The membrane activity of the amphibian Temporin B peptide analog TB_KKG6K sheds light on the mechanism that kills Candida albicans

Kakar, A.; Sastré-Velásquez, L. E.; Hess, M. W.; Galgóczy, L.; Papp, C.; Holzknecht, J.; Romanelli, A.; Váradi, G.; Malanovic, N.; Marx, F.

doi:10.1101/2022.06.15.496139

bioRxiv · 10.1101/2022.06.15.496139

The membrane activity of the amphibian Temporin B peptide analog TB_KKG6K sheds light on the mechanism that kills Candida albicans

Kakar, A.·Sastré-Velásquez, L. E.·Hess, M. W.·Galgóczy, L.·Papp, C.·Holzknecht, J.·Romanelli, A.·Váradi, G.·Malanovic, N.·Marx, F.

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Abstract

Temporin B (TB) is a 13 amino acid long, cationic peptide secreted by the granular glands of the European frog Rana temporaria. We could recently show that the modified TB peptide analog TB_KKG6K rapidly killed planktonic and sessile Candida albicans at low {micro}M 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 FITC-labelled 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 were disintegrated, suggesting that intracellular activity may form part of the killing mechanism of the peptide. Taken together, this study proved that the TB_KKG6K compromises C. albicans membrane function, which explains the previously observed rapid, fungicidal mode of action and promises its great potential as a future anti-Candida therapeutic. ImportanceFungal infections with the opportunistic human pathogen C. albicans are associated with high moratility rates in immunocompromised patients. This is partly due to the yeasts ability to rapidly develop resistance towards 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 prevously reported that the synthetic variant of the amphibian Temporin B 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, which may hinder the development of resistance.

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Kakar, A., Sastré-Velásquez, L. E., Hess, M. W., Galgóczy, L., Papp, C., Holzknecht, J., Romanelli, A., Váradi, G., Malanovic, N., Marx, F.. 2022-06-15. The membrane activity of the amphibian Temporin B peptide analog TB_KKG6K sheds light on the mechanism that kills Candida albicans. https://doi.org/10.1101/2022.06.15.496139

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