Mode of action of the antimicrobial peptide Os on Candida albicans (ATCC 90028) biofilm formation

Abstract

Antimicrobial resistance is one of the biggest risks to global health, with an increasing number of infections becoming harder to treat. These include fungal infections, especially Candida albicans biofilms. Fungal biofilms are microbial communities composed of various cell types, bound to a substrate surface that produces a protective extracellular matrix, which makes treatment challenging. Increasing resistance, and the lag in the development of novel antifungal agents, has created a need for novel and alternative anti-mycotic agents, of which antifungal peptides (AFPs) show promise. In a preliminary study, Os, a tick derived AFP was shown to possess anti-biofilm activity against C. albicans. In this study, the ability of the antimicrobial peptide Os to inhibit biofilm formation was further investigated and its mode of action was explored, and compared to a shorter, amidated analogue of Os, Os(11-22)NH2, which previously has been shown to inhibit planktonic C. albicans cells and biofilms. Os displayed no antifungal activity against planktonic C. albicans cells at the tested concentrations (0.78 µM to 100 µM) in RPMI-1640. In a 24h established C. albicans biofilm, Os exhibited a 50% biofilm inhibitory concentration (BIC50) value of 46 µM, which was approximately 2 fold better than reported for Os(11-22)NH2. At the BIC50, the mode of action was further investigated. Inverted light microscopy of crystal violet stained Os treated biofilms indicated that Os targets the growth and development of adhered yeast cells. Using the dichloro-dihydro-fluorescein diacetate assay, it was determined that treatment of C. albicans biofilms with Os induced reactive oxygen species (ROS) formation. However, the addition of the antioxidant ascorbic acid (AA) significantly (p < 0.0001) decreased the presence of endogenous ROS in treated cells, but this did not lead to a decrease in the biofilm inhibitiory activity of Os. Instead, addition of AA significantly (p < 0.05) enhanced the activity of Os. In contrast to Os(11-22)NH2, Os did not disrupt membrane structure, evaluated using propidium iodide staining. Using 5FAM-labelled Os and confocal laser scanning microscopy, Os was located to the plasma membrane or cell wall, bound to intracellular structures and accumulated in the cytoplasm. The effects of Os on the ultrastructure of yeast, pseudohyphae and hyphae in C. albicans biofilms was evaluated with scanning electron microscopy. Os caused cell shrinkage in yeast and pseudohyphal cells, as well as cell wall or plasma membrane cracks/tears in yeast and hyphal cells. The formation of pits was also observed in pseduohyphal cells. Surface protuberances were apparent on the surface of Os treated hyphal cells. In conclusion, although Os negatively affects the structures of the various cell types found within a C. albicans biofilm by associating with membranes and translocation into cells, its biofilm inhibitory activity is not due to membrane permeabilisation or endogenous ROS production. More investigation into the mode of action of this peptide is required.