further corrected by subtracting the fluorescence values of the C. albicans-SYTOX green combination without compound addition (untreated control). The values presented are the means ± SD determined from two independent experiments performed in technical duplicates.

FIG 3

Binding of FITC-TB_KKG6K to phosphoinositide phosphates. (A) The PIP strip was probed with 1.5 μg/mL of FITC-labeled TB_KKG6K, and binding of the peptide to the lipids was fluorometrically detected. The positive control for fluorescence signal detection is marked with an asterisk and represents 0.5 μg of peptide spotted onto the membrane. The PIP strip shown represents the result of one out of three independent experiments. (B) Relative quantification of signal intensities of the spots representing TB_KKG6K bound to lipids, compared to the blank, which was set as 0 AFU. AFU represent the means ± SD of fluorescence values quantified in three independent blots by ImageJ/FIJI. *, P ≤ 0.05. LPA, lysophosphatidic acid; LPC, lysophosphocholine; PIP, phosphoinositide phosphates; PE, phosphatidylethanolamine; PC, phosphatidylcholine; S1P, sphingosine-1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.

FIG 4

Results of in silico evaluation of peptide-membrane binding. (The helical wheel projections of TB_KKG6K and LL-37_13-37 [reference peptide] were performed with Membrane Protein Explorer mPEX [31]). The proposed partitioning of each peptide into the phospholipid bilayer (scheme in gray) is shown. The bilayer partitioning free energy (ΔG_W-OCT) is indicated in kilocalories per mole, and the hydrophobic moment (extent of amphipathicity) is indicated by μ for both peptides. Negatively charged residues are shown in red, positively charged are in blue, aliphatic are in green, polar are in yellow, and aromatic are in violet.

FIG 5

Laser scanning microscopic imaging of C. albicans stained with FM4-64 and treated with FITC-labeled TB_KKG6K in C. albicans. (A) The binding of 0.8 μM FM4-64 to the cell membrane and intracellular membranes was monitored in C. albicans incubated for 0 min and 60 min at 30°C. (B) Cells were exposed to 0.5 μM, 1 μM, and 2 μM peptide in the presence of 0.8 μM FM4-64 for 5 min at 30°C. Presumptive vacuoles and prevacuolar compartments are marked with arrowheads and small arrows, respectively. Scale bar, 5 μm (A) or 10 μm (B).

FIG 6

TB_KKG6K entry into C. albicans cells. Cells were exposed to 2 μM FITC-TB_KKG6K for 15 min at 30°C (standard condition), at 30°C in the presence of 100 μM CCCP (inhibition of respiration), or at 4°C (reduction of cellular metabolism) before fixation. As a control for energy-dependent uptake, C. albicans was incubated with 2 μM BODIPY-PAFB for 45 min under the same incubation conditions as described above and then fixed for fluorescence microscopy. BF, bright-field microscopy; FITC, fluorescence microscopy. Scale bar, 15 μm.

FIG 7

Changes in the cellular morphology of C. albicans in response to TB_KKG6K treatment. Electron micrographs of cryo-fixed C. albicans cells left untreated (controls) (A and B) or exposed to 30 μM TB_KKG6K (C to G) at 30°C for 60 min. Scale bars, 2 μm (A and C) or 500 nm (B, D, E, F, G). (A and B) Normal ultrastructure in untreated C. albicans serving as controls with nucleus (N), mitochondria (M), late endosomes (MVB), vacuoles (V), peroxisomes (PX), and abundant ribosomes and glycogen (not specifically highlighted) throughout the cytoplasm (the endoplasmic reticulum [ER] and Golgi apparatus are not depicted in those section planes). (C to G) Various patterns of subcellular degradation in TB analog-treated cells. (C) Overview showing only a few, visually intact cells (arrows). (D) Disintegrated mitochondria (M) are hardly recognizable, in contrast to numerous MVBs and strongly stained, small vesicles about 55 nm in width (arrowheads). (E) Damaged though recognizable mitochondria (M) with still-visible membrane remnants. (F) Ruptured envelope (double arrows) of the damaged nucleus (N). (G) Disintegrated mitochondria (M), MVB, and a vacuole (V), as well as strongly stained ≈55-nm-wide patches (arrowheads), which are presumably (endocytic) vesicles and/or glycogen rosettes and/or ribosomal aggregates.