Exploring the mode of action of an antimicrobial peptide, Os-C, in planktonic Candida albicans ATCC 90028

Abstract

Fungal pathogen resistance has increased rapidly in recent years, creating an urgent need for new antifungal drugs. Research has identified multifunctional antimicrobial peptides (AMPs) as promising candidates due to their broad-spectrum of cellular targets. The aim of this study was to further investigate the mode of action of a tick-derived AMP, Os-C, against planktonic Candida albicans (C. albicans). Os-C has previously shown inhibitory activity against Gram-positive and -negative bacteria as well as planktonic and biofilm forms of C. albicans but activity was reduced in the presence of salts. Preliminary mode of action studies indicated that Os-C kills C. albicans cells within 30 minutes by interacting with cell wall polysaccharides, translocating to the cytosol in an ATP-dependent manner and inducing the production of reactive oxygen species (ROS). For rapid screening and further mode of action studies of salt-sensitive AMPs a modified micro-broth dilution assay was developed. With this assay in 10% RPMI-1640 the minimum concentration of Amphotericin B (AmB; antifungal drug control) and melittin (peptide control) that inhibits 50% of planktonic C. albicans cells (MIC50) was 0.5 ± 0.06 µM and 0.98 ± 0.14 µM respectively, and under these conditions the MIC50 of Os-C was 4.69 ± 0.18 µM. The effect of AmB and Os-C at the MIC25, MIC50 and MIC75 on the morphology and ultrastructure of C. albicans was determined with scanning (SEM) and transmission electron microscopy (TEM) respectively. AmB treated C. albicans presented with an irregular cell wall structure, budding scars, bud formation and eventual collapse of cellular structure at high concentrations. In contrast, Os-C caused cytoplasmic retraction, depression in cell surfaces, cellular extravasation, the formation of buds, small vacuoles adjacent to the cell membrane and intracellular granulation. The differences between the ultrastructural effects of AmB and Os-C indicate different modes of action. The ability of Os-C to bind laminarin and mannan, components of the cell wall of C. albicans, was investigated with isothermal titration calorimetry (ITC). No binding occurred between Os-C and either of the polysaccharides at low, physiologically relevant concentrations. Using the modified micro-broth assay binding of Os-C (20 µM) to laminarin at 40 mM was observed. However, binding at such a high concentration of laminarin may not be of physiological value. The ability to induce membrane depolarization was evaluated using the probe, bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)]. Depolarization by Os-C was minimal compared with melittin, an AMP with well-described cell membrane depolarization and permeabilization activity. Flow cytometry and confocal laser scanning microscopy (CLSM) with 5-FAM-labelled-Os-C and a vacuole specific dye, Cell tracker blue (CTr) was undertaken to evaluate uptake and effects on the vacuoles. Os-C was able to enter and successfully carry the fluorescent 5-FAM tag into the cells. Os-C accumulated intracellularly and caused moderate vacuolar break down independent of concentration. This may be the result of ROS production, rather than the direct action of Os-C on the membrane or inhibition of associated biochemical processes. In conclusion, this study showed that Os-C does not bind laminarin or mannan but accumulates on the cell membrane before translocating into the cytoplasm via ATP-mediated endocytosis. Os-C accumulates in the cytoplasm and induces ROS that cause moderate vacuole degradation. ROS production also leads to changes in cellular morphology, moderate membrane permeabilization and pore formation, small vacuole accumulation associated with the cell membrane, degradation of intracellular organelles and the formation of hyphal filaments. All these effects eventually lead to cell death probably due to apoptosis.