Abstract:
Candida albicans is a fungus found in the normal biota of humans, but in immuno-compromised
individuals, C. albicans forms complex biofilms on the surface of medical prosthetics, skin, oral
cavities, the urinary tract, and other epithelial cell layers. Biofilms and the development of drug
resistance has limited treatment options. Antimicrobial peptides (AMPs) are increasingly becoming
attractive therapeutic agents for the treatment of these infections due to their multifunctional
properties, multiple cellular targets, and the lower incidence of resistance development.
Previous studies have shown that Os, an AMP derived from the tick defensin OsDef2, has antifungal
activity against C. albicans. Preliminary antifungal mode of action studies indicated that Os induces
the formation of reactive oxygen species although not a primary mode of killing. Os causes membrane
permeabilization, which is inhibited by an excess of free laminarin and mannan. Furthermore, Os was
shown to bind plasmid DNA but was inactive in high salt conditions.
The aim of this study was to further explore the mode of action of Os in planktonic C. albicans (ATCC
90028) cells. A modified microbroth dilution assay was developed to allow rapid screening of salt
sensitive AMPs such as Os. With this method the IC50 of the positive control, amphotericin B (AmpB),
and Os were determined as 0.547 ± 0.056 μM and 1.163 ± 0.116 μM, respectively.
The effects of AmpB and Os on cellular morphology were evaluated using scanning electron
microscopy and transmission electron microscopy at their respective IC25, IC50 and IC75 values. When
comparing the effects of Os with AmpB on the cell wall and membrane, Os had more severe and nonspecific
effects. Os induced the formation of pits on the cell surface and pores in the cell membrane,
as well as increased budding scars.
Using isothermal titration calorimetry, no interaction between Os and the fungal cell wall components,
mannan and laminarin, could be detected. Factors such as the lack of tryptophan and aspartate
residues as well as β-sheet secondary structures may account for the lack of interaction. However,
with the modified microbroth dilution assay in the presence of excess of mannan or laminarin
(20 mg/mL), reduced activity from Os was observed. The formation of soluble macro-complexes
between Os and the cell wall components at high concentrations may account for reduced activity.
The ability of Os to cause membrane depolarization was evaluated with bis-(1,3-dibutylbarbituric acid)
trimethine oxonol. The control, melittin, caused a linear increase in depolarization with a significant
increase at 0.63 μM, while Os caused a sigmoidal increase in depolarization with a significant increase
at 2.5 μM. Therefore, membrane depolarization occurs following membrane permeabilization which
occurs at 2 μM.
Finally, the localisation of 0.5 μM and 6.4 μM (IC25, IC75) 5-FAM-Os, and concurrently the effect on
vacuoles loaded with CellTracker Blue-CMAC, was determined with flow cytometry and confocal laser scanning microscopy (CLSM). Findings were that Os, at a concentration below its IC50, binds to the
cell membrane, then translocates and binds DNA. At a concentration above its IC50, Os accumulates
in the cytoplasm and causes destruction of membranes, including that of vacuoles, leading to cell
death.
In conclusion, this study shows that Os is a membrane acting AMP that can be further developed for
clinical application as an antifungal drug.
